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Biochemistry
An introduction
Contents
Articles
Cells and water
1
Biochemistry 1
Cells 11
Water 21
Structural Biochemistry
41
Nucleic acids
42
Nucleic acid 42
RNA 45
DNA 52
Proteins and amino acids
72
Protein 72
Amino acid 84
Properties of the twenty amino acids 97
Myoglobin 107
Hemoglobin 112
Enzyme mechanisms
129
Enzyme catalysis 129
Enzyme kinetics
137
Enzyme kinetics 137
Lipids and membranes
151
Lipid 151
Biological membrane 159
Membrane protein 161
Cell membrane 165
Carbohydrate structure
172
Carbohydrate 172
Polysaccharide 178
Intermediary metabolism
184
Metabolism
185
Overview of metabolism 185
Carbohydrate metabolism
200
Glycolysis 200
Gluconeogenesis 214
Glycogen 218
Pentose phosphate pathway 222
Citric acid cycle
226
Citric acid cycle 226
Oxidative phosphorylation
233
Oxidative phosphorylation 233
Photosynthesis
245
Photosynthesis 245
Lipid metabolism
259
Fatty acid synthesis 259
Lipogenesis 267
Acetyl-CoA carboxylase 269
Fatty acid degradation 276
Beta oxidation 278
Nitrogen metabolism
283
Nitrogen fixation 283
Amino acid synthesis 288
Nucleotide 295
Urea cycle 301
Integration of metabolism
305
Hormone 305
Signal transduction 309
Diabetes mellitus 316
Informational Macromolecules
327
DNA synthesis and repair
328
DNA replication 328
DNA repair 335
Oncogenes 346
RNA synthesis and processing
350
Transcription 350
Regulation of gene expression 356
Protein synthesis and modifications
362
Translation 362
Posttranslational modification 366
Proteolysis 370
Proteasome 375
References
Article Sources and Contributors 386
Image Sources, Licenses and Contributors 398
Article Licenses
License 406
1
Cells and water
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes within, and relating to,
living organisms.
[1]
By controlling information flow through biochemical signaling and the flow of chemical energy
through metabolism, biochemical processes give rise to the complexity of life. Over the last 40 years biochemistry
has become so successful at explaining living processes that now almost all areas of the life sciences from botany to
medicine are engaged in biochemical research.
[2]
Today the main focus of pure biochemistry is in understanding how
biological molecules give rise to the processes that occur within living cells, which in turn relates greatly to the study
and understanding of whole organisms.
Biochemistry is closely related to molecular biology, the study of the molecular mechanisms by which genetic
information encoded in DNA is able to result in the processes of life. Depending on the exact definition of the terms
used, molecular biology can be thought of as a branch of biochemistry, or biochemistry as a tool with which to
investigate and study molecular biology.
Much of biochemistry deals with the structures, functions and interactions of biological macromolecules, such as
proteins, nucleic acids, carbohydrates and lipids, which provide the structure of cells and perform many of the
functions associated with life. The chemistry of the cell also depends on the reactions of smaller molecules and ions.
These can be inorganic, for example water and metal ions, or organic, for example the amino acids which are used to
synthesise proteins. The mechanisms by which cells harness energy from their environment via chemical reactions
are known as metabolism.
History
Gerty Cori and Carl Cori jointly won the
Nobel Prize in 1947 for their discovery of
the Cori cycle at RPMI.
It once was generally believed that life and its materials had some essential
property or substance distinct from any found in non-living matter, and it
was thought that only living beings could produce the molecules of
life.
[citation needed]
Then, in 1828, Friedrich Whler published a paper on the
synthesis of urea, proving that organic compounds can be created
artificially.
[]
The dawn of biochemistry may have been the discovery of the first enzyme,
diastase (today called amylase), in 1833 by Anselme Payen.
[3]
Eduard
Buchner contributed the first demonstration of a complex biochemical
process outside of a cell in 1896: alcoholic fermentation in cell extracts of
yeast.
[4]
Although the term "biochemistry" seems to have been first used in
1882, it is generally accepted that the formal coinage of biochemistry
occurred in 1903 by Carl Neuberg, a German chemist.
[]
Since then,
biochemistry has advanced, especially since the mid-20th century, with the
development of new techniques such as chromatography, X-ray diffraction,
dual polarisation interferometry, NMR spectroscopy, radioisotopic labeling,
electron microscopy, and molecular dynamics simulations. These techniques allowed for the discovery and detailed
analysis of many molecules and metabolic pathways of the cell, such as glycolysis and the Krebs cycle (citric acid
cycle).
Biochemistry
2
Another significant historic event in biochemistry is the discovery of the gene and its role in the transfer of
information in the cell. This part of biochemistry is often called molecular biology.
[5]
In the 1950s, James D.
Watson, Francis Crick, Rosalind Franklin, and Maurice Wilkins were instrumental in solving DNA structure and
suggesting its relationship with genetic transfer of information.
[6]
In 1958, George Beadle and Edward Tatum
received the Nobel Prize for work in fungi showing that one gene produces one enzyme.
[]
In 1988, Colin Pitchfork
was the first person convicted of murder with DNA evidence, which led to growth of forensic science.
[]
More
recently, Andrew Z. Fire and Craig C. Mello received the 2006 Nobel Prize for discovering the role of RNA
interference (RNAi), in the silencing of gene expression.
[]
Starting materials: the chemical elements of life
Around two dozen of the 92 naturally occurring chemical elements are essential to various kinds of biological life.
Most rare elements on Earth are not needed by life (exceptions being selenium and iodine), while a few common
ones (aluminum and titanium) are not used. Most organisms share element needs, but there are a few differences
between plants and animals. For example ocean algae use bromine but land plants and animals seem to need none.
All animals require sodium, but some plants do not. Plants need boron and silicon, but animals may not (or may need
ultra-small amounts).
Just six elementscarbon, hydrogen, nitrogen, oxygen, calcium, and phosphorusmake up almost 99% of the mass
of a human body (see composition of the human body for a complete list). In addition to the six major elements that
compose most of the human body, humans require smaller amounts of possibly 18 more.
[7]
Biomolecules
The four main classes of molecules in biochemistry are carbohydrates, lipids, proteins, and nucleic acids. Many
biological molecules are polymers: in this terminology, monomers are relatively small micromolecules that are
linked together to create large macromolecules, which are known as polymers. When monomers are linked together
to synthesize a biological polymer, they undergo a process called dehydration synthesis. Different macromolecules
can assemble in larger complexes, often needed for biological activity.
Carbohydrates
A molecule of sucrose (glucose + fructose), a
disaccharide.
Carbohydrates are made from monomers called monosaccharides.
Some of these monosaccharides include glucose (C
6
H
12
O
6
), fructose
(C
6
H
12
O
6
), and deoxyribose (C
5
H
10
O
4
). When two monosaccharides
undergo dehydration synthesis, water is produced, as two hydrogen
atoms and one oxygen atom are lost from the two monosaccharides'
hydroxyl group.
Biochemistry
3
Lipids
A triglyceride with a glycerol molecule
on the left and three fatty acids coming
off it.
Lipids are usually made from one molecule of glycerol combined with other
molecules. In triglycerides, the main group of bulk lipids, there is one
molecule of glycerol and three fatty acids. Fatty acids are considered the
monomer in that case, and may be saturated (no double bonds in the carbon
chain) or unsaturated (one or more double bonds in the carbon chain).
Lipids, especially phospholipids, are also used in various pharmaceutical
products, either as co-solubilisers (e.g., in parenteral infusions) or else as drug
carrier components (e.g., in a liposome or transfersome).
Proteins
The general structure of an -amino acid,
with the amino group on the left and the
carboxyl group on the right.
Proteins are very large molecules macro-biopolymers made from
monomers called amino acids. There are 20 standard amino acids, each
containing a carboxyl group, an amino group, and a side-chain (known as
an "R" group). The "R" group is what makes each amino acid different, and
the properties of the side-chains greatly influence the overall
three-dimensional conformation of a protein. When amino acids combine,
they form a special bond called a peptide bond through dehydration
synthesis, and become a polypeptide, or protein.
In order to determine whether two proteins are related, or in other words to
decide whether they are homologous or not, scientists use
sequence-comparison methods. Methods like Sequence Alignments and
Structural Alignments are powerful tools that help scientists identify homologies between related molecules.
[]
The relevance of finding homologies among proteins goes beyond forming an evolutionary pattern of protein
families. By finding how similar two protein sequences are, we acquire knowledge about their structure and
therefore their function.
Biochemistry
4
Nucleic acids
The structure of deoxyribonucleic acid (DNA), the
picture shows the monomers being put together.
Nucleic acids are the molecules that make up DNA, an extremely
important substance that all cellular organisms use to store their
genetic information. The most common nucleic acids are
deoxyribonucleic acid and ribonucleic acid. Their monomers are
called nucleotides. The most common nucleotides are adenine,
cytosine, guanine, thymine, and uracil. Adenine binds with
thymine and uracil; Thymine binds only with adenine; and
cytosine and guanine can bind only with each other.
Carbohydrates
The function of carbohydrates includes energy storage and
providing structure. Sugars are carbohydrates, but not all
carbohydrates are sugars. There are more carbohydrates on Earth
than any other known type of biomolecule; they are used to store
energy and genetic information, as well as play important roles in
cell to cell interactions and communications.
Monosaccharides
Glucose
The simplest type of carbohydrate is a monosaccharide, which among
other properties contains carbon, hydrogen, and oxygen, mostly in a
ratio of 1:2:1 (generalized formula C
n
H
2n
O
n
, where n is at least 3).
Glucose, one of the most important carbohydrates, is an example of a
monosaccharide. So is fructose, the sugar commonly associated with
the sweet taste of fruits.
[][a]
Some carbohydrates (especially after
condensation to oligo- and polysaccharides) contain less carbon
relative to H and O, which still are present in 2:1 (H:O) ratio.
Monosaccharides can be grouped into aldoses (having an aldehyde group at the end of the chain, e.g. glucose) and
ketoses (having a keto group in their chain; e.g. fructose). Both aldoses and ketoses occur in an equilibrium (starting
with chain lengths of C4) cyclic forms. These are generated by bond formation between one of the hydroxyl groups
of the sugar chain with the carbon of the aldehyde or keto group to form a hemiacetal bond. This leads to saturated
five-membered (in furanoses) or six-membered (in pyranoses) heterocyclic rings containing one O as heteroatom.
Disaccharides
Sucrose: ordinary table sugar and probably the
most familiar carbohydrate.
Two monosaccharides can be joined together using dehydration
synthesis, in which a hydrogen atom is removed from the end of one
molecule and a hydroxyl group (OH) is removed from the other; the
remaining residues are then attached at the sites from which the atoms
were removed. The HOH or H
2
O is then released as a molecule of
water, hence the term dehydration. The new molecule, consisting of
two monosaccharides, is called a disaccharide and is conjoined
together by a glycosidic or ether bond. The reverse reaction can also
Biochemistry
5
occur, using a molecule of water to split up a disaccharide and break the glycosidic bond; this is termed hydrolysis.
The most well-known disaccharide is sucrose, ordinary sugar (in scientific contexts, called table sugar or cane sugar
to differentiate it from other sugars). Sucrose consists of a glucose molecule and a fructose molecule joined together.
Another important disaccharide is lactose, consisting of a glucose molecule and a galactose molecule. As most
humans age, the production of lactase, the enzyme that hydrolyzes lactose back into glucose and galactose, typically
decreases. This results in lactase deficiency, also called lactose intolerance.
Sugar polymers are characterised by having reducing or non-reducing ends. A reducing end of a carbohydrate is a
carbon atom that can be in equilibrium with the open-chain aldehyde or keto form. If the joining of monomers takes
place at such a carbon atom, the free hydroxy group of the pyranose or furanose form is exchanged with an
OH-side-chain of another sugar, yielding a full acetal. This prevents opening of the chain to the aldehyde or keto
form and renders the modified residue non-reducing. Lactose contains a reducing end at its glucose moiety, whereas
the galactose moiety form a full acetal with the C4-OH group of glucose. Saccharose does not have a reducing end
because of full acetal formation between the aldehyde carbon of glucose (C1) and the keto carbon of fructose (C2).
Oligosaccharides and polysaccharides
Cellulose as polymer of -D-glucose
When a few (around three to six) monosaccharides are joined together,
it is called an oligosaccharide (oligo- meaning "few"). These
molecules tend to be used as markers and signals, as well as having
some other uses. Many monosaccharides joined together make a
polysaccharide. They can be joined together in one long linear chain,
or they may be branched. Two of the most common polysaccharides
are cellulose and glycogen, both consisting of repeating glucose
monomers.
Cellulose is made by plants and is an important structural component of their cell walls. Humans can neither
manufacture nor digest it.
Glycogen, on the other hand, is an animal carbohydrate; humans and other animals use it as a form of energy
storage.
Use of carbohydrates as an energy source
Glucose is the major energy source in most life forms. For instance, polysaccharides are broken down into their
monomers (glycogen phosphorylase removes glucose residues from glycogen). Disaccharides like lactose or sucrose
are cleaved into their two component monosaccharides.
Glycolysis (anaerobic)
Glucose is mainly metabolized by a very important ten-step pathway called glycolysis, the net result of which is to
break down one molecule of glucose into two molecules of pyruvate; this also produces a net two molecules of ATP,
the energy currency of cells, along with two reducing equivalents in the form of converting NAD
+
to NADH. This
does not require oxygen; if no oxygen is available (or the cell cannot use oxygen), the NAD is restored by converting
the pyruvate to lactate (lactic acid) (e.g., in humans) or to ethanol plus carbon dioxide (e.g., in yeast). Other
monosaccharides like galactose and fructose can be converted into intermediates of the glycolytic pathway.
[8]
Biochemistry
6
Aerobic
In aerobic cells with sufficient oxygen, as in most human cells, the pyruvate is further metabolized. It is irreversibly
converted to acetyl-CoA, giving off one carbon atom as the waste product carbon dioxide, generating another
reducing equivalent as NADH. The two molecules acetyl-CoA (from one molecule of glucose) then enter the citric
acid cycle, producing two more molecules of ATP, six more NADH molecules and two reduced (ubi)quinones (via
FADH
2
as enzyme-bound cofactor), and releasing the remaining carbon atoms as carbon dioxide. The produced
NADH and quinol molecules then feed into the enzyme complexes of the respiratory chain, an electron transport
system transferring the electrons ultimately to oxygen and conserving the released energy in the form of a proton
gradient over a membrane (inner mitochondrial membrane in eukaryotes). Thus, oxygen is reduced to water and the
original electron acceptors NAD
+
and quinone are regenerated. This is why humans breathe in oxygen and breathe
out carbon dioxide. The energy released from transferring the electrons from high-energy states in NADH and quinol
is conserved first as proton gradient and converted to ATP via ATP synthase. This generates an additional 28
molecules of ATP (24 from the 8 NADH + 4 from the 2 quinols), totaling to 32 molecules of ATP conserved per
degraded glucose (two from glycolysis + two from the citrate cycle). It is clear that using oxygen to completely
oxidize glucose provides an organism with far more energy than any oxygen-independent metabolic feature, and this
is thought to be the reason why complex life appeared only after Earth's atmosphere accumulated large amounts of
oxygen.
Gluconeogenesis
In vertebrates, vigorously contracting skeletal muscles (during weightlifting or sprinting, for example) do not receive
enough oxygen to meet the energy demand, and so they shift to anaerobic metabolism, converting glucose to lactate.
The liver regenerates the glucose, using a process called gluconeogenesis. This process is not quite the opposite of
glycolysis, and actually requires three times the amount of energy gained from glycolysis (six molecules of ATP are
used, compared to the two gained in glycolysis). Analogous to the above reactions, the glucose produced can then
undergo glycolysis in tissues that need energy, be stored as glycogen (or starch in plants), or be converted to other
monosaccharides or joined into di- or oligosaccharides. The combined pathways of glycolysis during exercise,
lactate's crossing via the bloodstream to the liver, subsequent gluconeogenesis and release of glucose into the
bloodstream is called the Cori cycle.
[9]
Proteins
A schematic of hemoglobin. The red and
blue ribbons represent the protein globin;
the green structures are the heme groups.
Like carbohydrates, some proteins perform largely structural roles. For
instance, movements of the proteins actin and myosin ultimately are
responsible for the contraction of skeletal muscle. One property many
proteins have is that they specifically bind to a certain molecule or class of
moleculesthey may be extremely selective in what they bind. Antibodies
are an example of proteins that attach to one specific type of molecule. In
fact, the enzyme-linked immunosorbent assay (ELISA), which uses
antibodies, is currently one of the most sensitive tests modern medicine
uses to detect various biomolecules. Probably the most important proteins,
however, are the enzymes. These molecules recognize specific reactant
molecules called substrates; they then catalyze the reaction between them.
By lowering the activation energy, the enzyme speeds up that reaction by a
rate of 10
11
or more: a reaction that would normally take over 3,000 years
to complete spontaneously might take less than a second with an enzyme.
The enzyme itself is not used up in the process, and is free to catalyze the same reaction with a new set of substrates.
Using various modifiers, the activity of the enzyme can be regulated, enabling control of the biochemistry of the cell
Biochemistry
7
as a whole.
In essence, proteins are chains of amino acids. An amino acid consists of a carbon atom bound to four groups. One is
an amino group, NH
2
, and one is a carboxylic acid group, COOH (although these exist as NH
3
+
and COO

under physiologic conditions). The third is a simple hydrogen atom. The fourth is commonly denoted "R" and is
different for each amino acid. There are twenty standard amino acids. Some of these have functions by themselves or
in a modified form; for instance, glutamate functions as an important neurotransmitter.
Generic amino acids (1) in neutral form, (2) as they exist physiologically, and (3) joined
together as a dipeptide.
Amino acids can be joined together via
a peptide bond. In this dehydration
synthesis, a water molecule is removed
and the peptide bond connects the
nitrogen of one amino acid's amino
group to the carbon of the other's
carboxylic acid group. The resulting
molecule is called a dipeptide, and
short stretches of amino acids (usually,
fewer than thirty) are called peptides or polypeptides. Longer stretches merit the title proteins. As an example, the
important blood serum protein albumin contains 585 amino acid residues.
[]
The structure of proteins is traditionally described in a hierarchy of four levels. The primary structure of a protein
simply consists of its linear sequence of amino acids; for instance,
"alanine-glycine-tryptophan-serine-glutamate-asparagine-glycine-lysine-". Secondary structure is concerned with
local morphology (morphology being the study of structure). Some combinations of amino acids will tend to curl up
in a coil called an -helix or into a sheet called a -sheet; some -helixes can be seen in the hemoglobin schematic
above. Tertiary structure is the entire three-dimensional shape of the protein. This shape is determined by the
sequence of amino acids. In fact, a single change can change the entire structure. The alpha chain of hemoglobin
contains 146 amino acid residues; substitution of the glutamate residue at position 6 with a valine residue changes
the behavior of hemoglobin so much that it results in sickle-cell disease. Finally, quaternary structure is concerned
with the structure of a protein with multiple peptide subunits, like hemoglobin with its four subunits. Not all proteins
have more than one subunit.
[10]
Ingested proteins are usually broken up into single amino acids or dipeptides in the small intestine, and then
absorbed. They can then be joined together to make new proteins. Intermediate products of glycolysis, the citric acid
cycle, and the pentose phosphate pathway can be used to make all twenty amino acids, and most bacteria and plants
possess all the necessary enzymes to synthesize them. Humans and other mammals, however, can synthesize only
half of them. They cannot synthesize isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan,
and valine. These are the essential amino acids, since it is essential to ingest them. Mammals do possess the enzymes
to synthesize alanine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine, and tyrosine, the
nonessential amino acids. While they can synthesize arginine and histidine, they cannot produce it in sufficient
amounts for young, growing animals, and so these are often considered essential amino acids.
If the amino group is removed from an amino acid, it leaves behind a carbon skeleton called an -keto acid.
Enzymes called transaminases can easily transfer the amino group from one amino acid (making it an -keto acid) to
another -keto acid (making it an amino acid). This is important in the biosynthesis of amino acids, as for many of
the pathways, intermediates from other biochemical pathways are converted to the -keto acid skeleton, and then an
amino group is added, often via transamination. The amino acids may then be linked together to make a protein.
[11]
A similar process is used to break down proteins. It is first hydrolyzed into its component amino acids. Free
ammonia (NH
3
), existing as the ammonium ion (NH
4
+
) in blood, is toxic to life forms. A suitable method for
excreting it must therefore exist. Different strategies have evolved in different animals, depending on the animals'
needs. Unicellular organisms, of course, simply release the ammonia into the environment. Likewise, bony fish can
Biochemistry
8
release the ammonia into the water where it is quickly diluted. In general, mammals convert the ammonia into urea,
via the urea cycle.
[]
Lipids
The term lipid comprises a diverse range of molecules and to some extent is a catchall for relatively water-insoluble
or nonpolar compounds of biological origin, including waxes, fatty acids, fatty-acid derived phospholipids,
sphingolipids, glycolipids, and terpenoids (e.g., retinoids and steroids). Some lipids are linear aliphatic molecules,
while others have ring structures. Some are aromatic, while others are not. Some are flexible, while others are
rigid.
[12]
Most lipids have some polar character in addition to being largely nonpolar. In general, the bulk of their structure is
nonpolar or hydrophobic ("water-fearing"), meaning that it does not interact well with polar solvents like water.
Another part of their structure is polar or hydrophilic ("water-loving") and will tend to associate with polar solvents
like water. This makes them amphiphilic molecules (having both hydrophobic and hydrophilic portions). In the case
of cholesterol, the polar group is a mere -OH (hydroxyl or alcohol). In the case of phospholipids, the polar groups are
considerably larger and more polar, as described below.
Lipids are an integral part of our daily diet. Most oils and milk products that we use for cooking and eating like
butter, cheese, ghee etc., are composed of fats. Vegetable oils are rich in various polyunsaturated fatty acids (PUFA).
Lipid-containing foods undergo digestion within the body and are broken into fatty acids and glycerol, which are the
final degradation products of fats and lipids.
Nucleic acids
A nucleic acid is a complex, high-molecular-weight biochemical macromolecule composed of nucleotide chains that
convey genetic information. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA). Nucleic acids are found in all living cells and viruses. Aside from the genetic material of the cell, nucleic
acids often play a role as second messengers, as well as forming the base molecule for adenosine triphosphate, the
primary energy-carrier molecule found in all living organisms.
Nucleic acid, so called because of its prevalence in cellular nuclei, is the generic name of the family of biopolymers.
The monomers are called nucleotides, and each consists of three components: a nitrogenous heterocyclic base (either
a purine or a pyrimidine), a pentose sugar, and a phosphate group. Different nucleic acid types differ in the specific
sugar found in their chain (e.g., DNA or deoxyribonucleic acid contains 2-deoxyriboses). Also, the nitrogenous bases
possible in the two nucleic acids are different: adenine, cytosine, and guanine occur in both RNA and DNA, while
thymine occurs only in DNA and uracil occurs in RNA.
[13]
Biochemistry
9
Relationship to other "molecular-scale" biological sciences
Schematic relationship between biochemistry, genetics, and
molecular biology
Researchers in biochemistry use specific techniques
native to biochemistry, but increasingly combine these
with techniques and ideas developed in the fields of
genetics, molecular biology and biophysics. There has
never been a hard-line between these disciplines in
terms of content and technique. Today, the terms
molecular biology and biochemistry are nearly
interchangeable. The following figure is a schematic
that depicts one possible view of the relationship
between the fields:
Biochemistry is the study of the chemical substances
and vital processes occurring in living organisms.
Biochemists focus heavily on the role, function, and
structure of biomolecules. The study of the
chemistry behind biological processes and the
synthesis of biologically active molecules are
examples of biochemistry.
Genetics is the study of the effect of genetic differences on organisms. Often this can be inferred by the absence
of a normal component (e.g., one gene). The study of "mutants" organisms with a changed gene that leads to the
organism being different with respect to the so-called "wild type" or normal phenotype. Genetic interactions
(epistasis) can often confound simple interpretations of such "knock-out" or "knock-in" studies.
Molecular biology is the study of molecular underpinnings of the process of replication, transcription and
translation of the genetic material. The central dogma of molecular biology where genetic material is transcribed
into RNA and then translated into protein, despite being an oversimplified picture of molecular biology, still
provides a good starting point for understanding the field. This picture, however, is undergoing revision in light of
emerging novel roles for RNA.
[]
Chemical Biology seeks to develop new tools based on small molecules that allow minimal perturbation of
biological systems while providing detailed information about their function. Further, chemical biology employs
biological systems to create non-natural hybrids between biomolecules and synthetic devices (for example
emptied viral capsids that can deliver gene therapy or drug molecules).
Notes
a.
^
Fructose is not the only sugar found in fruits. Glucose and sucrose are also found in varying quantities in various
fruits, and indeed sometimes exceed the fructose present. For example, 32% of the edible portion of date is glucose,
compared with 23.70% fructose and 8.20% sucrose. However, peaches contain more sucrose (6.66%) than they do
fructose (0.93%) or glucose (1.47%).
[14]
References
[1] http:/ / portal. acs.org/ portal/ acs/ corg/ content?_nfpb=true& _pageLabel=PP_ARTICLEMAIN& node_id=1188&
content_id=CTP_003379& use_sec=true& sec_url_var=region1& __uuid=aa3f2aa3-8047-4fa2-88b8-32ffcad3a93e
[2] http:/ / www. biochemistry. org/ Education/ Careers/ Schoolsandcolleges/ Whatisbiochemistry. aspx
[3] [3] Hunter (2000), p. 75.
[4] Hunter (2000), pp. 9698.
[5] [5] Tropp (2012), p. 2.
[6] Tropp (2012), pp. 1920.
Biochemistry
10
[7] Ultratrace minerals. Authors: Nielsen, Forrest H. USDA, ARS Source: Modern nutrition in health and disease / editors, Maurice E. Shils ... et
al.. Baltimore : Williams & Wilkins, c1999., p. 283-303. Issue Date: 1999 URI: (http:/ / hdl. handle. net/ 10113/ 46493)
[8] Fromm and Hargrove (2012), pp. 163180.
[9] Fromm and Hargrove (2012), pp. 183194.
[10] Fromm and Hargrove (2012), pp. 3551.
[11] Fromm and Hargrove (2012), pp. 279292.
[12] Fromm and Hargrove (2012), pp. 2227.
[13] Tropp (2012), pp. 59.
[14] [14] Whiting, G.C. (1970), p.5
Cited literature
Fromm, Herbert J.; Hargrove, Mark (2012). Essentials of Biochemistry. Springer. ISBN978-3-642-19623-2.
Hunter, Graeme K. (2000). Vital Forces: The Discovery of the Molecular Basis of Life. Academic Press.
ISBN978-0-12-361811-5.
Tropp, Burton E. (2012). Molecular Biology (4th ed.). Jones & Bartlett Learning. ISBN978-1-4496-0091-4.
External links
The Virtual Library of Biochemistry and Cell Biology (http:/ / www. biochemweb. org/ )
Biochemistry, 5th ed. (http:/ / www. ncbi. nlm. nih. gov/ books/ bv. fcgi?call=bv. View. . ShowTOC& rid=stryer.
TOC& depth=2) Full text of Berg, Tymoczko, and Stryer, courtesy of NCBI.
Biochemistry, 2nd ed. (http:/ / www. web. virginia. edu/ Heidi/ home. htm) Full text of Garrett and Grisham.
Biochemistry Animation (http:/ / www. 1lec. com/ Biochemistry/ ) (Narrated Flash animations.)
SystemsX.ch - The Swiss Initiative in Systems Biology (http:/ / www. systemsX. ch/ )
Biochemistry Online Resources (http:/ / www. icademic. org/ 97445/ Biochemistry/ ) Lists of Biochemistry
departments, websites, journals, books and reviews, employment opportunities and events.
biochemical families: carbohydrates
alcohols
glycoproteins
glycosides
lipids
eicosanoids
fatty acids / intermediates
phospholipids
sphingolipids
steroids
nucleic acids
constituents / intermediates
proteins
amino acids / intermediates
tetrapyrroles / intermediates
Cells
11
Cells
Allium cells in different phases of the cell cycle
The cells of eukaryotes (left) and prokaryotes (right)
The cell is the basic structural,
functional and biological unit of all
known living organisms. It is the
smallest unit of life that is classified as
a living thing (except virus, which
consists only from DNA/RNA covered
by protein and lipids), and is often
called the building block of life.
It consists of a protoplasm enclosed
within a membrane, which contains
many biomolecules such as proteins
and nucleic acids.
[1]
Organisms can be
classified as unicellular (consisting of a
single cell; including most bacteria) or
multicellular (including plants and
animals).
While the number of cells in plants and
animals varies from species to species,
Humans contain about 100 trillion
(10
14
) cells.
[2]
Most plant and animal
cells are between 1 and
100micrometres and therefore are
visible only under the microscope.
[3]
The cell was discovered by Robert
Hooke in 1665. The cell theory, first
developed in 1839 by Matthias Jakob
Schleiden and Theodor Schwann, states that all organisms are composed of one or more cells, that all cells come
from preexisting cells, that vital functions of an organism occur within cells, and that all cells contain the hereditary
information necessary for regulating cell functions and for transmitting information to the next generation of cells.
[4]
Cells emerged on planet Earth at least 4.04.3 billion years ago.
The word cell comes from the Latin cella, meaning "small room".
[5]
The descriptive term for the smallest living
biological structure was coined by Robert Hooke in a book he published in 1665 when he compared the cork cells he
saw through his microscope to the small rooms monks lived in.
[6]
Cells
12
Anatomy
There are two types of cells: Eukaryote and prokaryote. Prokaryotic cells are usually independent, while eukaryotic
cells can either exist as a single celled organism or be found in multicellular organisms.
Table 1: Comparison of features of prokaryotic and eukaryotic cells
Prokaryotes Eukaryotes
Typical organisms bacteria, archaea protists, fungi, plants, animals
Typical size
~ 15 m
[]
~ 10100 m
[]
(sperm cells, apart from the tail, are smaller)
Type of nucleus nucleoid region; no real nucleus real nucleus with double membrane
DNA circular (usually) linear molecules (chromosomes) with histone proteins
RNA-/protein-synthesis coupled in cytoplasm RNA-synthesis inside the nucleus
protein synthesis in cytoplasm
Ribosomes 50S+30S 60S+40S
Cytoplasmatic structure very few structures highly structured by endomembranes and a cytoskeleton
Cell movement flagella made of flagellin flagella and cilia containing microtubules; lamellipodia and filopodia containing actin
Mitochondria none one to several thousand (though some lack mitochondria)
Chloroplasts none in algae and plants
Organization usually single cells single cells, colonies, higher multicellular organisms with specialized cells
Cell division Binary fission (simple division) Mitosis (fission or budding)
Meiosis
Prokaryotic cells
Diagram of a typical prokaryotic cell
The prokaryote cell is simpler, and
therefore smaller, than a eukaryote
cell, lacking a nucleus and most of the
other organelles of eukaryotes. There
are two kinds of prokaryotes: bacteria
and archaea; these share a similar
structure.
The nuclear material of a prokaryotic
cell consists of a single chromosome
that is in direct contact with the
cytoplasm. Here, the undefined nuclear
region in the cytoplasm is called the
nucleoid.
A prokaryotic cell has three
architectural regions:
On the outside, flagella and pili
project from the cell's surface.
These are structures (not present in
all prokaryotes) made of proteins that facilitate movement and communication between cells.
Cells
13
Enclosing the cell is the cell envelope generally consisting of a cell wall covering a plasma membrane though
some bacteria also have a further covering layer called a capsule. The envelope gives rigidity to the cell and
separates the interior of the cell from its environment, serving as a protective filter. Though most prokaryotes
have a cell wall, there are exceptions such as Mycoplasma (bacteria) and Thermoplasma (archaea). The cell wall
consists of peptidoglycan in bacteria, and acts as an additional barrier against exterior forces. It also prevents the
cell from expanding and finally bursting (cytolysis) from osmotic pressure against a hypotonic environment.
Some eukaryote cells (plant cells and fungal cells) also have a cell wall.
Inside the cell is the cytoplasmic region that contains the cell genome (DNA) and ribosomes and various sorts of
inclusions. A prokaryotic chromosome is usually a circular molecule (an exception is that of the bacterium
Borrelia burgdorferi, which causes Lyme disease).
[7]
Though not forming a nucleus, the DNA is condensed in a
nucleoid. Prokaryotes can carry extrachromosomal DNA elements called plasmids, which are usually circular.
Plasmids enable additional functions, such as antibiotic resistance.
Eukaryotic cells
Plants, animals, fungi, slime moulds, protozoa, and algae are all eukaryotic. These cells are about 15 times wider
than a typical prokaryote and can be as much as 1000 times greater in volume. The major difference between
prokaryotes and eukaryotes is that eukaryotic cells contain membrane-bound compartments in which specific
metabolic activities take place. Most important among these is a cell nucleus, a membrane-delineated compartment
that houses the eukaryotic cell's DNA. This nucleus gives the eukaryote its name, which means "true nucleus." Other
differences include:
The plasma membrane resembles that of prokaryotes in function, with minor differences in the setup. Cell walls
may or may not be present.
The eukaryotic DNA is organized in one or more linear molecules, called chromosomes, which are associated
with histone proteins. All chromosomal DNA is stored in the cell nucleus, separated from the cytoplasm by a
membrane. Some eukaryotic organelles such as mitochondria also contain some DNA.
Many eukaryotic cells are ciliated with primary cilia. Primary cilia play important roles in chemosensation,
mechanosensation, and thermosensation. Cilia may thus be "viewed as sensory cellular antennae that coordinate a
large number of cellular signaling pathways, sometimes coupling the signaling to ciliary motility or alternatively
to cell division and differentiation."
[]
Eukaryotes can move using motile cilia or flagella. The flagella are more complex than those of prokaryotes.
Structure of a typical animal cell Structure of a typical plant cell
Cells
14
Table 2: Comparison of structures between animal and plant cells
Typical animal cell Typical plant cell
Organelles Nucleus
Nucleolus (within nucleus)
Rough endoplasmic reticulum (ER)
Smooth ER
Ribosomes
Cytoskeleton
Golgi apparatus
Cytoplasm
Mitochondria
Vesicles
Lysosomes
Centrosome
Centrioles
Nucleus
Nucleolus (within nucleus)
Rough ER
Smooth ER
Ribosomes
Cytoskeleton
Golgi apparatus (dictiosomes)
Cytoplasm
Mitochondria
Plastids and its derivatives
Vacuole(s)
Cell wall
Subcellular components
All cells, whether prokaryotic or eukaryotic, have a membrane that envelops the cell, separates its interior from its
environment, regulates what moves in and out (selectively permeable), and maintains the electric potential of the
cell. Inside the membrane, a salty cytoplasm takes up most of the cell volume. All cells (except red blood cells which
lack a cell nucleus and most organelles to accommodate maximum space for hemoglobin) possess DNA, the
hereditary material of genes, and RNA, containing the information necessary to build various proteins such as
enzymes, the cell's primary machinery. There are also other kinds of biomolecules in cells. This article lists these
primary components of the cell, then briefly describe their function.
Membrane
The cytoplasm of a cell is surrounded by a cell membrane or plasma membrane. The plasma membrane in plants and
prokaryotes is usually covered by a cell wall. This membrane serves to separate and protect a cell from its
surrounding environment and is made mostly from a double layer of lipids (hydrophobic fat-like molecules) and
hydrophilic phosphorus molecules. Hence, the layer is called a phospholipid bilayer, or sometimes a fluid mosaic
membrane. Embedded within this membrane is a variety of protein molecules that act as channels and pumps that
move different molecules into and out of the cell. The membrane is said to be 'semi-permeable', in that it can either
let a substance (molecule or ion) pass through freely, pass through to a limited extent or not pass through at all. Cell
surface membranes also contain receptor proteins that allow cells to detect external signaling molecules such as
hormones.
Cells
15
Cytoskeleton
Bovine Pulmonary Artery Endothelial cell: nuclei stained blue,
mitochondria stained red, and F-actin, an important component in
microfilaments, stained green. Cell imaged on a fluorescent
microscope.
The cytoskeleton acts to organize and maintain the
cell's shape; anchors organelles in place; helps during
endocytosis, the uptake of external materials by a cell,
and cytokinesis, the separation of daughter cells after
cell division; and moves parts of the cell in processes of
growth and mobility. The eukaryotic cytoskeleton is
composed of microfilaments, intermediate filaments
and microtubules. There are a great number of proteins
associated with them, each controlling a cell's structure
by directing, bundling, and aligning filaments. The
prokaryotic cytoskeleton is less well-studied but is
involved in the maintenance of cell shape, polarity and
cytokinesis.
[8]
Genetic material
Two different kinds of genetic material exist:
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Most cells use DNA for their long-term information
storage. The biological information contained in an organism is encoded in its DNA sequence. RNA is used for
information transport (e.g., mRNA) and enzymatic functions (e.g., ribosomal RNA). Transfer RNA (tRNA)
molecules are used to add amino acids during protein translation.
Prokaryotic genetic material is organized in a simple circular DNA molecule (the bacterial chromosome) in the
nucleoid region of the cytoplasm. Eukaryotic genetic material is divided into different, linear molecules called
chromosomes inside a discrete nucleus, usually with additional genetic material in some organelles like mitochondria
and chloroplasts (see endosymbiotic theory).
A human cell has genetic material contained in the cell nucleus (the nuclear genome) and in the mitochondria (the
mitochondrial genome). In humans the nuclear genome is divided into 46 linear DNA molecules called
chromosomes, including 22 homologous chromosome pairs and a pair of sex chromosomes. The mitochondrial
genome is a circular DNA molecule distinct from the nuclear DNA. Although the mitochondrial DNA is very small
compared to nuclear chromosomes, it codes for 13 proteins involved in mitochondrial energy production and specific
tRNAs.
Foreign genetic material (most commonly DNA) can also be artificially introduced into the cell by a process called
transfection. This can be transient, if the DNA is not inserted into the cell's genome, or stable, if it is. Certain viruses
also insert their genetic material into the genome.
Organelles
The human body contains many different organs, such as the heart, lung, and kidney, with each organ performing a
different function. Cells also have a set of "little organs," called organelles, that are adapted and/or specialized for
carrying out one or more vital functions. Both eukaryotic and prokaryotic cells have organelles but organelles in
eukaryotes are generally more complex and may be membrane bound.
There are several types of organelles in a cell. Some (such as the nucleus and golgi apparatus) are typically solitary,
while others (such as mitochondria, peroxisomes and lysosomes) can be numerous (hundreds to thousands). The
cytosol is the gelatinous fluid that fills the cell and surrounds the organelles.
Cells
16
Diagram of a cell nucleus
Cell nucleus eukaryotes only - A cell's information center, the
cell nucleus is the most conspicuous organelle found in a eukaryotic
cell. It houses the cell's chromosomes, and is the place where almost
all DNA replication and RNA synthesis (transcription) occur. The
nucleus is spherical and separated from the cytoplasm by a double
membrane called the nuclear envelope. The nuclear envelope
isolates and protects a cell's DNA from various molecules that could
accidentally damage its structure or interfere with its processing.
During processing, DNA is transcribed, or copied into a special
RNA, called messenger RNA (mRNA). This mRNA is then
transported out of the nucleus, where it is translated into a specific
protein molecule. The nucleolus is a specialized region within the
nucleus where ribosome subunits are assembled. In prokaryotes,
DNA processing takes place in the cytoplasm.
Mitochondria and Chloroplasts eukaryotes only - the power generators: Mitochondria are self-replicating
organelles that occur in various numbers, shapes, and sizes in the cytoplasm of all eukaryotic cells. Mitochondria
play a critical role in generating energy in the eukaryotic cell. Mitochondria generate the cell's energy by
oxidative phosphorylation, using oxygen to release energy stored in cellular nutrients (typically pertaining to
glucose) to generate ATP. Mitochondria multiply by splitting in two. Respiration occurs in the cell mitochondria.
Chloroplasts can only be found in plants and algae, and they capture the sun's energy to make ATP.
Diagram of an endomembrane system
Endoplasmic reticulum eukaryotes only: The endoplasmic
reticulum (ER) is the transport network for molecules targeted for
certain modifications and specific destinations, as compared to
molecules that float freely in the cytoplasm. The ER has two forms:
the rough ER, which has ribosomes on its surface and secretes
proteins into the cytoplasm, and the smooth ER, which lacks them.
Smooth ER plays a role in calcium sequestration and release.
[citation
needed]
Golgi apparatus eukaryotes only : The primary function of the
Golgi apparatus is to process and package the macromolecules such
as proteins and lipids that are synthesized by the cell.
[citation needed]
Ribosomes: The ribosome is a large complex of RNA and protein
molecules. They each consist of two subunits, and act as an
assembly line where RNA from the nucleus is used to synthesise
proteins from amino acids. Ribosomes can be found either floating freely or bound to a membrane (the rough
endoplasmatic reticulum in eukaryotes, or the cell membrane in prokaryotes).
[9]
Lysosomes and Peroxisomes eukaryotes only: Lysosomes contain digestive enzymes (acid hydrolases). They
digest excess or worn-out organelles, food particles, and engulfed viruses or bacteria. Peroxisomes have enzymes
that rid the cell of toxic peroxides. The cell could not house these destructive enzymes if they were not contained
in a membrane-bound system.
[citation needed]
Centrosome the cytoskeleton organiser: The centrosome produces the microtubules of a cell a key
component of the cytoskeleton. It directs the transport through the ER and the Golgi apparatus. Centrosomes are
composed of two centrioles, which separate during cell division and help in the formation of the mitotic spindle.
A single centrosome is present in the animal cells. They are also found in some fungi and algae cells.
[citation
needed]
Cells
17
Vacuoles: Vacuoles store food and waste. Some vacuoles store extra water. They are often described as liquid
filled space and are surrounded by a membrane. Some cells, most notably Amoeba, have contractile vacuoles,
which can pump water out of the cell if there is too much water. The vacuoles of eukaryotic cells are usually
larger in those of plants than animals.
[citation needed]
Structures outside the cell membrane
Many cells also have structures which exist wholly or partially outside the cell membrane. These structures are
notable because they are not protected from the external environment by the impermeable cell membrane. In order to
assemble these structures export processes to carry macromolecules across the cell membrane must be used.
Cell wall
Many types of prokaryotic and eukaryotic cell have a cell wall. The cell wall acts to protect the cell mechanically
and chemically from its environment, and is an additional layer of protection to the cell membrane. Different types
of cell have cell walls made up of different materials; plant cell walls are primarily made up of pectin, fungi cell
walls are made up of chitin and bacteria cell walls are made up of peptidoglycan.
Prokaryotic
Capsule
A gelatinous capsule is present in some bacteria outside the cell membrane and cell wall. The capsule may be
polysaccharide as in pneumococci, meningococci or polypeptide as Bacillus anthracis or hyaluronic acid as in
streptococci.
[citation needed]
Capsules are not marked by normal staining protocols and can be detected by special
stain.
[citation needed]
Flagella
Flagella are organelles for cellular mobility. The bacterial flagellum stretches from cytoplasm through the cell
membrane(s) and extrudes through the cell wall. They are long and thick thread-like appendages, protein in nature.
Are most commonly found in bacteria cells but are found in animal cells as well.
Fimbriae (pili)
They are short and thin hair like filaments, formed of protein called pilin (antigenic). Fimbriae are responsible for
attachment of bacteria to specific receptors of human cell (adherence). There are special types of pili called (sex pili)
involved in conjunction.
[citation needed]
Growth and metabolism
Between successive cell divisions, cells grow through the functioning of cellular metabolism. Cell metabolism is the
process by which individual cells process nutrient molecules. Metabolism has two distinct divisions: catabolism, in
which the cell breaks down complex molecules to produce energy and reducing power, and anabolism, in which the
cell uses energy and reducing power to construct complex molecules and perform other biological functions.
Complex sugars consumed by the organism can be broken down into a less chemically complex sugar molecule
called glucose. Once inside the cell, glucose is broken down to make adenosine triphosphate (ATP), a form of
energy, through two different pathways.
The first pathway, glycolysis, requires no oxygen and is referred to as anaerobic metabolism. Each reaction is
designed to produce some hydrogen ions that can then be used to make energy packets (ATP). In prokaryotes,
glycolysis is the only method used for converting energy.
Cells
18
The second pathway, called the Krebs cycle, or citric acid cycle, occurs inside the mitochondria and can generate
enough ATP to run all the cell functions.
[citation needed]
An overview of protein
synthesis.Within the
nucleus of the cell (light
blue), genes (DNA, dark
blue) are transcribed into
RNA. This RNA is then
subject to
post-transcriptional
modification and control,
resulting in a mature
mRNA (red) that is then
transported out of the
nucleus and into the
cytoplasm (peach), where
it undergoes translation
into a protein. mRNA is
translated by ribosomes
(purple) that match the
three-base codons of the
mRNA to the three-base
anti-codons of the
appropriate tRNA. Newly
synthesized proteins
(black) are often further
modified, such as by
binding to an effector
molecule (orange), to
become fully active.
Self-replication
Cell division involves a single cell (called a mother cell) dividing into two daughter
cells. This leads to growth in multicellular organisms (the growth of tissue) and to
procreation (vegetative reproduction) in unicellular organisms.
Prokaryotic cells divide by binary fission. Eukaryotic cells usually undergo a process of
nuclear division, called mitosis, followed by division of the cell, called cytokinesis. A
diploid cell may also undergo meiosis to produce haploid cells, usually four. Haploid
cells serve as gametes in multicellular organisms, fusing to form new diploid cells.
DNA replication, or the process of duplicating a cell's genome, always happens when a
cell divides through mitosis or binary fission.
In meiosis, the DNA is replicated only once, while the cell divides twice. DNA
replication only occurs before meiosis I. DNA replication does not occur when the cells
divide the second time, in meiosis II.
[10]
Replication, like all cellular activities, requires
specialized proteins for carrying out the job.
Protein synthesis
Cells are capable of synthesizing new proteins, which are essential for the modulation
and maintenance of cellular activities. This process involves the formation of new
protein molecules from amino acid building blocks based on information encoded in
DNA/RNA. Protein synthesis generally consists of two major steps: transcription and
translation.
Transcription is the process where genetic information in DNA is used to produce a
complementary RNA strand. This RNA strand is then processed to give messenger RNA
(mRNA), which is free to migrate through the cell. mRNA molecules bind to
protein-RNA complexes called ribosomes located in the cytosol, where they are
translated into polypeptide sequences. The ribosome mediates the formation of a
polypeptide sequence based on the mRNA sequence. The mRNA sequence directly
relates to the polypeptide sequence by binding to transfer RNA (tRNA) adapter
molecules in binding pockets within the ribosome. The new polypeptide then folds into a
functional three-dimensional protein molecule.
Movement or motility
Cells can move during many processes: such as wound healing, the immune response and cancer metastasis. For
wound healing to occur, white blood cells and cells that ingest bacteria move to the wound site to kill the
microorganisms that cause infection.
At the same time fibroblasts (connective tissue cells) move there to remodel damaged structures. In the case of tumor
development, cells from a primary tumor move away and spread to other parts of the body. Cell motility involves
many receptors, crosslinking, bundling, binding, adhesion, motor and other proteins.
[11]
The process is divided into
three steps protrusion of the leading edge of the cell, adhesion of the leading edge and de-adhesion at the cell body
Cells
19
and rear, and cytoskeletal contraction to pull the cell forward. Each step is driven by physical forces generated by
unique segments of the cytoskeleton.
[12][13]
Origins
The origin of cells has to do with the origin of life, which began the history of life on Earth.
Origin of the first cell
There are several theories about the origin of small molecules that could lead to life in an early Earth. One is that
they came from meteorites (see Murchison meteorite). Another is that they were created at deep-sea vents. A third is
that they were synthesized by lightning in a reducing atmosphere (see MillerUrey experiment); although it is not
clear if Earth had such an atmosphere. There are essentially no experimental data defining what the first
self-replicating forms were. RNA is generally assumed the earliest self-replicating molecule, as it is capable of both
storing genetic information and catalyzing chemical reactions (see RNA world hypothesis). But some other entity
with the potential to self-replicate could have preceded RNA, like clay or peptide nucleic acid.
[]
Cells emerged at least 4.04.3 billion years ago. The current belief is that these cells were heterotrophs. An
important characteristic of cells is the cell membrane, composed of a bilayer of lipids. The early cell membranes
were probably more simple and permeable than modern ones, with only a single fatty acid chain per lipid. Lipids are
known to spontaneously form bilayered vesicles in water, and could have preceded RNA, but the first cell
membranes could also have been produced by catalytic RNA, or even have required structural proteins before they
could form.
[14]
Origin of eukaryotic cells
The eukaryotic cell seems to have evolved from a symbiotic community of prokaryotic cells. DNA-bearing
organelles like the mitochondria and the chloroplasts are almost certainly what remains of ancient symbiotic
oxygen-breathing proteobacteria and cyanobacteria, respectively, where the rest of the cell appears derived from an
ancestral archaean prokaryote cellan idea called the endosymbiotic theory.
There is still considerable debate about whether organelles like the hydrogenosome predated the origin of
mitochondria, or viceversa: see the hydrogen hypothesis for the origin of eukaryotic cells.
Sex, as the stereotyped choreography of meiosis and syngamy that persists in nearly all extant eukaryotes, may have
played a role in the transition from prokaryotes to eukaryotes. An 'origin of sex as vaccination' theory suggests that
the eukaryote genome accreted from prokaryan parasite genomes in numerous rounds of lateral gene transfer.
Sex-as-syngamy (fusion sex) arose when infected hosts began swapping nuclearized genomes containing co-evolved,
vertically transmitted symbionts that conveyed protection against horizontal infection by more virulent symbionts.
[]
History of research
16321723: Antonie van Leeuwenhoek teaches himself to make lenses, constructs simple microscopes and draws
protozoa, such as Vorticella from rain water, and bacteria from his own mouth.
1665: Robert Hooke discovers cells in cork, then in living plant tissue using an early compound microscope.
[6]
1839: Theodor Schwann and Matthias Jakob Schleiden elucidate the principle that plants and animals are made of
cells, concluding that cells are a common unit of structure and development, and thus founding the cell theory.
1855: Rudolf Virchow states that cells always emerge from cell divisions (omnis cellula ex cellula).
1859: The belief that life forms can occur spontaneously (generatio spontanea) is contradicted by Louis Pasteur
(18221895) (although Francesco Redi had performed an experiment in 1668 that suggested the same
conclusion).
Cells
20
1931: Ernst Ruska builds first transmission electron microscope (TEM) at the University of Berlin. By 1935, he
has built an EM with twice the resolution of a light microscope, revealing previously unresolvable organelles.
1953: Watson and Crick made their first announcement on the double-helix structure for DNA on February 28.
1981: Lynn Margulis published Symbiosis in Cell Evolution detailing the endosymbiotic theory.
References
[1] Cell Movements and the Shaping of the Vertebrate Body (http:/ / www. ncbi. nlm. nih. gov/ entrez/ query. fcgi?cmd=Search& db=books&
doptcmdl=GenBookHL& term=Cell+ Movements+ and+ the+ Shaping+ of+ the+ Vertebrate+ Body+ AND+ mboc4[book]+ AND+
374635[uid]& rid=mboc4. section. 3919) in Chapter 21 of Molecular Biology of the Cell (http:/ / www. ncbi. nlm. nih. gov/ entrez/ query.
fcgi?cmd=Search& db=books& doptcmdl=GenBookHL& term=cell+ biology+ AND+ mboc4[book]+ AND+ 373693[uid]& rid=mboc4)
fourth edition, edited by Bruce Alberts (2002) published by Garland Science.
The Alberts text discusses how the "cellular building blocks" move to shape developing embryos. It is also common to describe small
molecules such as amino acids as " molecular building blocks (http:/ / www. ncbi. nlm. nih. gov/ entrez/ query. fcgi?cmd=Search&
db=books& doptcmdl=GenBookHL& term="all+ cells"+ AND+ mboc4[book]+ AND+ 372023[uid]& rid=mboc4. section. 4#23)".
[6] "... I could exceedingly plainly perceive it to be all perforated and porous, much like a Honey-comb, but that the pores of it were not regular
[..] these pores, or cells, [..] were indeed the first microscopical pores I ever saw, and perhaps, that were ever seen, for I had not met with any
Writer or Person, that had made any mention of them before this. . ." Hooke describing his observations on a thin slice of cork. Robert
Hooke (http:/ / www. ucmp. berkeley.edu/ history/ hooke. html)
[7] European Bioinformatics Institute, Karyn's Genomes: Borrelia burgdorferi (http:/ / www. ebi. ac. uk/ 2can/ genomes/ bacteria/
Borrelia_burgdorferi. html), part of 2can on the EBI-EMBL database. Retrieved 5 August 2012
[12] [12] Alberts B, Johnson A, Lewis J. et al. Molecular Biology of the Cell, 4e. Garland Science. 2002
[13] Ananthakrishnan R, Ehrlicher A. The Forces Behind Cell Movement. Int J Biol Sci 2007; 3:303317. http:/ / www. biolsci. org/ v03p0303.
htm
This article incorporatespublic domain material from the NCBI document "Science Primer" (http:/ / www.
ncbi. nlm. nih. gov/ About/ primer/ index. html).
External links
Inside the Cell (http:/ / publications. nigms. nih. gov/ insidethecell/ ) - a science education booklet by National
Institutes of Health, in PDF and ePub.
Cells Alive! (http:/ / www. cellsalive. com/ )
Cell Biology (http:/ / www. biology. arizona. edu/ cell_bio/ cell_bio. html) in "The Biology Project" of University
of Arizona.
Centre of the Cell online (http:/ / www. centreofthecell. org/ )
The Image & Video Library of The American Society for Cell Biology (http:/ / cellimages. ascb. org/ ), a
collection of peer-reviewed still images, video clips and digital books that illustrate the structure, function and
biology of the cell.
HighMag Blog (http:/ / highmagblog. blogspot. com/ ), still images of cells from recent research articles.
New Microscope Produces Dazzling 3D Movies of Live Cells (http:/ / www. hhmi. org/ news/ betzig20110304.
html), March 4, 2011 - Howard Hughes Medical Institute.
WormWeb.org: Interactive Visualization of the C. elegans Cell lineage (http:/ / wormweb. org/ celllineage) -
Visualize the entire cell lineage tree of the nematode C. elegans
Cells
21
Textbooks
Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002). Molecular Biology of the Cell (http:/ / www.
ncbi. nlm. nih. gov/ books/ bv. fcgi?rid=mboc4. TOC& depth=2) (4th ed.). Garland. ISBN0-8153-3218-1.
Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipurksy SL, Darnell J (2004). Molecular
Cell Biology (http:/ / www. ncbi. nlm. nih. gov/ books/ bv. fcgi?rid=mcb. TOC) (5th ed.). WH Freeman: New
York, NY. ISBN978-0-7167-4366-8.
Cooper GM (2000). The cell: a molecular approach (http:/ / www. ncbi. nlm. nih. gov/ books/ bv.
fcgi?rid=cooper. TOC& depth=2) (2nd ed.). Washington, D.C: ASM Press. ISBN0-87893-102-3.
Water
Water in three states: liquid, solid (ice), and (invisible) water vapor in the air.
Clouds are accumulations of water droplets, condensed from vapor-saturated air.
Water is a chemical compound with the
chemical formula H
2
O. A water molecule
contains one oxygen and two hydrogen
atoms connected by covalent bonds. Water
is a liquid at standard ambient temperature
and pressure, but it often co-exists on Earth
with its solid state, ice, and gaseous state
(water vapor or steam). Water also exists in
a liquid crystal state near hydrophilic
surfaces.
[1][2]
Water covers 71% of the Earth's surface,
[3]
and is vital for all known forms of life.
[4]
On
Earth, 96.5% of the planet's water is found
in oceans, 1.7% in groundwater, 1.7% in glaciers and the ice caps of Antarctica and Greenland, a small fraction in
other large water bodies, and 0.001% in the air as vapor, clouds (formed of solid and liquid water particles
suspended in air), and precipitation.
[][5]
Only 2.5% of the Earth's water is freshwater, and 98.8% of that water is in
ice and groundwater. Less than 0.3% of all freshwater is in rivers, lakes, and the atmosphere, and an even smaller
amount of the Earth's freshwater (0.003%) is contained within biological bodies and manufactured products.
[]
Water on Earth moves continually through the hydrological cycle of evaporation and transpiration
(evapotranspiration), condensation, precipitation, and runoff, usually reaching the sea. Evaporation and transpiration
contribute to the precipitation over land.
Safe drinking water is essential to humans and other lifeforms even though it provides no calories or organic
nutrients. Access to safe drinking water has improved over the last decades in almost every part of the world, but
approximately one billion people still lack access to safe water and over 2.5 billion lack access to adequate
sanitation.
[]
There is a clear correlation between access to safe water and GDP per capita.
[6]
However, some
observers have estimated that by 2025 more than half of the world population will be facing water-based
vulnerability.
[7]
A recent report (November 2009) suggests that by 2030, in some developing regions of the world,
water demand will exceed supply by 50%.
[8]
Water plays an important role in the world economy, as it functions as a
solvent for a wide variety of chemical substances and facilitates industrial cooling and transportation. Approximately
70% of the fresh water used by humans goes to agriculture.
[]
Water
22
Chemical and physical properties
Model of hydrogen bonds (1) between molecules
of water
Impact from a water drop causes an upward
"rebound" jet surrounded by circular capillary
waves.
Snowflakes by Wilson Bentley, 1902
Water is the chemical substance with chemical formula H
2
O: one
molecule of water has two hydrogen atoms covalently bonded to a
single oxygen atom.
Water appears in nature in all three common states of matter (solid,
liquid, and gas) and may take many different forms on Earth: water
vapor and clouds in the sky; seawater in the oceans; icebergs in the
polar oceans; glaciers and rivers in the mountains; and the liquid in
aquifers in the ground.
The major chemical and physical properties of water are:
Water is a liquid at standard temperature and pressure. It is tasteless
and odorless. The intrinsic colour of water and ice is a very slight
blue hue, although both appear colorless in small quantities. Water
vapour is essentially invisible as a gas.
[9]
Water is transparent in the visible electromagnetic spectrum. Thus
aquatic plants can live in water because sunlight can reach them.
Infrared light is strongly absorbed by the hydrogen-oxygen or OH
bonds.
Since the water molecule is not linear and the oxygen atom has a
higher electronegativity than hydrogen atoms, it carries a slight
negative charge, whereas the hydrogen atoms are slightly positive.
As a result, water is a polar molecule with an electrical dipole
moment. Water also can form an unusually large number of
intermolecular hydrogen bonds (four) for a molecule of its size.
These factors lead to strong attractive forces between molecules of
water, giving rise to water's high surface tension
[10]
and capillary
forces. The capillary action refers to the tendency of water to move
up a narrow tube against the force of gravity. This property is relied
upon by all vascular plants, such as trees.
[11]
Water is a good polar solvent and is often referred to as the
universal solvent. Substances that dissolve in water, e.g., salts,
sugars, acids, alkalis, and some gases especially oxygen, carbon
dioxide (carbonation) are known as hydrophilic (water-loving)
substances, while those that are immiscible with water (e.g., fats and
oils), are known as hydrophobic (water-fearing) substances.
Most of the major components in cells (proteins, DNA and
polysaccharides) are also dissolved in water.
Pure water has a low electrical conductivity, but this increases with
the dissolution of a small amount of ionic material such as sodium
chloride.
The boiling point of water (and all other liquids) is dependent on the
barometric pressure. For example, on the top of Mt. Everest water
Water
23
Dew drops adhering to a spider web
Capillary action of water compared
to mercury
boils at 68 C (154F), compared to 100 C (212F) at sea level.
Conversely, water deep in the ocean near geothermal vents can
reach temperatures of hundreds of degrees and remain liquid.
At 4181.3 J/(kgK), water has a high specific heat capacity, as well
as a high heat of vaporization (40.65 kJmol
1
), both of which are a
result of the extensive hydrogen bonding between its molecules.
These two unusual properties allow water to moderate Earth's
climate by buffering large fluctuations in temperature.
The maximum density of water occurs at 3.98 C (39.16F).
[12]
It
has the anomalous property of becoming less dense, not more, when
it is cooled to its solid form, ice. During freezing, the 'open
structure' of ice is gradually broken and molecules enter cavities in
ice-like structure of low temperature water. There are two
competing effects: 1) Increasing volume of normal liquid and 2)
Decrease overall volume of the liquid. Between 0 and 3.98C, the
second effect will cancel off the first effect so the net effect is
shrinkage of volume with increasing temperature.
[13]
It expands to
occupy 9% greater volume in this solid state, which accounts for the
fact of ice floating on liquid water, as in icebergs.
The density of liquid water is 1,000kg/m
3
(62.43lb/cu ft) at 4C.
Ice has a density of 917kg/m
3
(57.25lb/cu ft).
ADR label for transporting goods dangerously
reactive with water
Water is miscible with many liquids, such as ethanol, in all
proportions, forming a single homogeneous liquid. On the other
hand, water and most oils are immiscible, usually forming layers
according to increasing density from the top. As a gas, water vapor
is completely miscible with air.
Water forms an azeotrope with many other solvents.
Water can be split by electrolysis into hydrogen and oxygen.
As an oxide of hydrogen, water is formed when hydrogen or
hydrogen-containing compounds burn or react with oxygen or
oxygen-containing compounds. Water is not a fuel, it is an
end-product of the combustion of hydrogen. The energy required to
split water into hydrogen and oxygen by electrolysis or any other
means is greater than the energy that can be collected when the
hydrogen and oxygen recombine.
[14]
Elements which are more electropositive than hydrogen such as lithium, sodium, calcium, potassium and caesium
displace hydrogen from water, forming hydroxides. Being a flammable gas, the hydrogen given off is dangerous
and the reaction of water with the more electropositive of these elements may be violently explosive.
Water
24
Taste and odor
Water can dissolve many different substances, giving it varying tastes and odors. Humans and other animals have
developed senses that enable them to evaluate the potability of water by avoiding water that is too salty or putrid.
The taste of spring water and mineral water, often advertised in marketing of consumer products, derives from the
minerals dissolved in it. However, pure H
2
O is tasteless and odorless. The advertised purity of spring and mineral
water refers to absence of toxins, pollutants and microbes, not the absence of naturally occurring minerals.
Distribution in nature
In the universe
Much of the universe's water is produced as a byproduct of star formation. When stars are born, their birth is
accompanied by a strong outward wind of gas and dust. When this outflow of material eventually impacts the
surrounding gas, the shock waves that are created compress and heat the gas. The water observed is quickly
produced in this warm dense gas.
[15]
On 22 July 2011 a report described the discovery of a gigantic cloud of water vapor containing "140 trillion times
more water than all of Earth's oceans combined" around a quasar located 12 billion light years from Earth.
According to the researchers, the "discovery shows that water has been prevalent in the universe for nearly its entire
existence".
[][]
Water has been detected in interstellar clouds within our galaxy, the Milky Way. Water probably exists in abundance
in other galaxies, too, because its components, hydrogen and oxygen, are among the most abundant elements in the
universe. Interstellar clouds eventually condense into solar nebulae and solar systems such as ours.
Water vapor is present in
Atmosphere of Mercury: 3.4%, and large amounts of water in Mercury's exosphere
[]
Atmosphere of Venus: 0.002%
Earth's atmosphere: ~0.40% over full atmosphere, typically 14% at surface
Atmosphere of Mars: 0.03%
Atmosphere of Jupiter: 0.0004%
Atmosphere of Saturn in ices only
Enceladus (moon of Saturn): 91%
exoplanets known as HD 189733 b
[16]
and HD 209458 b.
[17]
Liquid water is present on
Earth: 71% of surface
Europa: 100km deep subsurface ocean
Strong evidence suggests that liquid water is present just under the surface of Saturn's moon Enceladus.
Water ice is present on
Earth mainly as ice sheets
polar ice caps on Mars
Moon
Titan
Europa
Saturn's rings
[]
Enceladus
Pluto and Charon
[]
Comets and comet source populations (Kuiper belt and Oort cloud objects).
Water
25
Recent evidence points to the existence of water ice at the poles of Mercury.
[18]
Water ice may also be present on
Ceres and Tethys. Water and other volatiles probably comprise much of the internal structures of Uranus and
Neptune and the water in the deeper layers may be in the form of ionic water in which the molecules break down
into a soup of hydrogen and oxygen ions, and deeper down as superionic water in which the oxygen crystallises but
the hydrogen ions float around freely within the oxygen lattice.
[19]
Some of the Moon's minerals contain water molecules. For instance, in 2008 a laboratory device which ejects and
identifies particles found small amounts of the compound in the inside of volcanic rock brought from Moon to Earth
by the Apollo 15 crew in 1971.
[20]
NASA reported the detection of water molecules by NASA's Moon Mineralogy
Mapper aboard the Indian Space Research Organization's Chandrayaan-1 spacecraft in September 2009.
[21]
Water and habitable zone
The existence of liquid water, and to a lesser extent its gaseous and solid forms, on Earth are vital to the existence of
life on Earth as we know it. The Earth is located in the habitable zone of the solar system; if it were slightly closer to
or farther from the Sun (about 5%, or about 8 million kilometers), the conditions which allow the three forms to be
present simultaneously would be far less likely to exist.
[22][23]
Earth's gravity allows it to hold an atmosphere. Water vapor and carbon dioxide in the atmosphere provide a
temperature buffer (greenhouse effect) which helps maintain a relatively steady surface temperature. If Earth were
smaller, a thinner atmosphere would allow temperature extremes, thus preventing the accumulation of water except
in polar ice caps (as on Mars).
The surface temperature of Earth has been relatively constant through geologic time despite varying levels of
incoming solar radiation (insolation), indicating that a dynamic process governs Earth's temperature via a
combination of greenhouse gases and surface or atmospheric albedo. This proposal is known as the Gaia hypothesis.
The state of water on a planet depends on ambient pressure, which is determined by the planet's gravity. If a planet is
sufficiently massive, the water on it may be solid even at high temperatures, because of the high pressure caused by
gravity, as it was observed on exoplanets Gliese 436 b
[24]
and GJ 1214 b.
[25]
There are various theories about origin of water on Earth.
On Earth
A graphical distribution of the locations of water on Earth.
Hydrology is the study of the
movement, distribution, and quality of
water throughout the Earth. The study
of the distribution of water is
hydrography. The study of the
distribution and movement of
groundwater is hydrogeology, of
glaciers is glaciology, of inland waters
is limnology and distribution of oceans
is oceanography. Ecological processes
with hydrology are in focus of
ecohydrology.
The collective mass of water found on,
under, and over the surface of a planet
is called the hydrosphere. Earth's
Water
26
Water covers 71% of the Earth's surface; the
oceans contain 96.5% of the Earth's water. The
Antarctic ice sheet, which contains 61% of all
fresh water on Earth, is visible at the bottom.
Condensed atmospheric water can be seen as
clouds, contributing to the Earth's albedo.
approximate water volume (the total water supply of the world) is
1,338,000,000km
3
(321,000,000mi
3
).
[]
Liquid water is found in bodies of water, such as an ocean, sea, lake,
river, stream, canal, pond, or puddle. The majority of water on Earth is
sea water. Water is also present in the atmosphere in solid, liquid, and
vapor states. It also exists as groundwater in aquifers.
Water is important in many geological processes. Groundwater is
present in most rocks, and the pressure of this groundwater affects
patterns of faulting. Water in the mantle is responsible for the melt that
produces volcanoes at subduction zones. On the surface of the Earth,
water is important in both chemical and physical weathering processes.
Water and, to a lesser but still significant extent, ice, are also
responsible for a large amount of sediment transport that occurs on the
surface of the earth. Deposition of transported sediment forms many
types of sedimentary rocks, which make up the geologic record of
Earth history.
Water cycle
Water cycle
The water cycle (known scientifically
as the hydrologic cycle) refers to the
continuous exchange of water within
the hydrosphere, between the
atmosphere, soil water, surface water,
groundwater, and plants.
Water moves perpetually through each
of these regions in the water cycle
consisting of following transfer
processes:
evaporation from oceans and other
water bodies into the air and
transpiration from land plants and
animals into air.
precipitation, from water vapor
condensing from the air and falling to earth or ocean.
runoff from the land usually reaching the sea.
Most water vapor over the oceans returns to the oceans, but winds carry water vapor over land at the same rate as
runoff into the sea, about 47Tt per year. Over land, evaporation and transpiration contribute another 72Tt per year.
Precipitation, at a rate of 119 Tt per year over land, has several forms: most commonly rain, snow, and hail, with
some contribution from fog and dew.
[26]
Dew is small drops of water that are condensed when a high density of
water vapor meets a cool surface. Dew usually form in the morning when the temperature is the lowest, just before
sunrise and when the temperature of the earth's surface starts to increase.
[27]
Condensed water in the air may also
refract sunlight to produce rainbows.
Water
27
Water runoff often collects over watersheds flowing into rivers. A mathematical model used to simulate river or
stream flow and calculate water quality parameters is hydrological transport model. Some of water is diverted to
irrigation for agriculture. Rivers and seas offer opportunity for travel and commerce. Through erosion, runoff shapes
the environment creating river valleys and deltas which provide rich soil and level ground for the establishment of
population centers. A flood occurs when an area of land, usually low-lying, is covered with water. It is when a river
overflows its banks or flood from the sea. A drought is an extended period of months or years when a region notes a
deficiency in its water supply. This occurs when a region receives consistently below average precipitation.
Fresh water storage

The Bay of Fundy at high tide (left) and low tide (right)
Some runoff water is trapped for periods of time, for example in lakes. At high altitude, during winter, and in the far
north and south, snow collects in ice caps, snow pack and glaciers. Water also infiltrates the ground and goes into
aquifers. This groundwater later flows back to the surface in springs, or more spectacularly in hot springs and
geysers. Groundwater is also extracted artificially in wells. This water storage is important, since clean, fresh water
is essential to human and other land-based life. In many parts of the world, it is in short supply.
Sea water
Sea water contains about 3.5% salt on average, plus smaller amounts of other substances. The physical properties of
sea water differ from fresh water in some important respects. It freezes at a lower temperature (about 1.9 C) and its
density increases with decreasing temperature to the freezing point, instead of reaching maximum density at a
temperature above freezing. The salinity of water in major seas varies from about 0.7% in the Baltic Sea to 4.0% in
the Red Sea.
Tides
Tides are the cyclic rising and falling of local sea levels caused by the tidal forces of the Moon and the Sun acting on
the oceans. Tides cause changes in the depth of the marine and estuarine water bodies and produce oscillating
currents known as tidal streams. The changing tide produced at a given location is the result of the changing
positions of the Moon and Sun relative to the Earth coupled with the effects of Earth rotation and the local
bathymetry. The strip of seashore that is submerged at high tide and exposed at low tide, the intertidal zone, is an
important ecological product of ocean tides.
Water
28
Effects on life
Overview of photosynthesis and respiration.
Water (at right), together with carbon dioxide
(CO
2
), form oxygen and organic compounds (at
left), which can be respired to water and (CO
2
).
From a biological standpoint, water has many distinct properties that
are critical for the proliferation of life that set it apart from other
substances. It carries out this role by allowing organic compounds to
react in ways that ultimately allow replication. All known forms of life
depend on water. Water is vital both as a solvent in which many of the
body's solutes dissolve and as an essential part of many metabolic
processes within the body. Metabolism is the sum total of anabolism
and catabolism. In anabolism, water is removed from molecules
(through energy requiring enzymatic chemical reactions) in order to
grow larger molecules (e.g. starches, triglycerides and proteins for
storage of fuels and information). In catabolism, water is used to break
bonds in order to generate smaller molecules (e.g. glucose, fatty acids
and amino acids to be used for fuels for energy use or other purposes).
Without water, these particular metabolic processes could not exist.
Water is fundamental to photosynthesis and respiration. Photosynthetic
cells use the sun's energy to split off water's hydrogen from oxygen.
Hydrogen is combined with CO
2
(absorbed from air or water) to form
glucose and release oxygen. All living cells use such fuels and oxidize the hydrogen and carbon to capture the sun's
energy and reform water and CO
2
in the process (cellular respiration).
Water is also central to acid-base neutrality and enzyme function. An acid, a hydrogen ion (H
+
, that is, a proton)
donor, can be neutralized by a base, a proton acceptor such as hydroxide ion (OH

) to form water. Water is

considered to be neutral, with a pH (the negative log of the hydrogen ion concentration) of 7. Acids have pH values
less than 7 while bases have values greater than 7.
Aquatic life forms
Some of the biodiversity of a coral
reef
Earth surface waters are filled with life. The earliest life forms appeared in water;
nearly all fish live exclusively in water, and there are many types of marine
mammals, such as dolphins and whales. Some kinds of animals, such as
amphibians, spend portions of their lives in water and portions on land. Plants
such as kelp and algae grow in the water and are the basis for some underwater
ecosystems. Plankton is generally the foundation of the ocean food chain.
Aquatic vertebrates must obtain oxygen to survive, and they do so in various
ways. Fish have gills instead of lungs, although some species of fish, such as the
lungfish, have both. Marine mammals, such as dolphins, whales, otters, and seals
need to surface periodically to breathe air. Some amphibians are able to absorb
oxygen through their skin. Invertebrates exhibit a wide range of modifications to
survive in poorly oxygenated waters including breathing tubes (see insect and
mollusc siphons) and gills (Carcinus). However as invertebrate life evolved in an
aquatic habitat most have little or no specialisation for respiration in water.
Water
29
Some marine diatoms a key phytoplankton
group
Effects on human civilization
Water fountain
Civilization has historically flourished around rivers and major
waterways; Mesopotamia, the so-called cradle of civilization, was
situated between the major rivers Tigris and Euphrates; the ancient
society of the Egyptians depended entirely upon the Nile. Large
metropolises like Rotterdam, London, Montreal, Paris, New York City,
Buenos Aires, Shanghai, Tokyo, Chicago, and Hong Kong owe their
success in part to their easy accessibility via water and the resultant
expansion of trade. Islands with safe water ports, like Singapore, have
flourished for the same reason. In places such as North Africa and the
Middle East, where water is more scarce, access to clean drinking
water was and is a major factor in human development.
Health and pollution
An environmental science program - a student
from Iowa State University sampling water
Water fit for human consumption is called drinking water or potable
water. Water that is not potable may be made potable by filtration or
distillation, or by a range of other methods.
Water that is not fit for drinking but is not harmful for humans when
used for swimming or bathing is called by various names other than
potable or drinking water, and is sometimes called safe water, or "safe
for bathing". Chlorine is a skin and mucous membrane irritant that is
used to make water safe for bathing or drinking. Its use is highly
technical and is usually monitored by government regulations
(typically 1 part per million (ppm) for drinking water, and 12 ppm of
chlorine not yet reacted with impurities for bathing water). Water for
bathing may be maintained in satisfactory microbiological condition using chemical disinfectants such as chlorine or
ozone or by the use of ultraviolet light.
In the USA, non-potable forms of wastewater generated by humans may be referred to as greywater, which is
treatable and thus easily able to be made potable again, and blackwater, which generally contains sewage and other
forms of waste which require further treatment in order to be made reusable. Greywater composes 5080% of
residential wastewater generated by a household's sanitation equipment (sinks, showers and kitchen runoff, but not
toilets, which generate blackwater.) These terms may have different meanings in other countries and cultures.
Water
30
This natural resource is becoming scarcer in certain places, and its availability is a major social and economic
concern. Currently, about a billion people around the world routinely drink unhealthy water. Most countries accepted
the goal of halving by 2015 the number of people worldwide who do not have access to safe water and sanitation
during the 2003 G8 Evian summit.
[28]
Even if this difficult goal is met, it will still leave more than an estimated half
a billion people without access to safe drinking water and over a billion without access to adequate sanitation. Poor
water quality and bad sanitation are deadly; some five million deaths a year are caused by polluted drinking water.
The World Health Organization estimates that safe water could prevent 1.4 million child deaths from diarrhea each
year.
[29]
Water, however, is not a finite resource, but rather re-circulated as potable water in precipitation in
quantities many degrees of magnitude higher than human consumption. Therefore, it is the relatively small quantity
of water in reserve in the earth (about 1% of our drinking water supply, which is replenished in aquifers around
every 1 to 10 years), that is a non-renewable resource, and it is, rather, the distribution of potable and irrigation water
which is scarce, rather than the actual amount of it that exists on the earth. Water-poor countries use importation of
goods as the primary method of importing water (to leave enough for local human consumption), since the
manufacturing process uses around 10 to 100 times products' masses in water.
In the developing world, 90% of all wastewater still goes untreated into local rivers and streams.
[30]
Some 50
countries, with roughly a third of the world's population, also suffer from medium or high water stress, and 17 of
these extract more water annually than is recharged through their natural water cycles.
[31]
The strain not only affects
surface freshwater bodies like rivers and lakes, but it also degrades groundwater resources.
Human uses
Agriculture
Water distribution in subsurface drip irrigation.
Irrigation of field crops
The most important use of water in agriculture is for irrigation, which
is a key component to produce enough food. Irrigation takes up to 90%
of water withdrawn in some developing countries
[32]
and significant
proportions in more economically developed countries (United States,
30% of freshwater usage is for irrigation).
[33]
It takes around 3,000
litres of water, converted from liquid to vapour, to produce enough
food to satisfy one person's daily dietary need. This is a considerable
amount, when compared to that required for drinking, which is
between two and five litres. To produce food for the 6.5 billion or so
people who inhabit the planet today requires the water that would fill a
canal ten metres deep, 100 metres wide and 7.1 million kilometres long
that's enough to circle the globe 180 times.
Fifty years ago, the common perception was that water was an infinite
resource. At this time, there were fewer than half the current number of
people on the planet. People were not as wealthy as today, consumed
fewer calories and ate less meat, so less water was needed to produce
their food. They required a third of the volume of water we presently
take from rivers. Today, the competition for the fixed amount of water
resources is much more intense, giving rise to the concept of peak
water.
[34]
This is because there are now nearly seven billion people on
the planet, their consumption of water-thirsty meat and vegetables is
rising, and there is increasing competition for water from industry, urbanisation and biofuel crops. In future, even
more water will be needed to produce food because the Earth's population is forecast to rise to 9 billion by 2050.
[35]
Water
31
An additional 2.5 or 3 billion people, choosing to eat fewer cereals and more meat and vegetables could add an
additional five million kilometres to the virtual canal mentioned above.
An assessment of water management in agriculture was conducted in 2007 by the International Water Management
Institute in Sri Lanka to see if the world had sufficient water to provide food for its growing population.
[36]
It
assessed the current availability of water for agriculture on a global scale and mapped out locations suffering from
water scarcity. It found that a fifth of the world's people, more than 1.2 billion, live in areas of physical water
scarcity, where there is not enough water to meet all demands. A further 1.6 billion people live in areas experiencing
economic water scarcity, where the lack of investment in water or insufficient human capacity make it impossible for
authorities to satisfy the demand for water. The report found that it would be possible to produce the food required in
future, but that continuation of today's food production and environmental trends would lead to crises in many parts
of the world. To avoid a global water crisis, farmers will have to strive to increase productivity to meet growing
demands for food, while industry and cities find ways to use water more efficiently.
[37]
As a scientific standard
On 7 April 1795, the gram was defined in France to be equal to "the absolute weight of a volume of pure water equal
to a cube of one hundredth of a meter, and to the temperature of the melting ice."
[38]
For practical purposes though, a
metallic reference standard was required, one thousand times more massive, the kilogram. Work was therefore
commissioned to determine precisely the mass of one liter of water. In spite of the fact that the decreed definition of
the gram specified water at 0C a highly reproducible temperature the scientists chose to redefine the standard
and to perform their measurements at the temperature of highest water density, which was measured at the time as 4
C (39F).
[39]
The Kelvin temperature scale of the SI system is based on the triple point of water, defined as exactly 273.16K or
0.01C. The scale is an absolute temperature scale with the same increment as the Celsius temperature scale, which
was originally defined according the boiling point (set to 100C) and melting point (set to 0C) of water.
Natural water consists mainly of the isotopes hydrogen-1 and oxygen-16, but there is also small quantity of heavier
isotopes such as hydrogen-2 (deuterium). The amount of deuterium oxides or heavy water is very small, but it still
affects the properties of water. Water from rivers and lakes tends to contain less deuterium than seawater. Therefore,
standard water is defined in the Vienna Standard Mean Ocean Water specification.
For drinking
A young girl drinking bottled water
The human body contains from 55% to 78% water, depending on body
size.
[40]
To function properly, the body requires between one and
seven liters of water per day to avoid dehydration; the precise amount
depends on the level of activity, temperature, humidity, and other
factors. Most of this is ingested through foods or beverages other than
drinking straight water. It is not clear how much water intake is needed
by healthy people, though most advocates agree that approximately 2
liters (6 to 7 glasses) of water daily is the minimum to maintain proper
hydration.
[41]
Medical literature favors a lower consumption, typically
1 liter of water for an average male, excluding extra requirements due
to fluid loss from exercise or warm weather.
[]
For those who have
healthy kidneys, it is rather difficult to drink too much water, but (especially in warm humid weather and while
exercising) it is dangerous to drink too little. People can drink far more water than
Water
32
Water availability: fraction of population using
improved water sources by country.
necessary while exercising, however, putting them at risk of water
intoxication (hyperhydration), which can be fatal.
[42][43]
The popular
claim that "a person should consume eight glasses of water per day"
seems to have no real basis in science.
[44]
Similar misconceptions
concerning the effect of water on weight loss and constipation have
also been dispelled.
[45]
Hazard symbol for non-potable water
An original recommendation for water intake in 1945 by the Food and
Nutrition Board of the United States National Research Council read:
"An ordinary standard for diverse persons is 1 milliliter for each calorie
of food. Most of this quantity is contained in prepared foods."
[46]
The
latest dietary reference intake report by the United States National
Research Council in general recommended (including food sources):
3.7 liters for men and 2.7 liters of water total for women.
[47]
Specifically, pregnant and breastfeeding women need additional fluids
to stay hydrated. The Institute of Medicine (U.S.) recommends that, on
average, men consume 3.0 liters and women 2.2 liters; pregnant
women should increase intake to 2.4 liters (10 cups) and breastfeeding
women should get 3 liters (12 cups), since an especially large amount
of fluid is lost during nursing.
[48]
Also noted is that normally, about
20% of water intake comes from food, while the rest comes from drinking water and beverages (caffeinated
included). Water is excreted from the body in multiple forms; through urine and feces, through sweating, and by
exhalation of water vapor in the breath. With physical exertion and heat exposure, water loss will increase and daily
fluid needs may increase as well.
Humans require water with few impurities. Common impurities include metal salts and oxides, including copper,
iron, calcium and lead,
[49]
and/or harmful bacteria, such as Vibrio. Some solutes are acceptable and even desirable
for taste enhancement and to provide needed electrolytes.
[50]
The single largest (by volume) freshwater resource suitable for drinking is Lake Baikal in Siberia.
[51]
Washing
The propensity of water to form solutions and emulsions is useful in various washing processes. Many industrial
processes rely on reactions using chemicals dissolved in water, suspension of solids in water slurries or using water
to dissolve and extract substances. Washing is also an important component of several aspects of personal body
hygiene.
Transportation
The use of water for transportation of materials through rivers and canals as well as the international shipping lanes
is an important part of the world economy.
Water
33
Chemical uses
Water is widely used in chemical reactions as a solvent or reactant and less commonly as a solute or catalyst. In
inorganic reactions, water is a common solvent, dissolving many ionic compounds. In organic reactions, it is not
usually used as a reaction solvent, because it does not dissolve the reactants well and is amphoteric (acidic and basic)
and nucleophilic. Nevertheless, these properties are sometimes desirable. Also, acceleration of Diels-Alder reactions
by water has been observed. Supercritical water has recently been a topic of research. Oxygen-saturated supercritical
water combusts organic pollutants efficiently.
Heat exchange
Water and steam are used as heat transfer fluids in diverse heat exchange systems, due to its availability and high
heat capacity, both as a coolant and for heating. Cool water may even be naturally available from a lake or the sea.
Condensing steam is a particularly efficient heating fluid because of the large heat of vaporization. A disadvantage is
that water and steam are somewhat corrosive. In almost all electric power stations, water is the coolant, which
vaporizes and drives steam turbines to drive generators. In the U.S., cooling power plants is the largest use of
water.
[33]
In the nuclear power industry, water can also be used as a neutron moderator. In most nuclear reactors, water is both
a coolant and a moderator. This provides something of a passive safety measure, as removing the water from the
reactor also slows the nuclear reaction down however other methods are favored for stopping a reaction and it is
preferred to keep the nuclear core covered with water so as to ensure adequate cooling.
Fire extinction
Water is used for fighting wildfires.
Water has a high heat of vaporization and is relatively inert, which
makes it a good fire extinguishing fluid. The evaporation of water
carries heat away from the fire. It is dangerous to use water on fires
involving oils and organic solvents, because many organic materials
float on water and the water tends to spread the burning liquid.
Use of water in fire fighting should also take into account the hazards
of a steam explosion, which may occur when water is used on very hot
fires in confined spaces, and of a hydrogen explosion, when substances
which react with water, such as certain metals or hot carbon such as
coal, charcoal, coke graphite, decompose the water, producing water
gas.
The power of such explosions was seen in the Chernobyl disaster, although the water involved did not come from
fire-fighting at that time but the reactor's own water cooling system. A steam explosion occurred when the extreme
overheating of the core caused water to flash into steam. A hydrogen explosion may have occurred as a result of
reaction between steam and hot zirconium.
Water
34
Recreation
Grand Anse Beach, St. George's, Grenada, West
Indies.
Humans use water for many recreational purposes, as well as for
exercising and for sports. Some of these include swimming,
waterskiing, boating, surfing and diving. In addition, some sports, like
ice hockey and ice skating, are played on ice. Lakesides, beaches and
water parks are popular places for people to go to relax and enjoy
recreation. Many find the sound and appearance of flowing water to be
calming, and fountains and other water features are popular
decorations. Some keep fish and other life in aquariums or ponds for
show, fun, and companionship. Humans also use water for snow sports
i.e. skiing, sledding, snowmobiling or snowboarding, which requires
the water to be frozen.
Water industry
A water-carrier in India, 1882. In
many places where running water is
not available, water has to be
transported by people.
A manual water pump in China
The water industry provides drinking water and wastewater services
(including sewage treatment) to households and industry. Water supply
facilities include water wells cisterns for rainwater harvesting, water
supply network, water purification facilities, water tanks, water towers,
water pipes including old aqueducts. Atmospheric water generators are
in development.
Drinking water is often collected at springs, extracted from artificial
borings (wells) in the ground, or pumped from lakes and rivers.
Building more wells in adequate places is thus a possible way to
produce more water, assuming the aquifers can supply an adequate
flow. Other water sources include rainwater collection. Water may
require purification for human consumption. This may involve removal
of undissolved substances, dissolved substances and harmful microbes.
Popular methods are filtering with sand which only removes
undissolved material, while chlorination and boiling kill harmful
microbes. Distillation does all three functions. More advanced
techniques exist, such as reverse osmosis. Desalination of abundant
seawater is a more expensive solution used in coastal arid climates.
The distribution of drinking water is done through municipal water
systems, tanker delivery or as bottled water. Governments in many
countries have programs to distribute water to the needy at no charge.
Reducing usage by using drinking (potable) water only for human
consumption is another option. In some cities such as Hong Kong, sea
water is extensively used for flushing toilets citywide in order to
conserve fresh water resources.
Polluting water may be the biggest single misuse of water; to the extent
that a pollutant limits other uses of the water, it becomes a waste of the resource, regardless of benefits to the
polluter. Like other types of pollution, this does not enter standard accounting of market costs,
Water
35
Water purification facility
being conceived as externalities for which the market cannot account.
Thus other people pay the price of water pollution, while the private
firms' profits are not redistributed to the local population victim of this
pollution. Pharmaceuticals consumed by humans often end up in the
waterways and can have detrimental effects on aquatic life if they
bioaccumulate and if they are not biodegradable.
Wastewater facilities are storm sewers and wastewater treatment
plants. Another way to remove pollution from surface runoff water is
bioswale.
Industrial applications
Water is used in power generation. Hydroelectricity is electricity obtained from hydropower. Hydroelectric power
comes from water driving a water turbine connected to a generator. Hydroelectricity is a low-cost, non-polluting,
renewable energy source. The energy is supplied by the motion of water. Typically a dam is constructed on a river,
creating an artificial lake behind it. Water flowing out of the lake is forced through turbines that turn generators.
Three Gorges Dam is the largest hydro-electric power station.
Pressurized water is used in water blasting and water jet cutters. Also, very high pressure water guns are used for
precise cutting. It works very well, is relatively safe, and is not harmful to the environment. It is also used in the
cooling of machinery to prevent overheating, or prevent saw blades from overheating.
Water is also used in many industrial processes and machines, such as the steam turbine and heat exchanger, in
addition to its use as a chemical solvent. Discharge of untreated water from industrial uses is pollution. Pollution
includes discharged solutes (chemical pollution) and discharged coolant water (thermal pollution). Industry requires
pure water for many applications and utilizes a variety of purification techniques both in water supply and discharge.
Water
36
Food processing
Water can be used to cook foods such as noodles.
Water plays many critical roles within the field of food science. It is
important for a food scientist to understand the roles that water plays
within food processing to ensure the success of their products.
Solutes such as salts and sugars found in water affect the physical
properties of water. The boiling and freezing points of water are
affected by solutes, as well as air pressure, which is in turn affected by
altitude. Water boils at lower temperatures with the lower air pressure
which occurs at higher elevations. One mole of sucrose (sugar) per
kilogram of water raises the boiling point of water by 0.51 C, and one
mole of salt per kg raises the boiling point by 1.02 C; similarly,
increasing the number of dissolved particles lowers water's freezing
point.
[]
Solutes in water also affect water activity which affects many chemical reactions and the growth of microbes
in food.
[]
Water activity can be described as a ratio of the vapor pressure of water in a solution to the vapor pressure
of pure water.
[]
Solutes in water lower water activity. This is important to know because most bacterial growth
ceases at low levels of water activity.
[]
Not only does microbial growth affect the safety of food but also the
preservation and shelf life of food.
Water hardness is also a critical factor in food processing. It can dramatically affect the quality of a product as well
as playing a role in sanitation. Water hardness is classified based on the amounts of removable calcium carbonate
salt it contains per gallon. Water hardness is measured in grains; 0.064 g calcium carbonate is equivalent to one grain
of hardness.
[]
Water is classified as soft if it contains 1 to 4 grains, medium if it contains 5 to 10 grains and hard if it
contains 11 to 20 grains. Wikipedia:Vagueness
[]
The hardness of water may be altered or treated by using a
chemical ion exchange system. The hardness of water also affects its pH balance which plays a critical role in food
processing. For example, hard water prevents successful production of clear beverages. Water hardness also affects
sanitation; with increasing hardness, there is a loss of effectiveness for its use as a sanitizer.
[]
Boiling, steaming, and simmering are popular cooking methods that often require immersing food in water or its
gaseous state, steam. Water is also used for dishwashing.
Water
37
Water law, water politics and water crisis
An estimate of the share of people in developing countries with access to potable
water 19702000
Water politics is politics affected by water
and water resources. For this reason, water
is a strategic resource in the globe and an
important element in many political
conflicts. It causes health impacts and
damage to biodiversity.
1.6 billion people have gained access to a
safe water source since 1990.
[52]
The
proportion of people in developing countries
with access to safe water is calculated to
have improved from 30% in 1970
[]
to 71%
in 1990, 79% in 2000 and 84% in 2004.
This trend is projected to continue.
[]
To
halve, by 2015, the proportion of people
without sustainable access to safe drinking
water is one of the Millennium
Development Goals. This goal is projected
to be reached.
A 2006 United Nations report stated that "there is enough water for everyone", but that access to it is hampered by
mismanagement and corruption.
[53]
In addition, global initiatives to improve the efficiency of aid delivery, such as
the Paris Declaration on Aid Effectiveness, have not been taken up by water sector donors as effectively as they have
in education and health, potentially leaving multiple donors working on overlapping projects and recipient
governments without empowerment to act.
[54]
The authors of the 2007 Comprehensive Assessment of Water Management in Agriculture cited poor governance as
one reason for some forms of water scarcity. Water governance is the set of formal and informal processes through
which decisions related to water management are made. Good water governance is primarily about knowing what
processes work best in a particular physical and socioeconomic context. Mistakes have sometimes been made by
trying to apply 'blueprints' that work in the developed world to developing world locations and contexts. The
Mekong river is one example; a review by the International Water Management Institute of policies in six countries
that rely on the Mekong river for water found that thorough and transparent cost-benefit analyses and environmental
impact assessments were rarely undertaken. They also discovered that Cambodia's draft water law was much more
complex than it needed to be.
[55]
The UN World Water Development Report (WWDR, 2003) from the World Water Assessment Program indicates
that, in the next 20 years, the quantity of water available to everyone is predicted to decrease by 30%. 40% of the
world's inhabitants currently have insufficient fresh water for minimal hygiene. More than 2.2 million people died in
2000 from waterborne diseases (related to the consumption of contaminated water) or drought. In 2004, the UK
charity WaterAid reported that a child dies every 15 seconds from easily preventable water-related diseases; often
this means lack of sewage disposal; see toilet.
Organizations concerned with water protection include International Water Association (IWA), WaterAid, Water 1st,
American Water Resources Association
[56]
. The International Water Management Institute undertakes projects with
the aim of using effective water management to reduce poverty. Water related conventions are United Nations
Convention to Combat Desertification (UNCCD), International Convention for the Prevention of Pollution from
Ships, United Nations Convention on the Law of the Sea and Ramsar Convention. World Day for Water takes place
on 22 March and World Ocean Day on 8 June.
Water
38
Water used in the production of a good or service is virtual water.
In culture
Religion
Water is considered a purifier in most religions. Major faiths that incorporate ritual washing (ablution) include
Christianity, Hinduism, Islam, Judaism, Rastafari movement, Shinto, Taoism, and Wicca. Immersion (or aspersion or
affusion) of a person in water is a central sacrament of Christianity (where it is called baptism); it is also a part of the
practice of other religions, including Islam (Ghusl), Judaism (mikvah) and Sikhism (Amrit Sanskar). In addition, a
ritual bath in pure water is performed for the dead in many religions including Islam and Judaism. In Islam, the five
daily prayers can be done in most cases after completing washing certain parts of the body using clean water (wudu),
unless water is unavailable (see Tayammum). In Shinto, water is used in almost all rituals to cleanse a person or an
area (e.g., in the ritual of misogi). Water is mentioned numerous times in the Bible, for example: "The earth was
formed out of water and by water" (NIV). In the Qur'an it is stated that "Living things are made of water" and it is
often used to describe paradise.
Philosophy
The Ancient Greek philosopher Empedocles held that water is one of the four classical elements along with fire,
earth and air, and was regarded as the ylem, or basic substance of the universe. Water was considered cold and moist.
In the theory of the four bodily humors, water was associated with phlegm. The classical element of Water was also
one of the five elements in traditional Chinese philosophy, along with earth, fire, wood, and metal.
Water is also taken as a role model in some parts of traditional and popular Asian philosophy. James Legge's 1891
translation of the Dao De Jing states "The highest excellence is like (that of) water. The excellence of water appears
in its benefiting all things, and in its occupying, without striving (to the contrary), the low place which all men
dislike. Hence (its way) is near to (that of) the Tao" and "There is nothing in the world more soft and weak than
water, and yet for attacking things that are firm and strong there is nothing that can take precedence of itfor there
is nothing (so effectual) for which it can be changed."
[57]
Literature
Water is used in literature as a symbol of purification. Examples include the critical importance of a river in As I Lay
Dying by William Faulkner and the drowning of Ophelia in Hamlet.
Sherlock Holmes held that "From a drop of water, a logician could infer the possibility of an Atlantic or a Niagara
without having seen or heard of one or the other."
[58]
References
[5] Water Vapor in the Climate System (http:/ / www.agu. org/ sci_soc/ mockler. html), Special Report, [AGU], December 1995 (linked 4/2007).
Vital Water (http:/ / www. unep.org/ dewa/ assessments/ ecosystems/ water/ vitalwater/ ) UNEP.
[6] "Public Services" (http:/ / www. gapminder. org/ videos/ gapcasts/ gapcast-9-public-services/ ), Gapminder video
[11] Capillary Action Liquid, Water, Force, and Surface JRank Articles (http:/ / science. jrank. org/ pages/ 1182/ Capillary-Action. html)
[15] Melnick, Gary, Harvard-Smithsonian Center for Astrophysics and Neufeld, David, Johns Hopkins University quoted in:
(linked 4/2007)
[16] Water Found on Distant Planet (http:/ / www.time. com/ time/ health/ article/ 0,8599,1642811,00. html) July 12, 2007 By Laura Blue, Time
[17] Water Found in Extrasolar Planet's Atmosphere (http:/ / www. space. com/ scienceastronomy/ 070410_water_exoplanet. html) Space.com
[18] NASA, " MESSENGER Finds New Evidence for Water Ice at Mercury's Poles (http:/ / www. nasa. gov/ mission_pages/ messenger/ media/
PressConf20121129.html)", 29 November 2012.
[19] Weird water lurking inside giant planets (http:/ / www. newscientist. com/ article/ mg20727764.
500-weird-water-lurking-inside-giant-planets.html), New Scientist, 1 September 2010, Magazine issue 2776.
Water
39
[20] Versteckt in Glasperlen: Auf dem Mond gibt es Wasser Wissenschaft [[Der Spiegel (http:/ / www. spiegel. de/ wissenschaft/ weltall/
0,1518,564911,00.html)] Nachrichten]
[21] Water Molecules Found on the Moon (http:/ / science. nasa. gov/ headlines/ y2009/ 24sep_moonwater. htm), NASA, 24 September 2009
[33] Water Use in the United States (http:/ / nationalatlas. gov/ articles/ water/ a_wateruse. html), National Atlas.gov
[35] United Nations Press Release POP/952, 13 March 2007. World population will increase by 2.5 billion by 2050 (http:/ / www. un. org/ News/
Press/ docs/ 2007/ pop952. doc. htm)
[36] Molden, D. (Ed). Water for food, Water for life: A Comprehensive Assessment of Water Management in Agriculture. Earthscan/IWMI, 2007.
[37] Chartres, C. and Varma, S. Out of water. From Abundance to Scarcity and How to Solve the World's Water Problems FT Press (USA), 2010
[38] Dcret relatif aux poids et aux mesures. 18 germinal an 3 (7 avril 1795) (http:/ / smdsi. quartier-rural. org/ histoire/ 18germ_3. htm). Decree
relating to the weights and measurements (in French). quartier-rural.org
[39] here L'Histoire Du Mtre, La Dtermination De L'Unit De Poids (http:/ / histoire. du. metre. free. fr/ fr/ index. htm).
histoire.du.metre.free.fr
[40] Re: What percentage of the human body is composed of water? (http:/ / www. madsci. org/ posts/ archives/ 2000-05/ 958588306. An. r.
html) Jeffrey Utz, M.D., The MadSci Network
[44] "Drink at least eight glasses of water a day." Really? Is there scientific evidence for "8 8"? (http:/ / ajpregu. physiology. org/ cgi/ content/
full/ 283/ 5/ R993) by Heinz Valdin, Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire
[45] Drinking Water How Much? (http:/ / www.factsmart. org/ h2o/ h2o. htm), Factsmart.org web site and references within
[47] Dietary Reference Intakes: Water, Potassium, Sodium, Chloride, and Sulfate (http:/ / www. iom. edu/ report. asp?id=18495), Food and
Nutrition Board
[49] [49] "Conquering Chemistry" 4th Ed. Published 2008
[52] The Millennium Development Goals Report (http:/ / mdgs. un. org/ unsd/ mdg/ Resources/ Static/ Products/ Progress2008/
MDG_Report_2008_En. pdf#page=44), United Nations, 2008
[53] UNESCO, (2006), Water, a shared responsibility. The United Nations World Water Development Report 2 (http:/ / unesdoc. unesco. org/
images/ 0014/ 001444/ 144409E. pdf).
[54] Welle, Katharina; Evans, Barbara; Tucker, Josephine and Nicol, Alan (2008) Is water lagging behind on Aid Effectiveness? (http:/ / www.
odi. org. uk/ resources/ download/ 1894.pdf)
[55] Water governance (http:/ / www.iwmi.cgiar.org/ Publications/ Water_Issue_Briefs/ index. aspx), Water Issue Brief, Issue 5, 2010, IWMI
[56] http:/ / www.awra. org/
[58] Arthur Conan Doyle, A Study in Scarlet, Chapter 2, "The Science of Deduction"
Further reading
Debenedetti,PG., and HE Stanley, "Supercooled and Glassy Water", Physics Today 56 (6), p.4046 (2003).
Downloadable PDF (1.9 MB) (http:/ / polymer. bu. edu/ hes/ articles/ ds03. pdf)
Franks, F (Ed), Water, A comprehensive treatise, Plenum Press, New York, 19721982
Gleick, PH., (editor), The World's Water: The Biennial Report on Freshwater Resources. Island Press,
Washington, D.C. (published every two years, beginning in 1998.) The World's Water, Island Press (http:/ / www.
worldwater. org/ )
Jones, OA., JN Lester and N Voulvoulis, Pharmaceuticals: a threat to drinking water? TRENDS in Biotechnology
23(4): 163, 2005
Journal of Contemporary Water Resources and Education (http:/ / ucowr. org/ updates/ index. html)
Postel,S., Last Oasis: Facing Water Scarcity. W.W. Norton and Company, New York. 1992
Reisner,M., Cadillac Desert: The American West and Its Disappearing Water. Penguin Books, New York. 1986.
United Nations World Water Development Report. Produced every three years. UN World Water Development
Report (http:/ / www. unesco. org/ water/ wwap/ wwdr/ )
Water
40
External links
OECD Water statistics (http:/ / stats. oecd. org/ wbos/ Index. aspx?DataSetCode=ENV_WAT)
The World's Water Data Page (http:/ / www. worldwater. org/ )
FAO Comprehensive Water Database, AQUASTAT (http:/ / www. fao. org/ nr/ water/ aquastat/ main/ index. stm)
The Water Conflict Chronology: Water Conflict Database (http:/ / worldwater. org/ conflict. html)
US Geological Survey Water for Schools information (http:/ / ga. water. usgs. gov/ edu/ )
Portal to The World Bank's strategy, work and associated publications on water resources (http:/ / water.
worldbank. org/ )
41
Structural Biochemistry
42
Nucleic acids
Nucleic acid
A comparison of the two principal nucleic acids: RNA (left) and DNA (right),
showing the helices and nucleobases each employs.
Nucleic acids are large biological molecules
essential for all known forms of life. They
include DNA (deoxyribonucleic acid) and
RNA (ribonucleic acid). Together with
proteins, nucleic acids are the most
important biological macromolecules; each
is found in abundance in all living things,
where they function in encoding,
transmitting and expressing genetic
information.
Nucleic acids were discovered by Friedrich
Miescher in 1869.
[1]
Experimental studies of
nucleic acids constitute a major part of
modern biological and medical research, and
form a foundation for genome and forensic
science, as well as the biotechnology and
pharmaceutical industries.
[][][]
Occurrence and
nomenclature
[]
The term nucleic acid is the overall name for DNA and RNA, members of a family of biopolymers,
[2]
and is
synonymous with polynucleotide. Nucleic acids were named for their initial discovery within the nucleus, and for the
presence of phosphate groups (related to phosphoric acid). Although first discovered within the nucleus of
eukaryotic cells, nucleic acids are now known to be found in all life forms as well as some nonliving entities,
including within bacteria, archaea, mitochondria, chloroplasts, viruses and viroids. All living cells contain both DNA
and RNA (except some cells such as mature red blood cells), while viruses contain either DNA or RNA, but usually
not both.
[]
The basic component of biological nucleic acids is the nucleotide, each of which contains a pentose sugar
(ribose or deoxyribose), a phosphate group, and a nucleobase. Nucleic acids are also generated within the laboratory,
through the use of enzymes
[3]
(DNA and RNA polymerases) and by solid-phase chemical synthesis. The chemical
methods also enable the generation of altered nucleic acids that are not found in nature,
[4]
for example peptide
nucleic acids.
Nucleic acid
43
Molecular composition and size
[]
Nucleic acids can vary in size, but are generally very large molecules. Indeed, DNA molecules are probably the
largest individual molecules known. Well-studied biological nucleic acid molecules range in size from 21
nucleotides (small interfering RNA) to large chromosomes (human chromosome 1 is a single molecule that contains
247 million base pairs
[5]
).
In most cases, naturally occurring DNA molecules are double-stranded and RNA molecules are single-stranded.
There are numerous exceptions, howeversome viruses have genomes made of double-stranded RNA and other
viruses have single-stranded DNA genomes, and, in some circumstances, nucleic acid structures with three or four
strands can form.
Nucleic acids are linear polymers (chains) of nucleotides. Each nucleotide consists of three components: a purine or
pyrimidine nucleobase (sometimes termed nitrogenous base or simply base), a pentose sugar, and a phosphate
group. The substructure consisting of a nucleobase plus sugar is termed a nucleoside. Nucleic acid types differ in the
structure of the sugar in their nucleotides - DNA contains 2'-deoxyribose while RNA contains ribose (where the only
difference is the presence of a hydroxyl group). Also, the nucleobases found in the two nucleic acid types are
different: adenine, cytosine, and guanine are found in both RNA and DNA, while thymine occurs in DNA and uracil
occurs in RNA.
The sugars and phosphates in nucleic acids are connected to each other in an alternating chain (sugar-phosphate
backbone) through phosphodiester linkages.
[]
In conventional nomenclature, the carbons to which the phosphate
groups attach are the 3'-end and the 5'-end carbons of the sugar. This gives nucleic acids directionality, and the ends
of nucleic acid molecules are referred to as 5'-end and 3'-end. The nucleobases are joined to the sugars via an
N-glycosidic linkage involving a nucleobase ring nitrogen (N-1 for pyrimidines and N-9 for purines) and the 1'
carbon of the pentose sugar ring.
Non-standard nucleosides are also found in both RNA and DNA and usually arise from modification of the standard
nucleosides within the DNA molecule or the primary (initial) RNA transcript. Transfer RNA (tRNA) molecules
contain a particularly large number of modified nucleosides.
[6]
Topology
Double-stranded nucleic acids are made up of complementary sequences, in which extensive Watson-Crick base
pairing results in the a highly repeated and quite uniform double-helical three-dimensional structure.
[7]
In contrast,
single-stranded RNA and DNA molecules are not constrained to a regular double helix, and can adopt highly
complex three-dimensional structures that are based on short stretches of intramolecular base-paired sequences that
include both Watson-Crick and noncanonical base pairs, as well as a wide range of complex tertiary interactions.
[8]
Nucleic acid molecules are usually unbranched, and may occur as linear and circular molecules. For example,
bacterial chromosomes, plasmids, mitochondrial DNA and chloroplast DNA are usually circular double-stranded
DNA molecules, while chromosomes of the eukaryotic nucleus are usually linear double-stranded DNA molecules.
[]
Most RNA molecules are linear, single-stranded molecules, but both circular and branched molecules can result from
RNA splicing reactions.
[]
Nucleic acid sequences
One DNA or RNA molecule differs from another primarily in the sequence of nucleotides. Nucleotide sequences are
of great importance in biology, since they carry the ultimate instructions that encode all biological molecules,
molecular assemblies, subcellular and cellular structures, organs and organisms, and directly enable cognition,
memory and behavior (See: Genetics). Enormous efforts have gone into the development of experimental methods to
determine the nucleotide sequence of biological DNA and RNA molecules,
[9][10]
and today hundreds of millions of
nucleotides are sequenced daily at genome centers and smaller laboratories worldwide.
Nucleic acid
44
Types of nucleic acids
Deoxyribonucleic acid
Deoxyribonucleic acid (/diksirab.njukle.k sd/; DNA) is a nucleic acid containing the genetic instructions
used in the development and functioning of all known living organisms (with the exception of RNA viruses). The
DNA segments carrying this genetic information are called genes. Likewise, other DNA sequences have structural
purposes, or are involved in regulating the use of this genetic information. Along with RNA and proteins, DNA is
one of the three major macromolecules that are essential for all known forms of life. DNA consists of two long
polymers of simple units called nucleotides, with backbones made of sugars and phosphate groups joined by ester
bonds. These two strands run in opposite directions to each other and are therefore anti-parallel. Attached to each
sugar is one of four types of molecules called nucleobases (informally, bases). It is the sequence of these four
nucleobases along the backbone that encodes information. This information is read using the genetic code, which
specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the
related nucleic acid RNA in a process called transcription. Within cells DNA is organized into long structures called
chromosomes. During cell division these chromosomes are duplicated in the process of DNA replication, providing
each cell its own complete set of chromosomes. Eukaryotic organisms (animals, plants, fungi, and protists) store
most of their DNA inside the cell nucleus and some of their DNA in organelles, such as mitochondria or
chloroplasts.[1] In contrast, prokaryotes (bacteria and archaea) store their DNA only in the cytoplasm. Within the
chromosomes, chromatin proteins such as histones compact and organize DNA. These compact structures guide the
interactions between DNA and other proteins, helping control which parts of the DNA are transcribed.
Ribonucleic acid
Ribonucleic acid (RNA) functions in converting genetic information from genes into the amino acid sequences of
proteins. The three universal types of RNA include transfer RNA (tRNA), messenger RNA (mRNA), and ribosomal
RNA (rRNA). Messenger RNA acts to carry genetic sequence information between DNA and ribosomes, directing
protein synthesis. Ribosomal RNA is a major component of the ribosome, and catalyzes peptide bond formation.
Transfer RNA serves as the carrier molecule for amino acids to be used in protein synthesis, and is responsible for
decoding the mRNA. In addition, many other classes of RNA are now known.
Artificial nucleic acid analogs
Artificial nucleic acid analogs have been designed and synthesized by chemists, and include peptide nucleic acid,
morpholino- and locked nucleic acid, as well as glycol nucleic acid and threose nucleic acid. Each of these is
distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule.
References
[3] Mullis, Kary B. The Polymerase Chain Reaction (Nobel Lecture). 1993. (retrieved December 1, 2010) http:/ / nobelprize. org/ nobel_prizes/
chemistry/ laureates/ 1993/ mullis-lecture.html
[9] Gilbert, Walter G. 1980. DNA Sequencing and Gene Structure (Nobel Lecture) http:/ / nobelprize. org/ nobel_prizes/ chemistry/ laureates/
1980/ gilbert-lecture.html
[10] Sanger, Frederick. 1980. Determination of Nucleotide Sequences in DNA (Nobel Lecture) http:/ / nobelprize. org/ nobel_prizes/ chemistry/
laureates/ 1980/ sanger-lecture. html
Nucleic acid
45
Further reading
Wolfram Saenger, Principles of Nucleic Acid Structure, 1984, Springer-Verlag New York Inc.
Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter Molecular Biology
of the Cell, 2007, ISBN 978-0-8153-4105-5. Fourth edition is available online through the NCBI Bookshelf: link
(http:/ / www. ncbi. nlm. nih. gov/ bookshelf/ br. fcgi?book=mboc4)
Jeremy M Berg, John L Tymoczko, and Lubert Stryer, Biochemistry 5th edition, 2002, W H Freeman. Available
online through the NCBI Bookshelf: link (http:/ / www. ncbi. nlm. nih. gov/ bookshelf/ br. fcgi?book=stryer)
Astrid Sigel, Helmut Sigel and Roland K. O. Sigel, ed. (2012). Interplay between Metal Ions and Nucleic Acids.
Metal Ions in Life Sciences 10. Springer. doi: 10.1007/978-94-007-2172-2 (http:/ / dx. doi. org/ 10. 1007/
978-94-007-2172-2). ISBN978-94-007-2171-5.
External links
Interview with Aaron Klug, Nobel Laureate for structural elucidation of biologically important nucleic-acid
protein complexes (http:/ / www. vega. org. uk/ video/ programme/ 122) provided by the Vega Science Trust.
Nucleic Acids Research (Journal) (http:/ / nar. oxfordjournals. org/ )
Nucleic Acids Book (free online book on the chemistry and biology of nucleic acids) (http:/ / www. atdbio. com/
nucleic-acids-book)
RNA
RNA
46
A hairpin loop from a pre-mRNA. Highlighted
are the nucleobases (green) and the
ribose-phosphate backbone (blue). Note that this
is a single strand of RNA that folds back upon
itself.
Ribonucleic acid (RNA) is a ubiquitous family of large biological
molecules that perform multiple vital roles in the coding, decoding,
regulation, and expression of genes. Together with DNA, RNA
comprises the nucleic acids, which, along with proteins, constitute the
three major macromolecules essential for all known forms of life. Like
DNA, RNA is assembled as a chain of nucleotides, but is usually
single-stranded. Cellular organisms use messenger RNA (mRNA) to
convey genetic information (often notated using the letters G, A, U,
and C for the nucleotides guanine, adenine, uracil and cytosine) that
directs synthesis of specific proteins, while many viruses encode their
genetic information using an RNA genome.
Some RNA molecules play an active role within cells by catalyzing
biological reactions, controlling gene expression, or sensing and
communicating responses to cellular signals. One of these active
processes is protein synthesis, a universal function whereby mRNA
molecules direct the assembly of proteins on ribosomes. This process
uses transfer RNA (tRNA) molecules to deliver amino acids to the
ribosome, where ribosomal RNA (rRNA) links amino acids together to
form proteins.
Comparison with DNA
Three-dimensional representation of the 50S
ribosomal subunit. RNA is in ochre, protein in
blue. The active site is in the middle (red).
The chemical structure of RNA is very similar to that of DNA, but
differs in three main ways:
Unlike double-stranded DNA, RNA is a single-stranded molecule in
many of its biological roles and has a much shorter chain of
nucleotides. However, RNA can, by complementary base pairing,
form intrastrand double helixes, as in tRNA.
While DNA contains deoxyribose, RNA contains ribose (in
deoxyribose there is no hydroxyl group attached to the pentose ring
in the 2' position). These hydroxyl groups make RNA less stable
than DNA because it is more prone to hydrolysis.
The complementary base to adenine is not thymine, as it is in DNA,
but rather uracil, which is an unmethylated form of thymine.
[]
Like DNA, most biologically active RNAs, including mRNA, tRNA,
rRNA, snRNAs, and other non-coding RNAs, contain
self-complementary sequences that allow parts of the RNA to fold
[1]
and pair with itself to form double helices.
Analysis of these RNAs has revealed that they are highly structured. Unlike DNA, their structures do not consist of
RNA
47
long double helices but rather collections of short helices packed together into structures akin to proteins. In this
fashion, RNAs can achieve chemical catalysis, like enzymes.
[2]
For instance, determination of the structure of the
ribosomean enzyme that catalyzes peptide bond formationrevealed that its active site is composed entirely of
RNA.
[]
Structure
Watson-Crick base pairs in a siRNA (hydrogen atoms are not
shown)
Each nucleotide in RNA contains a ribose sugar, with
carbons numbered 1' through 5'. A base is attached to the 1'
position, in general, adenine (A), cytosine (C), guanine (G),
or uracil (U). Adenine and guanine are purines, cytosine,
and uracil are pyrimidines. A phosphate group is attached to
the 3' position of one ribose and the 5' position of the next.
The phosphate groups have a negative charge each at
physiological pH, making RNA a charged molecule
(polyanion). The bases may form hydrogen bonds between
cytosine and guanine, between adenine and uracil and
between guanine and uracil.
[]
However, other interactions
are possible, such as a group of adenine bases binding to
each other in a bulge,
[3]
or the GNRA tetraloop that has a
guanineadenine base-pair.
[]
Chemical structure of RNA
An important structural feature of RNA that distinguishes it from DNA
is the presence of a hydroxyl group at the 2' position of the ribose
sugar. The presence of this functional group causes the helix to adopt
the A-form geometry rather than the B-form most commonly observed
in DNA.
[4]
This results in a very deep and narrow major groove and a
shallow and wide minor groove.
[5]
A second consequence of the
presence of the 2'-hydroxyl group is that in conformationally flexible
regions of an RNA molecule (that is, not involved in formation of a
double helix), it can chemically attack the adjacent phosphodiester
bond to cleave the backbone.
[6]
Secondary structure of a telomerase RNA.
RNA is transcribed with only four bases (adenine, cytosine, guanine
and uracil),
[7]
but these bases and attached sugars can be modified in
numerous ways as the RNAs mature. Pseudouridine (), in which the
linkage between uracil and ribose is changed from a CN bond to a
CC bond, and ribothymidine (T) are found in various places (the most
notable ones being in the TC loop of tRNA).
[8]
Another notable
modified base is hypoxanthine, a deaminated adenine base whose
nucleoside is called inosine (I). Inosine plays a key role in the wobble
hypothesis of the genetic code.
[9]
RNA
48
There are nearly 100 other naturally occurring modified nucleosides,
[10]
of which pseudouridine and nucleosides
with 2'-O-methylribose are the most common.
[11]
The specific roles of many of these modifications in RNA are not
fully understood. However, it is notable that, in ribosomal RNA, many of the post-transcriptional modifications
occur in highly functional regions, such as the peptidyl transferase center and the subunit interface, implying that
they are important for normal function.
[12]
The functional form of single-stranded RNA molecules, just like proteins, frequently requires a specific tertiary
structure. The scaffold for this structure is provided by secondary structural elements that are hydrogen bonds within
the molecule. This leads to several recognizable "domains" of secondary structure like hairpin loops, bulges, and
internal loops.
[13]
Since RNA is charged, metal ions such as Mg
2+
are needed to stabilise many secondary and
tertiary structures.
[14]
Synthesis
Synthesis of RNA is usually catalyzed by an enzymeRNA polymeraseusing DNA as a template, a process
known as transcription. Initiation of transcription begins with the binding of the enzyme to a promoter sequence in
the DNA (usually found "upstream" of a gene). The DNA double helix is unwound by the helicase activity of the
enzyme. The enzyme then progresses along the template strand in the 3 to 5 direction, synthesizing a
complementary RNA molecule with elongation occurring in the 5 to 3 direction. The DNA sequence also dictates
where termination of RNA synthesis will occur.
[15]
RNAs are often modified by enzymes after transcription. For example, a poly(A) tail and a 5' cap are added to
eukaryotic pre-mRNA and introns are removed by the spliceosome.
There are also a number of RNA-dependent RNA polymerases that use RNA as their template for synthesis of a new
strand of RNA. For instance, a number of RNA viruses (such as poliovirus) use this type of enzyme to replicate their
genetic material.
[16]
Also, RNA-dependent RNA polymerase is part of the RNA interference pathway in many
organisms.
[17]
RNA
49
Types of RNA
Overview
Structure of a hammerhead ribozyme, a ribozyme
that cuts RNA
Messenger RNA (mRNA) is the RNA that carries information from
DNA to the ribosome, the sites of protein synthesis (translation) in the
cell. The coding sequence of the mRNA determines the amino acid
sequence in the protein that is produced.
[]
Many RNAs do not code for
protein however (about 97% of the transcriptional output is
non-protein-coding in eukaryotes
[18][19][20][21]
).
These so-called non-coding RNAs ("ncRNA") can be encoded by their
own genes (RNA genes), but can also derive from mRNA introns.
[]
The most prominent examples of non-coding RNAs are transfer RNA
(tRNA) and ribosomal RNA (rRNA), both of which are involved in the
process of translation.
[]
There are also non-coding RNAs involved in
gene regulation, RNA processing and other roles. Certain RNAs are
able to catalyse chemical reactions such as cutting and ligating other
RNA molecules,
[22]
and the catalysis of peptide bond formation in the
ribosome;
[]
these are known as ribozymes.
In translation
Messenger RNA (mRNA) carries information about a protein sequence
to the ribosomes, the protein synthesis factories in the cell. It is coded
so that every three nucleotides (a codon) correspond to one amino acid.
In eukaryotic cells, once precursor mRNA (pre-mRNA) has been transcribed from DNA, it is processed to mature
mRNA. This removes its intronsnon-coding sections of the pre-mRNA. The mRNA is then exported from the
nucleus to the cytoplasm, where it is bound to ribosomes and translated into its corresponding protein form with the
help of tRNA. In prokaryotic cells, which do not have nucleus and cytoplasm compartments, mRNA can bind to
ribosomes while it is being transcribed from DNA. After a certain amount of time the message degrades into its
component nucleotides with the assistance of ribonucleases.
[]
Transfer RNA (tRNA) is a small RNA chain of about 80 nucleotides that transfers a specific amino acid to a growing
polypeptide chain at the ribosomal site of protein synthesis during translation. It has sites for amino acid attachment
and an anticodon region for codon recognition that binds to a specific sequence on the messenger RNA chain
through hydrogen bonding.
[]
Ribosomal RNA (rRNA) is the catalytic component of the ribosomes. Eukaryotic ribosomes contain four different
rRNA molecules: 18S, 5.8S, 28S and 5S rRNA. Three of the rRNA molecules are synthesized in the nucleolus, and
one is synthesized elsewhere. In the cytoplasm, ribosomal RNA and protein combine to form a nucleoprotein called
a ribosome. The ribosome binds mRNA and carries out protein synthesis. Several ribosomes may be attached to a
single mRNA at any time.
[]
Nearly all the RNA found in a typical eukaryotic cell is rRNA.
Transfer-messenger RNA (tmRNA) is found in many bacteria and plastids. It tags proteins encoded by mRNAs that
lack stop codons for degradation and prevents the ribosome from stalling.
[23]
RNA
50
Regulatory RNAs
Several types of RNA can downregulate gene expression by being complementary to a part of an mRNA or a gene's
DNA. MicroRNAs (miRNA; 21-22nt) are found in eukaryotes and act through RNA interference (RNAi), where an
effector complex of miRNA and enzymes can cleave complementary mRNA, block the mRNA from being
translated, or accelerate its degradation.
[24][25]
While small interfering RNAs (siRNA; 20-25nt) are often produced by breakdown of viral RNA, there are also
endogenous sources of siRNAs.
[26][27]
siRNAs act through RNA interference in a fashion similar to miRNAs. Some
miRNAs and siRNAs can cause genes they target to be methylated, thereby decreasing or increasing transcription of
those genes.
[28][29][30]
Animals have Piwi-interacting RNAs (piRNA; 29-30nt) that are active in germline cells and
are thought to be a defense against transposons and play a role in gametogenesis.
[][31]
Many prokaryotes have CRISPR RNAs, a regulatory system similar to RNA interference.
[32]
Antisense RNAs are
widespread; most downregulate a gene, but a few are activators of transcription.
[33]
One way antisense RNA can act
is by binding to an mRNA, forming double-stranded RNA that is enzymatically degraded.
[34]
There are many long
noncoding RNAs that regulate genes in eukaryotes,
[35]
one such RNA is Xist, which coats one X chromosome in
female mammals and inactivates it.
[36]
An mRNA may contain regulatory elements itself, such as riboswitches, in the 5' untranslated region or 3'
untranslated region; these cis-regulatory elements regulate the activity of that mRNA.
[37]
The untranslated regions
can also contain elements that regulate other genes.
[38]
In RNA processing
Uridine to pseudouridine is a common RNA
modification.
Many RNAs are involved in modifying other RNAs. Introns are
spliced out of pre-mRNA by spliceosomes, which contain several
small nuclear RNAs (snRNA),
[]
or the introns can be ribozymes that
are spliced by themselves.
[39]
RNA can also be altered by having its
nucleotides modified to other nucleotides than A, C, G and U. In
eukaryotes, modifications of RNA nucleotides are in general directed
by small nucleolar RNAs (snoRNA; 60-300nt),
[]
found in the
nucleolus and cajal bodies. snoRNAs associate with enzymes and
guide them to a spot on an RNA by basepairing to that RNA. These
enzymes then perform the nucleotide modification. rRNAs and tRNAs are extensively modified, but snRNAs and
mRNAs can also be the target of base modification.
[40][41]
RNA can also be methylated.
[42][43]
RNA genomes
Like DNA, RNA can carry genetic information. RNA viruses have genomes composed of RNA that encodes a
number of proteins. The viral genome is replicated by some of those proteins, while other proteins protect the
genome as the virus particle moves to a new host cell. Viroids are another group of pathogens, but they consist only
of RNA, do not encode any protein and are replicated by a host plant cell's polymerase.
[44]
In reverse transcription
Reverse transcribing viruses replicate their genomes by reverse transcribing DNA copies from their RNA; these
DNA copies are then transcribed to new RNA. Retrotransposons also spread by copying DNA and RNA from one
another,
[45]
and telomerase contains an RNA that is used as template for building the ends of eukaryotic
chromosomes.
[46]
RNA
51
Double-stranded RNA
Double-stranded RNA (dsRNA) is RNA with two complementary strands, similar to the DNA found in all cells.
dsRNA forms the genetic material of some viruses (double-stranded RNA viruses). Double-stranded RNA such as
viral RNA or siRNA can trigger RNA interference in eukaryotes, as well as interferon response in
vertebrates.
[47][48][49][50]
Key discoveries in RNA biology
Robert W. Holley, left, poses with his research team.
Research on RNA has led to many important biological
discoveries and numerous Nobel Prizes. Nucleic acids were
discovered in 1868 by Friedrich Miescher, who called the
material 'nuclein' since it was found in the nucleus.
[51]
It was
later discovered that prokaryotic cells, which do not have a
nucleus, also contain nucleic acids. The role of RNA in protein
synthesis was suspected already in 1939.
[52]
Severo Ochoa won
the 1959 Nobel Prize in Medicine (shared with Arthur
Kornberg) after he discovered an enzyme that can synthesize
RNA in the laboratory.
[53]
However, the enzyme discovered by
Ochoa (polynucleotide phosphorylase) was later shown to be
responsible for RNA degradation, not RNA synthesis.
The sequence of the 77 nucleotides of a yeast tRNA was found by Robert W. Holley in 1965,
[54]
winning Holley the
1968 Nobel Prize in Medicine (shared with Har Gobind Khorana and Marshall Nirenberg). In 1967, Carl Woese
hypothesized that RNA might be catalytic and suggested that the earliest forms of life (self-replicating molecules)
could have relied on RNA both to carry genetic information and to catalyze biochemical reactionsan RNA
world.
[55][56]
During the early 1970s retroviruses and reverse transcriptase were discovered, showing for the first time that
enzymes could copy RNA into DNA (the opposite of the usual route for transmission of genetic information). For
this work, David Baltimore, Renato Dulbecco and Howard Temin were awarded a Nobel Prize in 1975. In 1976,
Walter Fiers and his team determined the first complete nucleotide sequence of an RNA virus genome, that of
bacteriophage MS2.
[57]
In 1977, introns and RNA splicing were discovered in both mammalian viruses and in cellular genes, resulting in a
1993 Nobel to Philip Sharp and Richard Roberts. Catalytic RNA molecules (ribozymes) were discovered in the early
1980s, leading to a 1989 Nobel award to Thomas Cech and Sidney Altman. In 1990 it was found in petunia that
introduced genes can silence similar genes of the plant's own, now known to be a result of RNA interference.
[58][59]
At about the same time, 22 nt long RNAs, now called microRNAs, were found to have a role in the development of
C. elegans.
[60]
Studies on RNA interference gleaned a Nobel Prize for Andrew Fire and Craig Mello in 2006, and
another Nobel was awarded for studies on transcription of RNA to Roger Kornberg in the same year. The discovery
of gene regulatory RNAs has led to attempts to develop drugs made of RNA, such as siRNA, to silence genes.
[61]
RNA
52
References
[1] Papercore summary (http:/ / papercore.org/ Tinoco1999)
External links
RNA World website (http:/ / www. imb-jena. de/ RNA. html) Link collection (structures, sequences, tools,
journals)
Nucleic Acid Database (http:/ / ndbserver. rutgers. edu/ atlas/ xray/ ) Images of DNA, RNA and complexes.
EteRNA (http:/ / eterna. cmu. edu/ content/ EteRNA) a game forming RNA by pairing bases.
DNA
The structure of the DNA double helix. The atoms in the structure are colour-coded by
element and the detailed structure of two base pairs are shown in the bottom right.
Deoxyribonucleic acid (DNA) is a
molecule that encodes the genetic
instructions used in the development
and functioning of all known living
organisms and many viruses. Along
with RNA and proteins, DNA is one of
the three major macromolecules
essential for all known forms of life.
Genetic information is encoded as a
sequence of nucleotides (guanine,
adenine, thymine, and cytosine)
recorded using the letters G, A, T, and
C. Most DNA molecules are
double-stranded helices, consisting of
two long polymers of simple units
called nucleotides, molecules with
backbones made of alternating sugars
(deoxyribose) and phosphate groups
(related to phosphoric acid), with the
nucleobases (G, A, T, C) attached to
the sugars. DNA is well-suited for
biological information storage, since
the DNA backbone is resistant to cleavage and the double-stranded structure provides the molecule with a built-in
duplicate of the encoded information.
These two strands run in opposite directions to each other and are therefore anti-parallel, one backbone being 3
(three prime) and the other 5
DNA
53
The structure of part of a DNA double helix
(five prime). This refers to the direction the 3rd and 5th carbon on the
sugar molecule is facing. Attached to each sugar is one of four types of
molecules called nucleobases (informally, bases). It is the sequence of
these four nucleobases along the backbone that encodes information.
This information is read using the genetic code, which specifies the
sequence of the amino acids within proteins. The code is read by
copying stretches of DNA into the related nucleic acid RNA in a
process called transcription.
Within cells, DNA is organized into long structures called
chromosomes. During cell division these chromosomes are duplicated
in the process of DNA replication, providing each cell its own
complete set of chromosomes. Eukaryotic organisms (animals, plants,
fungi, and protists) store most of their DNA inside the cell nucleus and
some of their DNA in organelles, such as mitochondria or
chloroplasts.
[1]
In contrast, prokaryotes (bacteria and archaea) store
their DNA only in the cytoplasm. Within the chromosomes, chromatin
proteins such as histones compact and organize DNA. These compact
structures guide the interactions between DNA and other proteins,
helping control which parts of the DNA are transcribed.
DNA
54
Properties
Chemical structure of DNA. Hydrogen bonds shown as dotted lines.
DNA is a long polymer made from
repeating units called nucleotides.
[2][][3]
DNA was first identified and isolated by
Friedrich Miescher and the double helix
structure of DNA was first discovered by
James Watson and Francis Crick. The
structure of DNA of all species comprises
two helical chains each coiled round the
same axis, and each with a pitch of
34ngstrms (3.4nanometres) and a radius
of 10ngstrms (1.0nanometres).
[]
According to another study, when measured
in a particular solution, the DNA chain
measured 22 to 26ngstrms wide (2.2 to
2.6nanometres), and one nucleotide unit
measured 3.3 (0.33nm) long.
[4]
Although
each individual repeating unit is very small,
DNA polymers can be very large molecules
containing millions of nucleotides. For
instance, the largest human chromosome,
chromosome number 1, consists of
approximately 220 million base pairs
[5]
and
is 85 mm long.
In living organisms DNA does not usually exist as a single molecule, but instead as a pair of molecules that are held
tightly together.
[][6]
These two long strands entwine like vines, in the shape of a double helix. The nucleotide repeats
contain both the segment of the backbone of the molecule, which holds the chain together, and a nucleobase, which
interacts with the other DNA strand in the helix. A nucleobase linked to a sugar is called a nucleoside and a base
linked to a sugar and one or more phosphate groups is called a nucleotide. A polymer comprising multiple linked
nucleotides (as in DNA) is called a polynucleotide.
[7]
The backbone of the DNA strand is made from alternating phosphate and sugar residues.
[]
The sugar in DNA is
2-deoxyribose, which is a pentose (five-carbon) sugar. The sugars are joined together by phosphate groups that form
phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. These asymmetric bonds
mean a strand of DNA has a direction. In a double helix the direction of the nucleotides in one strand is opposite to
their direction in the other strand: the strands are antiparallel. The asymmetric ends of DNA strands are called the 5
(five prime) and 3 (three prime) ends, with the 5 end having a terminal phosphate group and the 3 end a terminal
hydroxyl group. One major difference between DNA and RNA is the sugar, with the 2-deoxyribose in DNA being
replaced by the alternative pentose sugar ribose in RNA.
[6]
DNA
55
A section of DNA. The bases lie
horizontally between the two
spiraling strands.
[8]
(animated
version).
The DNA double helix is stabilized primarily by two forces: hydrogen bonds
between nucleotides and base-stacking interactions among aromatic
nucleobases.
[]
In the aqueous environment of the cell, the conjugated bonds of
nucleotide bases align perpendicular to the axis of the DNA molecule,
minimizing their interaction with the solvation shell and therefore, the Gibbs free
energy. The four bases found in DNA are adenine (abbreviated A), cytosine (C),
guanine (G) and thymine (T). These four bases are attached to the
sugar/phosphate to form the complete nucleotide, as shown for adenosine
monophosphate.
Nucleobase classification
The nucleobases are classified into two types: the purines, A and G, being fused
five- and six-membered heterocyclic compounds, and the pyrimidines, the
six-membered rings C and T.
[6]
A fifth pyrimidine nucleobase, uracil (U), usually
takes the place of thymine in RNA and differs from thymine by lacking a methyl
group on its ring. In addition to RNA and DNA a large number of artificial
nucleic acid analogues have also been created to study the properties of nucleic
acids, or for use in biotechnology.
[9]
Uracil is not usually found in DNA, occurring only as a breakdown product of
cytosine. However in a number of bacteriophages Bacillus subtilis bacteriophages PBS1 and PBS2 and Yersinia
bacteriophage piR1-37 thymine has been replaced by uracil.
[]
Base J (beta-d-glucopyranosyloxymethyluracil), a
modified form of uracil, is also found in a number of organisms: the flagellates Diplonema and Euglena, and all the
kinetoplastid genera
[]
Biosynthesis of J occurs in two steps: in the first step a specific thymidine in DNA is converted
into hydroxymethyldeoxyuridine; in the second HOMedU is glycosylated to form J.
[]
Proteins that bind specifically
to this base have been identified.
[][][]
These proteins appear to be distant relatives of the Tet1 oncogene that is
involved in the pathogenesis of acute myeloid leukemia.
[]
J appears to act as a termination signal for RNA
polymerase II.
[][]
Major and minor grooves of DNA. Minor groove
is a binding site for the dye Hoechst 33258.
Grooves
Twin helical strands form the DNA backbone. Another double helix
may be found tracing the spaces, or grooves, between the strands.
These voids are adjacent to the base pairs and may provide a binding
site. As the strands are not symmetrically located with respect to each
other, the grooves are unequally sized. One groove, the major groove,
is 22 wide and the other, the minor groove, is 12 wide.
[10]
The
narrowness of the minor groove means that the edges of the bases are
more accessible in the major groove. As a result, proteins like
transcription factors that can bind to specific sequences in
double-stranded DNA usually make contacts to the sides of the bases exposed in the major groove.
[]
This situation
varies in unusual conformations of DNA within the cell (see below), but the major and minor grooves are always
named to reflect the differences in size that would be seen if the DNA is twisted back into the ordinary B form.
DNA
56
Base pairing
In a DNA double helix, each type of nucleobase on one strand bonds with just one type of nucleobase on the other
strand. This is called complementary base pairing. Here, purines form hydrogen bonds to pyrimidines, with adenine
bonding only to thymine in two hydrogen bonds, and cytosine bonding only to guanine in three hydrogen bonds.
This arrangement of two nucleotides binding together across the double helix is called a base pair. As hydrogen
bonds are not covalent, they can be broken and rejoined relatively easily. The two strands of DNA in a double helix
can therefore be pulled apart like a zipper, either by a mechanical force or high temperature.
[11]
As a result of this
complementarity, all the information in the double-stranded sequence of a DNA helix is duplicated on each strand,
which is vital in DNA replication. Indeed, this reversible and specific interaction between complementary base pairs
is critical for all the functions of DNA in living organisms.
[]

Top, a GC base pair with three hydrogen bonds. Bottom, an AT base pair with two hydrogen bonds. Non-covalent
hydrogen bonds between the pairs are shown as dashed lines.
The two types of base pairs form different numbers of hydrogen bonds, AT forming two hydrogen bonds, and GC
forming three hydrogen bonds (see figures, right). DNA with high GC-content is more stable than DNA with low
GC-content.
As noted above, most DNA molecules are actually two polymer strands, bound together in a helical fashion by
noncovalent bonds; this double stranded structure (dsDNA) is maintained largely by the intrastrand base stacking
interactions, which are strongest for G,C stacks. The two strands can come apart a process known as melting to
form two ssDNA molecules. Melting occurs when conditions favor ssDNA; such conditions are high temperature,
low salt and high pH (low pH also melts DNA, but since DNA is unstable due to acid depurination, low pH is rarely
used).
The stability of the dsDNA form depends not only on the GC-content (% G,C basepairs) but also on sequence (since
stacking is sequence specific) and also length (longer molecules are more stable). The stability can be measured in
various ways; a common way is the "melting temperature", which is the temperature at which 50% of the ds
molecules are converted to ss molecules; melting temperature is dependent on ionic strength and the concentration of
DNA. As a result, it is both the percentage of GC base pairs and the overall length of a DNA double helix that
determines the strength of the association between the two strands of DNA. Long DNA helices with a high
GC-content have stronger-interacting strands, while short helices with high AT content have weaker-interacting
strands.
[12]
In biology, parts of the DNA double helix that need to separate easily, such as the TATAAT Pribnow
box in some promoters, tend to have a high AT content, making the strands easier to pull apart.
[13]
In the laboratory, the strength of this interaction can be measured by finding the temperature necessary to break the
hydrogen bonds, their melting temperature (also called T
m
value). When all the base pairs in a DNA double helix
melt, the strands separate and exist in solution as two entirely independent molecules. These single-stranded DNA
molecules (ssDNA) have no single common shape, but some conformations are more stable than others.
[14]
DNA
57
Sense and antisense
A DNA sequence is called "sense" if its sequence is the same as that of a messenger RNA copy that is translated into
protein.
[15]
The sequence on the opposite strand is called the "antisense" sequence. Both sense and antisense
sequences can exist on different parts of the same strand of DNA (i.e. both strands contain both sense and antisense
sequences). In both prokaryotes and eukaryotes, antisense RNA sequences are produced, but the functions of these
RNAs are not entirely clear.
[16]
One proposal is that antisense RNAs are involved in regulating gene expression
through RNA-RNA base pairing.
[17]
A few DNA sequences in prokaryotes and eukaryotes, and more in plasmids and viruses, blur the distinction between
sense and antisense strands by having overlapping genes.
[18]
In these cases, some DNA sequences do double duty,
encoding one protein when read along one strand, and a second protein when read in the opposite direction along the
other strand. In bacteria, this overlap may be involved in the regulation of gene transcription,
[19]
while in viruses,
overlapping genes increase the amount of information that can be encoded within the small viral genome.
[20]
Supercoiling
DNA can be twisted like a rope in a process called DNA supercoiling. With DNA in its "relaxed" state, a strand
usually circles the axis of the double helix once every 10.4 base pairs, but if the DNA is twisted the strands become
more tightly or more loosely wound.
[21]
If the DNA is twisted in the direction of the helix, this is positive
supercoiling, and the bases are held more tightly together. If they are twisted in the opposite direction, this is
negative supercoiling, and the bases come apart more easily. In nature, most DNA has slight negative supercoiling
that is introduced by enzymes called topoisomerases.
[]
These enzymes are also needed to relieve the twisting stresses
introduced into DNA strands during processes such as transcription and DNA replication.
[]
From left to right, the structures of A, B and Z
DNA
Alternate DNA structures
DNA exists in many possible conformations that include A-DNA,
B-DNA, and Z-DNA forms, although, only B-DNA and Z-DNA have
been directly observed in functional organisms.
[]
The conformation
that DNA adopts depends on the hydration level, DNA sequence, the
amount and direction of supercoiling, chemical modifications of the
bases, the type and concentration of metal ions, as well as the presence
of polyamines in solution.
[22]
The first published reports of A-DNA X-ray diffraction patterns and
also B-DNA used analyses based on Patterson transforms that
provided only a limited amount of structural information for oriented fibers of DNA.
[23][]
An alternate analysis was
then proposed by Wilkins et al., in 1953, for the in vivo B-DNA X-ray diffraction/scattering patterns of highly
hydrated DNA fibers in terms of squares of Bessel functions.
[]
In the same journal, James Watson and Francis Crick
presented their molecular modeling analysis of the DNA X-ray diffraction patterns to suggest that the structure was a
double-helix.
[]
Although the `B-DNA form' is most common under the conditions found in cells,
[24]
it is not a well-defined
conformation but a family of related DNA conformations
[25]
that occur at the high hydration levels present in living
cells. Their corresponding X-ray diffraction and scattering patterns are characteristic of molecular paracrystals with a
significant degree of disorder.
[26][27]
Compared to B-DNA, the A-DNA form is a wider right-handed spiral, with a shallow, wide minor groove and a
narrower, deeper major groove. The A form occurs under non-physiological conditions in partially dehydrated
samples of DNA, while in the cell it may be produced in hybrid pairings of DNA and RNA strands, as well as in
enzyme-DNA complexes.
[28][29]
Segments of DNA where the bases have been chemically modified by methylation
DNA
58
may undergo a larger change in conformation and adopt the Z form. Here, the strands turn about the helical axis in a
left-handed spiral, the opposite of the more common B form.
[30]
These unusual structures can be recognized by
specific Z-DNA binding proteins and may be involved in the regulation of transcription.
[31]
Alternate DNA chemistry
For a number of years exobiologists have proposed the existence of a shadow biosphere, a postulated microbial
biosphere of Earth that uses radically different biochemical and molecular processes than currently known life. One
of the proposals was the existence of lifeforms that use arsenic instead of phosphorus in DNA. A report in 2010 of
the possibility in the bacterium GFAJ-1, was announced,
[][][]
though the research was disputed,
[][32]
and evidence
suggests the bacterium actively prevents the incorporation of arsenic into the DNA backbone and other
biomolecules.
[]
Quadruplex structures
At the ends of the linear chromosomes are specialized regions of DNA called telomeres. The main function of these
regions is to allow the cell to replicate chromosome ends using the enzyme telomerase, as the enzymes that normally
replicate DNA cannot copy the extreme 3 ends of chromosomes.
[]
These specialized chromosome caps also help
protect the DNA ends, and stop the DNA repair systems in the cell from treating them as damage to be corrected.
[]
In
human cells, telomeres are usually lengths of single-stranded DNA containing several thousand repeats of a simple
TTAGGG sequence.
[33]
DNA quadruplex formed by telomere repeats.
The looped conformation of the DNA backbone
is very different from the typical DNA helix.
[34]
These guanine-rich sequences may stabilize chromosome ends by
forming structures of stacked sets of four-base units, rather than the
usual base pairs found in other DNA molecules. Here, four guanine
bases form a flat plate and these flat four-base units then stack on top
of each other, to form a stable G-quadruplex structure.
[]
These
structures are stabilized by hydrogen bonding between the edges of the
bases and chelation of a metal ion in the centre of each four-base
unit.
[35]
Other structures can also be formed, with the central set of
four bases coming from either a single strand folded around the bases,
or several different parallel strands, each contributing one base to the
central structure.
In addition to these stacked structures, telomeres also form large loop
structures called telomere loops, or T-loops. Here, the single-stranded
DNA curls around in a long circle stabilized by telomere-binding
proteins.
[36]
At the very end of the T-loop, the single-stranded telomere DNA is held onto a region of
double-stranded DNA by the telomere strand disrupting the double-helical DNA and base pairing to one of the two
strands. This triple-stranded structure is called a displacement loop or D-loop.
[]
Single branch Multiple branches
Branched DNA can form networks containing multiple branches.
DNA
59
Branched DNA
In DNA fraying occurs when non-complementary regions exist at the end of an otherwise complementary
double-strand of DNA. However, branched DNA can occur if a third strand of DNA is introduced and contains
adjoining regions able to hybridize with the frayed regions of the pre-existing double-strand. Although the simplest
example of branched DNA involves only three strands of DNA, complexes involving additional strands and multiple
branches are also possible.
[37]
Branched DNA can be used in nanotechnology to construct geometric shapes, see the
section on uses in technology below.
Vibration
DNA may carry out low-frequency collective motion as observed by the Raman spectroscopy
[][]
and analyzed with a
quasi-continuum model.
[][]
Chemical modifications and altered DNA packaging
cytosine 5-methylcytosine thymine
Structure of cytosine with and without the 5-methyl group. Deamination converts 5-methylcytosine into thymine.
Base modifications and DNA packaging
The expression of genes is influenced by how the DNA is packaged in chromosomes, in a structure called chromatin.
Base modifications can be involved in packaging, with regions that have low or no gene expression usually
containing high levels of methylation of cytosine bases. DNA packaging and its influence on gene expression can
also occur by covalent modifications of the histone protein core around which DNA is wrapped in the chromatin
structure or else by remodeling carried out by chromatin remodeling complexes (see Chromatin remodeling). There
is, further, crosstalk between DNA methylation and histone modification, so they can coordinately affect chromatin
and gene expression.
[38]
For one example, cytosine methylation, produces 5-methylcytosine, which is important for X-chromosome
inactivation.
[39]
The average level of methylation varies between organisms the worm Caenorhabditis elegans
lacks cytosine methylation, while vertebrates have higher levels, with up to 1% of their DNA containing
5-methylcytosine.
[40]
Despite the importance of 5-methylcytosine, it can deaminate to leave a thymine base, so
methylated cytosines are particularly prone to mutations.
[41]
Other base modifications include adenine methylation in
bacteria, the presence of 5-hydroxymethylcytosine in the brain,
[42]
and the glycosylation of uracil to produce the
"J-base" in kinetoplastids.
[43][44]
DNA
60
Damage
A covalent adduct between a metabolically
activated form of benzo[a]pyrene, the major
mutagen in tobacco smoke, and DNA
[45]
DNA can be damaged by many sorts of mutagens, which change the
DNA sequence. Mutagens include oxidizing agents, alkylating agents
and also high-energy electromagnetic radiation such as ultraviolet light
and X-rays. The type of DNA damage produced depends on the type of
mutagen. For example, UV light can damage DNA by producing
thymine dimers, which are cross-links between pyrimidine bases.
[46]
On the other hand, oxidants such as free radicals or hydrogen peroxide
produce multiple forms of damage, including base modifications,
particularly of guanosine, and double-strand breaks.
[47]
A typical
human cell contains about 150,000 bases that have suffered oxidative
damage.
[48]
Of these oxidative lesions, the most dangerous are
double-strand breaks, as these are difficult to repair and can produce
point mutations, insertions and deletions from the DNA sequence, as
well as chromosomal translocations.
[49]
These mutations can cause
cancer. Because of inherent limitations in the DNA repair mechanisms,
if humans lived long enough, they would all eventually develop
cancer.
[][50]
DNA damages that are naturally occurring, due to normal
cellular processes that produce reactive oxygen species, the hydrolytic
activities of cellular water, etc., also occur frequently. Although most
of these damages are repaired, in any cell some DNA damage may remain despite the action of repair processes.
These remaining DNA damages accumulate with age in mammalian postmitotic tissues. This accumulation appears
to be an important underlying cause of aging.
[51][52][53]
Many mutagens fit into the space between two adjacent base pairs, this is called intercalation. Most intercalators are
aromatic and planar molecules; examples include ethidium bromide, acridines, daunomycin, and doxorubicin. For an
intercalator to fit between base pairs, the bases must separate, distorting the DNA strands by unwinding of the
double helix. This inhibits both transcription and DNA replication, causing toxicity and mutations.
[54]
As a result,
DNA intercalators may be carcinogens, and in the case of thalidomide, a teratogen.
[55]
Others such as
benzo[a]pyrene diol epoxide and aflatoxin form DNA adducts that induce errors in replication.
[56]
Nevertheless, due
to their ability to inhibit DNA transcription and replication, other similar toxins are also used in chemotherapy to
inhibit rapidly growing cancer cells.
[57]
Biological functions
DNA usually occurs as linear chromosomes in eukaryotes, and circular chromosomes in prokaryotes. The set of
chromosomes in a cell makes up its genome; the human genome has approximately 3 billion base pairs of DNA
arranged into 46 chromosomes.
[58]
The information carried by DNA is held in the sequence of pieces of DNA called
genes. Transmission of genetic information in genes is achieved via complementary base pairing. For example, in
transcription, when a cell uses the information in a gene, the DNA sequence is copied into a complementary RNA
sequence through the attraction between the DNA and the correct RNA nucleotides. Usually, this RNA copy is then
used to make a matching protein sequence in a process called translation, which depends on the same interaction
between RNA nucleotides. In alternative fashion, a cell may simply copy its genetic information in a process called
DNA replication. The details of these functions are covered in other articles; here we focus on the interactions
between DNA and other molecules that mediate the function of the genome.
DNA
61
Genes and genomes
Genomic DNA is tightly and orderly packed in the process called DNA condensation to fit the small available
volumes of the cell. In eukaryotes, DNA is located in the cell nucleus, as well as small amounts in mitochondria and
chloroplasts. In prokaryotes, the DNA is held within an irregularly shaped body in the cytoplasm called the
nucleoid.
[59]
The genetic information in a genome is held within genes, and the complete set of this information in an
organism is called its genotype. A gene is a unit of heredity and is a region of DNA that influences a particular
characteristic in an organism. Genes contain an open reading frame that can be transcribed, as well as regulatory
sequences such as promoters and enhancers, which control the transcription of the open reading frame.
In many species, only a small fraction of the total sequence of the genome encodes protein. For example, only about
1.5% of the human genome consists of protein-coding exons, with over 50% of human DNA consisting of
non-coding repetitive sequences.
[60]
The reasons for the presence of so much noncoding DNA in eukaryotic
genomes and the extraordinary differences in genome size, or C-value, among species represent a long-standing
puzzle known as the "C-value enigma".
[61]
However, some DNA sequences that do not code protein may still encode
functional non-coding RNA molecules, which are involved in the regulation of gene expression.
[62]
T7 RNA polymerase (blue) producing a mRNA
(green) from a DNA template (orange).
[63]
Some noncoding DNA sequences play structural roles in
chromosomes. Telomeres and centromeres typically contain few genes,
but are important for the function and stability of chromosomes.
[][64]
An abundant form of noncoding DNA in humans are pseudogenes,
which are copies of genes that have been disabled by mutation.
[65]
These sequences are usually just molecular fossils, although they can
occasionally serve as raw genetic material for the creation of new
genes through the process of gene duplication and divergence.
[66]
Transcription and translation
A gene is a sequence of DNA that contains genetic information and
can influence the phenotype of an organism. Within a gene, the sequence of bases along a DNA strand defines a
messenger RNA sequence, which then defines one or more protein sequences. The relationship between the
nucleotide sequences of genes and the amino-acid sequences of proteins is determined by the rules of translation,
known collectively as the genetic code. The genetic code consists of three-letter 'words' called codons formed from a
sequence of three nucleotides (e.g. ACT, CAG, TTT).
In transcription, the codons of a gene are copied into messenger RNA by RNA polymerase. This RNA copy is then
decoded by a ribosome that reads the RNA sequence by base-pairing the messenger RNA to transfer RNA, which
carries amino acids. Since there are 4 bases in 3-letter combinations, there are 64 possible codons (
combinations). These encode the twenty standard amino acids, giving most amino acids more than one possible
codon. There are also three 'stop' or 'nonsense' codons signifying the end of the coding region; these are the TAA,
TGA and TAG codons.
DNA
62
DNA replication. The double helix is unwound by a helicase and topoisomerase. Next,
one DNA polymerase produces the leading strand copy. Another DNA polymerase binds
to the lagging strand. This enzyme makes discontinuous segments (called Okazaki
fragments) before DNA ligase joins them together.
Replication
Cell division is essential for an
organism to grow, but, when a cell
divides, it must replicate the DNA in
its genome so that the two daughter
cells have the same genetic
information as their parent. The
double-stranded structure of DNA
provides a simple mechanism for DNA
replication. Here, the two strands are
separated and then each strand's
complementary DNA sequence is
recreated by an enzyme called DNA
polymerase. This enzyme makes the
complementary strand by finding the correct base through complementary base pairing, and bonding it onto the
original strand. As DNA polymerases can only extend a DNA strand in a 5 to 3 direction, different mechanisms are
used to copy the antiparallel strands of the double helix.
[67]
In this way, the base on the old strand dictates which
base appears on the new strand, and the cell ends up with a perfect copy of its DNA.
Interactions with proteins
All the functions of DNA depend on interactions with proteins. These protein interactions can be non-specific, or the
protein can bind specifically to a single DNA sequence. Enzymes can also bind to DNA and of these, the
polymerases that copy the DNA base sequence in transcription and DNA replication are particularly important.
DNA-binding proteins
Interaction of DNA (shown in orange) with histones (shown in blue). These proteins' basic amino acids bind to the
acidic phosphate groups on DNA.
Structural proteins that bind DNA are well-understood examples of non-specific DNA-protein interactions. Within
chromosomes, DNA is held in complexes with structural proteins. These proteins organize the DNA into a compact
structure called chromatin. In eukaryotes this structure involves DNA binding to a complex of small basic proteins
called histones, while in prokaryotes multiple types of proteins are involved.
[68][69]
The histones form a disk-shaped
complex called a nucleosome, which contains two complete turns of double-stranded DNA wrapped around its
surface. These non-specific interactions are formed through basic residues in the histones making ionic bonds to the
acidic sugar-phosphate backbone of the DNA, and are therefore largely independent of the base sequence.
[70]
Chemical modifications of these basic amino acid residues include methylation, phosphorylation and acetylation.
[71]
These chemical changes alter the strength of the interaction between the DNA and the histones, making the DNA
more or less accessible to transcription factors and changing the rate of transcription.
[72]
Other non-specific
DNA
63
DNA-binding proteins in chromatin include the high-mobility group proteins, which bind to bent or distorted
DNA.
[73]
These proteins are important in bending arrays of nucleosomes and arranging them into the larger
structures that make up chromosomes.
[74]
A distinct group of DNA-binding proteins are the DNA-binding proteins that specifically bind single-stranded DNA.
In humans, replication protein A is the best-understood member of this family and is used in processes where the
double helix is separated, including DNA replication, recombination and DNA repair.
[75]
These binding proteins
seem to stabilize single-stranded DNA and protect it from forming stem-loops or being degraded by nucleases.
The lambda repressor
helix-turn-helix transcription factor
bound to its DNA target
[76]
In contrast, other proteins have evolved to bind to particular DNA sequences.
The most intensively studied of these are the various transcription factors, which
are proteins that regulate transcription. Each transcription factor binds to one
particular set of DNA sequences and activates or inhibits the transcription of
genes that have these sequences close to their promoters. The transcription
factors do this in two ways. Firstly, they can bind the RNA polymerase
responsible for transcription, either directly or through other mediator proteins;
this locates the polymerase at the promoter and allows it to begin
transcription.
[77]
Alternatively, transcription factors can bind enzymes that
modify the histones at the promoter. This changes the accessibility of the DNA
template to the polymerase.
[78]
As these DNA targets can occur throughout an organism's genome, changes in
the activity of one type of transcription factor can affect thousands of genes.
[79]
Consequently, these proteins are often the targets of the signal transduction
processes that control responses to environmental changes or cellular
differentiation and development. The specificity of these transcription factors'
interactions with DNA come from the proteins making multiple contacts to the edges of the DNA bases, allowing
them to "read" the DNA sequence. Most of these base-interactions are made in the major groove, where the bases are
most accessible.
[]
The restriction enzyme EcoRV (green) in a
complex with its substrate DNA
[80]
DNA-modifying enzymes
Nucleases and ligases
Nucleases are enzymes that cut DNA strands by catalyzing the
hydrolysis of the phosphodiester bonds. Nucleases that hydrolyse
nucleotides from the ends of DNA strands are called exonucleases,
while endonucleases cut within strands. The most frequently used
nucleases in molecular biology are the restriction endonucleases, which
cut DNA at specific sequences. For instance, the EcoRV enzyme
shown to the left recognizes the 6-base sequence 5-GATATC-3 and
makes a cut at the vertical line. In nature, these enzymes protect bacteria against phage infection by digesting the
phage DNA when it enters the bacterial cell, acting as part of the restriction modification system.
[81]
In technology,
these sequence-specific nucleases are used in molecular cloning and DNA fingerprinting.
Enzymes called DNA ligases can rejoin cut or broken DNA strands.
[]
Ligases are particularly important in lagging
strand DNA replication, as they join together the short segments of DNA produced at the replication fork into a
complete copy of the DNA template. They are also used in DNA repair and genetic recombination.
[]
DNA
64
Topoisomerases and helicases
Topoisomerases are enzymes with both nuclease and ligase activity. These proteins change the amount of
supercoiling in DNA. Some of these enzymes work by cutting the DNA helix and allowing one section to rotate,
thereby reducing its level of supercoiling; the enzyme then seals the DNA break.
[]
Other types of these enzymes are
capable of cutting one DNA helix and then passing a second strand of DNA through this break, before rejoining the
helix.
[82]
Topoisomerases are required for many processes involving DNA, such as DNA replication and
transcription.
[]
Helicases are proteins that are a type of molecular motor. They use the chemical energy in nucleoside triphosphates,
predominantly ATP, to break hydrogen bonds between bases and unwind the DNA double helix into single
strands.
[83]
These enzymes are essential for most processes where enzymes need to access the DNA bases.
Polymerases
Polymerases are enzymes that synthesize polynucleotide chains from nucleoside triphosphates. The sequence of their
products are copies of existing polynucleotide chainswhich are called templates. These enzymes function by
adding nucleotides onto the 3 hydroxyl group of the previous nucleotide in a DNA strand. As a consequence, all
polymerases work in a 5 to 3 direction.
[]
In the active site of these enzymes, the incoming nucleoside triphosphate
base-pairs to the template: this allows polymerases to accurately synthesize the complementary strand of their
template. Polymerases are classified according to the type of template that they use.
In DNA replication, a DNA-dependent DNA polymerase makes a copy of a DNA sequence. Accuracy is vital in this
process, so many of these polymerases have a proofreading activity. Here, the polymerase recognizes the occasional
mistakes in the synthesis reaction by the lack of base pairing between the mismatched nucleotides. If a mismatch is
detected, a 3 to 5 exonuclease activity is activated and the incorrect base removed.
[84]
In most organisms, DNA
polymerases function in a large complex called the replisome that contains multiple accessory subunits, such as the
DNA clamp or helicases.
[85]
RNA-dependent DNA polymerases are a specialized class of polymerases that copy the sequence of an RNA strand
into DNA. They include reverse transcriptase, which is a viral enzyme involved in the infection of cells by
retroviruses, and telomerase, which is required for the replication of telomeres.
[][86]
Telomerase is an unusual
polymerase because it contains its own RNA template as part of its structure.
[]
Transcription is carried out by a DNA-dependent RNA polymerase that copies the sequence of a DNA strand into
RNA. To begin transcribing a gene, the RNA polymerase binds to a sequence of DNA called a promoter and
separates the DNA strands. It then copies the gene sequence into a messenger RNA transcript until it reaches a
region of DNA called the terminator, where it halts and detaches from the DNA. As with human DNA-dependent
DNA polymerases, RNA polymerase II, the enzyme that transcribes most of the genes in the human genome,
operates as part of a large protein complex with multiple regulatory and accessory subunits.
[87]
DNA
65
Genetic recombination

Structure of the Holliday junction intermediate in genetic recombination. The four separate DNA strands are
coloured red, blue, green and yellow.
[88]
Recombination involves the breakage and rejoining of two
chromosomes (M and F) to produce two re-arranged chromosomes
(C1 and C2).
A DNA helix usually does not interact with other
segments of DNA, and in human cells the different
chromosomes even occupy separate areas in the
nucleus called "chromosome territories".
[89]
This
physical separation of different chromosomes is
important for the ability of DNA to function as a stable
repository for information, as one of the few times
chromosomes interact is during chromosomal crossover
when they recombine. Chromosomal crossover is when
two DNA helices break, swap a section and then rejoin.
Recombination allows chromosomes to exchange
genetic information and produces new combinations of
genes, which increases the efficiency of natural
selection and can be important in the rapid evolution of
new proteins.
[90]
Genetic recombination can also be involved in DNA repair, particularly in the cell's response to
double-strand breaks.
[91]
The most common form of chromosomal crossover is homologous recombination, where the two chromosomes
involved share very similar sequences. Non-homologous recombination can be damaging to cells, as it can produce
chromosomal translocations and genetic abnormalities. The recombination reaction is catalyzed by enzymes known
as recombinases, such as RAD51.
[92]
The first step in recombination is a double-stranded break caused by either an
endonuclease or damage to the DNA.
[93]
A series of steps catalyzed in part by the recombinase then leads to joining
of the two helices by at least one Holliday junction, in which a segment of a single strand in each helix is annealed to
the complementary strand in the other helix. The Holliday junction is a tetrahedral junction structure that can be
moved along the pair of chromosomes, swapping one strand for another. The recombination reaction is then halted
by cleavage of the junction and re-ligation of the released DNA.
[94]
DNA
66
Evolution
DNA contains the genetic information that allows all modern living things to function, grow and reproduce.
However, it is unclear how long in the 4-billion-year history of life DNA has performed this function, as it has been
proposed that the earliest forms of life may have used RNA as their genetic material.
[][95]
RNA may have acted as
the central part of early cell metabolism as it can both transmit genetic information and carry out catalysis as part of
ribozymes.
[96]
This ancient RNA world where nucleic acid would have been used for both catalysis and genetics
may have influenced the evolution of the current genetic code based on four nucleotide bases. This would occur,
since the number of different bases in such an organism is a trade-off between a small number of bases increasing
replication accuracy and a large number of bases increasing the catalytic efficiency of ribozymes.
[97]
However, there is no direct evidence of ancient genetic systems, as recovery of DNA from most fossils is impossible.
This is because DNA survives in the environment for less than one million years, and slowly degrades into short
fragments in solution.
[98]
Claims for older DNA have been made, most notably a report of the isolation of a viable
bacterium from a salt crystal 250 million years old,
[99]
but these claims are controversial.
[100][101]
On 8 August 2011, a report, based on NASA studies with meteorites found on Earth, was published suggesting
building blocks of DNA (adenine, guanine and related organic molecules) may have been formed extraterrestrially in
outer space.
[][][]
Uses in technology
Genetic engineering
Methods have been developed to purify DNA from organisms, such as phenol-chloroform extraction, and to
manipulate it in the laboratory, such as restriction digests and the polymerase chain reaction. Modern biology and
biochemistry make intensive use of these techniques in recombinant DNA technology. Recombinant DNA is a
man-made DNA sequence that has been assembled from other DNA sequences. They can be transformed into
organisms in the form of plasmids or in the appropriate format, by using a viral vector.
[102]
The genetically modified
organisms produced can be used to produce products such as recombinant proteins, used in medical research,
[103]
or
be grown in agriculture.
[104][105]
Forensics
Forensic scientists can use DNA in blood, semen, skin, saliva or hair found at a crime scene to identify a matching
DNA of an individual, such as a perpetrator. This process is formally termed DNA profiling, but may also be called
"genetic fingerprinting". In DNA profiling, the lengths of variable sections of repetitive DNA, such as short tandem
repeats and minisatellites, are compared between people. This method is usually an extremely reliable technique for
identifying a matching DNA.
[106]
However, identification can be complicated if the scene is contaminated with DNA
from several people.
[107]
DNA profiling was developed in 1984 by British geneticist Sir Alec Jeffreys,
[108]
and first
used in forensic science to convict Colin Pitchfork in the 1988 Enderby murders case.
[109]
The development of forensic science, and the ability to now obtain genetic matching on minute samples of blood,
skin, saliva or hair has led to a re-examination of a number of cases. Evidence can now be uncovered that was not
scientifically possible at the time of the original examination. Combined with the removal of the double jeopardy law
in some places, this can allow cases to be reopened where previous trials have failed to produce sufficient evidence
to convince a jury. People charged with serious crimes may be required to provide a sample of DNA for matching
purposes. The most obvious defence to DNA matches obtained forensically is to claim that cross-contamination of
evidence has taken place. This has resulted in meticulous strict handling procedures with new cases of serious crime.
DNA profiling is also used to identify victims of mass casualty incidents.
[110]
As well as positively identifying
bodies or body parts in serious accidents, DNA profiling is being successfully used to identify individual victims in
mass war graves matching to family members.
DNA
67
Bioinformatics
Bioinformatics involves the manipulation, searching, and data mining of biological data, and this includes DNA
sequence data. The development of techniques to store and search DNA sequences have led to widely applied
advances in computer science, especially string searching algorithms, machine learning and database theory.
[111]
String searching or matching algorithms, which find an occurrence of a sequence of letters inside a larger sequence
of letters, were developed to search for specific sequences of nucleotides.
[112]
The DNA sequence may be aligned
with other DNA sequences to identify homologous sequences and locate the specific mutations that make them
distinct. These techniques, especially multiple sequence alignment, are used in studying phylogenetic relationships
and protein function.
[113]
Data sets representing entire genomes' worth of DNA sequences, such as those produced
by the Human Genome Project, are difficult to use without the annotations that identify the locations of genes and
regulatory elements on each chromosome. Regions of DNA sequence that have the characteristic patterns associated
with protein- or RNA-coding genes can be identified by gene finding algorithms, which allow researchers to predict
the presence of particular gene products and their possible functions in an organism even before they have been
isolated experimentally.
[]
Entire genomes may also be compared, which can shed light on the evolutionary history of
particular organism and permit the examination of complex evolutionary events.
DNA nanotechnology
The DNA structure at left (schematic shown) will self-assemble into the structure
visualized by atomic force microscopy at right. DNA nanotechnology is the field that
seeks to design nanoscale structures using the molecular recognition properties of DNA
molecules. Image from Strong, 2004
[114]
.
DNA nanotechnology uses the unique
molecular recognition properties of
DNA and other nucleic acids to create
self-assembling branched DNA
complexes with useful properties.
[115]
DNA is thus used as a structural
material rather than as a carrier of
biological information. This has led to
the creation of two-dimensional
periodic lattices (both tile-based as
well as using the "DNA origami"
method) as well as three-dimensional
structures in the shapes of
polyhedra.
[116]
Nanomechanical
devices and algorithmic self-assembly
have also been demonstrated,
[117]
and
these DNA structures have been used to template the arrangement of other molecules such as gold nanoparticles and
streptavidin proteins.
[118]
History and anthropology
Because DNA collects mutations over time, which are then inherited, it contains historical information, and, by
comparing DNA sequences, geneticists can infer the evolutionary history of organisms, their phylogeny.
[119]
This
field of phylogenetics is a powerful tool in evolutionary biology. If DNA sequences within a species are compared,
population geneticists can learn the history of particular populations. This can be used in studies ranging from
ecological genetics to anthropology; For example, DNA evidence is being used to try to identify the Ten Lost Tribes
of Israel.
[120][121]
DNA has also been used to look at modern family relationships, such as establishing family relationships between
the descendants of Sally Hemings and Thomas Jefferson. This usage is closely related to the use of DNA in criminal
DNA
68
investigations detailed above. Indeed, some criminal investigations have been solved when DNA from crime scenes
has matched relatives of the guilty individual.
[122]
Information storage
In a paper published in Nature in January, 2013, scientists from the European Bioinformatics Institute and Agilent
Technologies proposed a mechanism to use DNA's ability to code information as a means of digital data storage. The
group was able to encode 739 kilobytes of data into DNA code, synthesize the actual DNA, then sequence the DNA
and decode the information back to its original form, with a reported 100% accuracy. The encoded information
consisted of text files and audio files. A prior experiment was published in August 2012. It was conducted by
researchers at Harvard University, where the text of a 54,000-word book was encoded in DNA.
[123][124]
History of DNA research
James Watson and Francis Crick (right),
co-originators of the double-helix model, with
Maclyn McCarty (left).
DNA was first isolated by the Swiss physician Friedrich Miescher
who, in 1869, discovered a microscopic substance in the pus of
discarded surgical bandages. As it resided in the nuclei of cells, he
called it "nuclein".
[125]
In 1878, Albrecht Kossel isolated the
non-protein component of "nuclein", nucleic acid, and later isolated its
five primary nucleobases.
[]
In 1919, Phoebus Levene identified the
base, sugar and phosphate nucleotide unit.
[126]
Levene suggested that
DNA consisted of a string of nucleotide units linked together through
the phosphate groups. However, Levene thought the chain was short
and the bases repeated in a fixed order. In 1937 William Astbury
produced the first X-ray diffraction patterns that showed that DNA had
a regular structure.
[127]
In 1927, Nikolai Koltsov proposed that inherited traits would be inherited via a "giant hereditary molecule" made up
of "two mirror strands that would replicate in a semi-conservative fashion using each strand as a template".
[]
In 1928,
Frederick Griffith discovered that traits of the "smooth" form of Pneumococcus could be transferred to the "rough"
form of the same bacteria by mixing killed "smooth" bacteria with the live "rough" form.
[128]
This system provided
the first clear suggestion that DNA carries genetic informationthe AveryMacLeodMcCarty experimentwhen
Oswald Avery, along with coworkers Colin MacLeod and Maclyn McCarty, identified DNA as the transforming
principle in 1943.
[129]
DNA's role in heredity was confirmed in 1952, when Alfred Hershey and Martha Chase in the
HersheyChase experiment showed that DNA is the genetic material of the T2 phage.
[130]
In 1953, James Watson and Francis Crick suggested what is now accepted as the first correct double-helix model of
DNA structure in the journal Nature.
[]
Their double-helix, molecular model of DNA was then based on a single
X-ray diffraction image (labeled as "Photo 51")
[131]
taken by Rosalind Franklin and Raymond Gosling in May 1952,
as well as the information that the DNA bases are paired also obtained through private communications from
Erwin Chargaff in the previous years. Chargaff's rules played a very important role in establishing double-helix
configurations for B-DNA as well as A-DNA.
Experimental evidence supporting the Watson and Crick model was published in a series of five articles in the same
issue of Nature.
[132]
Of these, Franklin and Gosling's paper was the first publication of their own X-ray diffraction
data and original analysis method that partially supported the Watson and Crick model;
[][133]
this issue also
contained an article on DNA structure by Maurice Wilkins and two of his colleagues, whose analysis and in vivo
B-DNA X-ray patterns also supported the presence in vivo of the double-helical DNA configurations as proposed by
Crick and Watson for their double-helix molecular model of DNA in the previous two pages of Nature.
[]
In 1962,
after Franklin's death, Watson, Crick, and Wilkins jointly received the Nobel Prize in Physiology or Medicine.
[134]
DNA
69
Nobel Prizes were awarded only to living recipients at the time. A debate continues about who should receive credit
for the discovery.
[135]
In an influential presentation in 1957, Crick laid out the central dogma of molecular biology, which foretold the
relationship between DNA, RNA, and proteins, and articulated the "adaptor hypothesis".
[136]
Final confirmation of
the replication mechanism that was implied by the double-helical structure followed in 1958 through the
MeselsonStahl experiment.
[137]
Further work by Crick and coworkers showed that the genetic code was based on
non-overlapping triplets of bases, called codons, allowing Har Gobind Khorana, Robert W. Holley and Marshall
Warren Nirenberg to decipher the genetic code.
[138]
These findings represent the birth of molecular biology.
References
[3] pp. 1415.
[6] Berg J., Tymoczko J. and Stryer L. (2002) Biochemistry. W. H. Freeman and Company ISBN 0-7167-4955-6
[7] Abbreviations and Symbols for Nucleic Acids, Polynucleotides and their Constituents (http:/ / www. chem. qmul. ac. uk/ iupac/ misc/ naabb.
html) IUPAC-IUB Commission on Biochemical Nomenclature (CBN). Retrieved 3 January 2006.
[8] Created from PDB 1D65 (http:/ / www. rcsb.org/ pdb/ cgi/ explore. cgi?pdbId=1D65)
[15] Designation of the two strands of DNA (http:/ / www. chem. qmul. ac. uk/ iubmb/ newsletter/ misc/ DNA. html) JCBN/NC-IUB Newsletter
1989. Retrieved 7 May 2008
[25] http:/ / cogprints.org/ 3822/
[26] Hosemann R., Bagchi R.N., Direct analysis of diffraction by matter, North-Holland Publs., Amsterdam New York, 1962.
[34] Created from NDB UD0017 (http:/ / ndbserver. rutgers. edu/ atlas/ xray/ structures/ U/ ud0017/ ud0017. html)
[38] [38] Hu Q, Rosenfeld MG. (2012) Epigenetic regulation of human embryonic stem cells. Front Genet. 3:238. doi: 10.3389/fgene.2012.00238.
PMID 23133442
[45] Created from PDB 1JDG (http:/ / www.rcsb. org/ pdb/ cgi/ explore. cgi?pdbId=1JDG)
[51] Bernstein H, Payne CM, Bernstein C, Garewal H, Dvorak K. (2008) Cancer and aging as consequences of un-repaired DNA damage. In:
New Research on DNA Damage (Editors: Honoka Kimura And Aoi Suzuki) Nova Science Publishers, Inc., New York, Chapter 1, pp. 147.
ISBN 978-1-60456-581-2
[63] Created from PDB 1MSW (http:/ / www. rcsb.org/ pdb/ explore/ explore. do?structureId=1MSW)
[76] Created from PDB 1LMB (http:/ / www. rcsb.org/ pdb/ explore/ explore. do?structureId=1LMB)
[80] Created from PDB 1RVA (http:/ / www. rcsb.org/ pdb/ explore/ explore. do?structureId=1RVA)
[88] Created from PDB 1M6G (http:/ / www. rcsb.org/ pdb/ explore/ explore. do?structureId=1M6G)
[109] Colin Pitchfork first murder conviction on DNA evidence also clears the prime suspect (http:/ / web. archive. org/ web/
20061214004903/ http:/ / www.forensic.gov.uk/ forensic_t/ inside/ news/ list_casefiles. php?case=1) Forensic Science Service Accessed 23
December 2006
[112] Gusfield, Dan. Algorithms on Strings, Trees, and Sequences: Computer Science and Computational Biology. Cambridge University Press,
15 January 1997. ISBN 978-0-521-58519-4.
[114] http:/ / dx. doi. org/ 10.1371/ journal.pbio.0020073
[120] Lost Tribes of Israel, NOVA, PBS airdate: 22 February 2000. Transcript available from PBS.org (http:/ / www. pbs. org/ wgbh/ nova/
transcripts/ 2706israel.html). Retrieved 4 March 2006.
[121] Kleiman, Yaakov. "The Cohanim/DNA Connection: The fascinating story of how DNA studies confirm an ancient biblical tradition".
(http:/ / www. aish. com/ societywork/ sciencenature/ the_cohanim_-_dna_connection. asp) aish.com (13 January 2000). Retrieved 4 March
2006.
[122] Bhattacharya, Shaoni. "Killer convicted thanks to relative's DNA". (http:/ / www. newscientist. com/ article. ns?id=dn4908)
newscientist.com (20 April 2004). Retrieved 22 December 06.
[131] The B-DNA X-ray pattern on the right of this linked image (http:/ / osulibrary. oregonstate. edu/ specialcollections/ coll/ pauling/ dna/
pictures/ sci9. 001.5.html) was obtained by Rosalind Franklin and Raymond Gosling in May 1952 at high hydration levels of DNA and it has
been labeled as "Photo 51"
[132] Nature Archives Double Helix of DNA: 50 Years (http:/ / www. nature. com/ nature/ dna50/ archive. html)
[134] The Nobel Prize in Physiology or Medicine 1962 (http:/ / nobelprize. org/ nobel_prizes/ medicine/ laureates/ 1962/ ) Nobelprize .org
Accessed 22 December 06
[136] Crick, F.H.C. On degenerate templates and the adaptor hypothesis (PDF). (http:/ / genome. wellcome. ac. uk/ assets/ wtx030893. pdf)
genome.wellcome.ac.uk (Lecture, 1955). Retrieved 22 December 2006.
[138] The Nobel Prize in Physiology or Medicine 1968 (http:/ / nobelprize. org/ nobel_prizes/ medicine/ laureates/ 1968/ ) Nobelprize.org
Accessed 22 December 06
DNA
70
Further reading
Berry, Andrew; Watson, James. (2003). DNA: the secret of life. New York: Alfred A. Knopf.
ISBN0-375-41546-7.
Calladine, Chris R.; Drew, Horace R.; Luisi, Ben F. and Travers, Andrew A. (2003). Understanding DNA: the
molecule & how it works. Amsterdam: Elsevier Academic Press. ISBN0-12-155089-3.
Dennis, Carina; Julie Clayton (2003). 50 years of DNA. Basingstoke: Palgrave Macmillan. ISBN1-4039-1479-6.
Judson, Horace F. 1979. The Eighth Day of Creation: Makers of the Revolution in Biology. Touchstone Books,
ISBN 0-671-22540-5. 2nd edition: Cold Spring Harbor Laboratory Press, 1996 paperback: ISBN 0-87969-478-5.
Olby, Robert C. (1994). The path to the double helix: the discovery of DNA. New York: Dover Publications.
ISBN0-486-68117-3., first published in October 1974 by MacMillan, with foreword by Francis Crick;the
definitive DNA textbook,revised in 1994 with a 9 page postscript
Micklas, David. 2003. DNA Science: A First Course. Cold Spring Harbor Press: ISBN 978-0-87969-636-8
Ridley, Matt (2006). Francis Crick: discoverer of the genetic code. Ashland, OH: Eminent Lives, Atlas Books.
ISBN0-06-082333-X.
Olby, Robert C. (2009). Francis Crick: A Biography. Plainview, N.Y: Cold Spring Harbor Laboratory Press.
ISBN0-87969-798-9.
Rosenfeld, Israel. 2010. DNA: A Graphic Guide to the Molecule that Shook the World. Columbia University
Press: ISBN 978-0-231-14271-7
Schultz, Mark and Zander Cannon. 2009. The Stuff of Life: A Graphic Guide to Genetics and DNA. Hill and
Wang: ISBN 0-8090-8947-5
Stent, Gunther Siegmund; Watson, James. (1980). The double helix: a personal account of the discovery of the
structure of DNA. New York: Norton. ISBN0-393-95075-1.
Watson, James. 2004. DNA: The Secret of Life. Random House: ISBN 978-0-09-945184-6
Wilkins, Maurice (2003). The third man of the double helix the autobiography of Maurice Wilkins. Cambridge,
Eng: University Press. ISBN0-19-860665-6.
External links
Books about DNA:
Online books (http:/ / onlinebooks. library. upenn. edu/ webbin/ ftl?st=& su=DNA& library=OLBP),
Resources in your library (http:/ / onlinebooks. library. upenn. edu/ webbin/ ftl?st=& su=DNA),
Resources in other libraries (http:/ / onlinebooks. library. upenn. edu/ webbin/ ftl?st=& su=DNA&
library=0CHOOSE0)
DNA (http:/ / www. dmoz. org/ Science/ Biology/ Biochemistry_and_Molecular_Biology/ Biomolecules/
Nucleic_Acids/ DNA/ / ) at the Open Directory Project
DNA binding site prediction on protein (http:/ / pipe. scs. fsu. edu/ displar. html)
DNA the Double Helix Game (http:/ / nobelprize. org/ educational_games/ medicine/ dna_double_helix/ ) From
the official Nobel Prize web site
DNA under electron microscope (http:/ / www. fidelitysystems. com/ Unlinked_DNA. html)
Dolan DNA Learning Center (http:/ / www. dnalc. org/ )
Double Helix: 50 years of DNA (http:/ / www. nature. com/ nature/ dna50/ archive. html), Nature
Proteopedia DNA (http:/ / www. proteopedia. org/ wiki/ index. php/ DNA)
ENCODE threads explorer (http:/ / www. nature. com/ encode/ ) ENCODE Home page. Nature (journal)
Double Helix 19532003 (http:/ / www. ncbe. reading. ac. uk/ DNA50/ ) National Centre for Biotechnology
Education
DNA
71
Genetic Education Modules for Teachers (http:/ / www. genome. gov/ 10506718)DNA from the Beginning
Study Guide
PDB Molecule of the Month pdb23_1 (http:/ / www. rcsb. org/ pdb/ static. do?p=education_discussion/
molecule_of_the_month/ pdb23_1. html)
Rosalind Franklin's contributions to the study of DNA (http:/ / mason. gmu. edu/ ~emoody/ rfranklin. html)
U.S. National DNA Day (http:/ / www. genome. gov/ 10506367)watch videos and participate in real-time chat
with top scientists
Clue to chemistry of heredity found (http:/ / www. nytimes. com/ packages/ pdf/ science/ dna-article. pdf) The
New York Times June 1953. First American newspaper coverage of the discovery of the DNA structure
Olby R (2003). "Quiet debut for the double helix". Nature 421 (6921): 4025. doi: 10.1038/nature01397 (http:/ /
dx. doi. org/ 10. 1038/ nature01397). PMID 12540907 (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 12540907).
DNA from the Beginning (http:/ / www. dnaftb. org/ ) Another DNA Learning Center site on DNA, genes, and
heredity from Mendel to the human genome project.
The Register of Francis Crick Personal Papers 1938 2007 (http:/ / orpheus. ucsd. edu/ speccoll/ testing/ html/
mss0660a. html#abstract) at Mandeville Special Collections Library, University of California, San Diego
Seven-page, handwritten letter that Crick sent to his 12-year-old son Michael in 1953 describing the structure of
DNA. (http:/ / www. nature. com/ polopoly_fs/ 7. 9746!/ file/ Crick letter to Michael. pdf) See Cricks medal goes
under the hammer (http:/ / www. nature. com/ news/ crick-s-medal-goes-under-the-hammer-1. 12705), Nature, 5
April 2013.
72
Proteins and amino acids
Protein
A representation of the 3D structure of the protein
myoglobin showing turquoise alpha helices. This
protein was the first to have its structure solved
by X-ray crystallography. Towards the
right-center among the coils, a prosthetic group
called a heme group (shown in gray) with a
bound oxygen molecule (red).
Proteins (pron.: /protinz/ or /proti.nz/) are large biological
molecules consisting of one or more chains of amino acids. Proteins
perform a vast array of functions within living organisms, including
catalyzing metabolic reactions, replicating DNA, responding to stimuli,
and transporting molecules from one location to another. Proteins
differ from one another primarily in their sequence of amino acids,
which is dictated by the nucleotide sequence of their genes, and which
usually results in folding of the protein into a specific
three-dimensional structure that determines its activity.
A polypeptide is a single linear polymer chain of amino acids bonded
together by peptide bonds between the carboxyl and amino groups of
adjacent amino acid residues. The sequence of amino acids in a protein
is defined by the sequence of a gene, which is encoded in the genetic
code. In general, the genetic code specifies 20 standard amino acids;
however, in certain organisms the genetic code can include
selenocysteine andin certain archaeapyrrolysine. Shortly after or
even during synthesis, the residues in a protein are often chemically
modified by posttranslational modification, which alters the physical
and chemical properties, folding, stability, activity, and ultimately, the function of the proteins. Sometimes proteins
have non-peptide groups attached, which can be called prosthetic groups or cofactors. Proteins can also work
together to achieve a particular function, and they often associate to form stable protein complexes.
Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts of
organisms and participate in virtually every process within cells. Many proteins are enzymes that catalyze
biochemical reactions and are vital to metabolism. Proteins also have structural or mechanical functions, such as
actin and myosin in muscle and the proteins in the cytoskeleton, which form a system of scaffolding that maintains
cell shape. Other proteins are important in cell signaling, immune responses, cell adhesion, and the cell cycle.
Proteins are also necessary in animals' diets, since animals cannot synthesize all the amino acids they need and must
obtain essential amino acids from food. Through the process of digestion, animals break down ingested protein into
free amino acids that are then used in metabolism.
Proteins may be purified from other cellular components using a variety of techniques such as ultracentrifugation,
precipitation, electrophoresis, and chromatography; the advent of genetic engineering has made possible a number of
methods to facilitate purification. Methods commonly used to study protein structure and function include
immunohistochemistry, site-directed mutagenesis, nuclear magnetic resonance and mass spectrometry.
Protein
73
Biochemistry
Chemical structure of the peptide bond (bottom) and the three-dimensional
structure of a peptide bond between an alanine and an adjacent amino acid
(top/inset)
Resonance structures of the peptide bond that links individual amino acids to form
a protein polymer
Most proteins consist of linear polymers
built from series of up to 20 different
L--amino acids. All proteinogenic amino
acids possess common structural features,
including an -carbon to which an amino
group, a carboxyl group, and a variable side
chain are bonded. Only proline differs from
this basic structure as it contains an unusual
ring to the N-end amine group, which forces
the CONH amide moiety into a fixed
conformation.
[]
The side chains of the
standard amino acids, detailed in the list of
standard amino acids, have a great variety of
chemical structures and properties; it is the
combined effect of all of the amino acid side
chains in a protein that ultimately
determines its three-dimensional structure
and its chemical reactivity.
[]
The amino
acids in a polypeptide chain are linked by
peptide bonds. Once linked in the protein
chain, an individual amino acid is called a
residue, and the linked series of carbon,
nitrogen, and oxygen atoms are known as
the main chain or protein backbone.
[1]
The peptide bond has two resonance forms
that contribute some double-bond character and inhibit rotation around its axis, so that the alpha carbons are roughly
coplanar. The other two dihedral angles in the peptide bond determine the local shape assumed by the protein
backbone.
[2]
The end of the protein with a free carboxyl group is known as the C-terminus or carboxy terminus,
whereas the end with a free amino group is known as the N-terminus or amino terminus. The words protein,
polypeptide, and peptide are a little ambiguous and can overlap in meaning. Protein is generally used to refer to the
complete biological molecule in a stable conformation, whereas peptide is generally reserved for a short amino acid
oligomers often lacking a stable three-dimensional structure. However, the boundary between the two is not well
defined and usually lies near 2030 residues.
[]
Polypeptide can refer to any single linear chain of amino acids,
usually regardless of length, but often implies an absence of a defined conformation.
Protein
74
Synthesis
A ribosome produces a protein using mRNA as
template.
The DNA sequence of a gene encodes the amino
acid sequence of a protein.
Proteins are assembled from amino acids using information encoded in
genes. Each protein has its own unique amino acid sequence that is
specified by the nucleotide sequence of the gene encoding this protein.
The genetic code is a set of three-nucleotide sets called codons and
each three-nucleotide combination designates an amino acid, for
example AUG (adenine-uracil-guanine) is the code for methionine.
Because DNA contains four nucleotides, the total number of possible
codons is 64; hence, there is some redundancy in the genetic code, with
some amino acids specified by more than one codon.
[3]
Genes encoded
in DNA are first transcribed into pre-messenger RNA (mRNA) by
proteins such as RNA polymerase. Most organisms then process the
pre-mRNA (also known as a primary transcript) using various forms
of Post-transcriptional modification to form the mature mRNA, which
is then used as a template for protein synthesis by the ribosome. In
prokaryotes the mRNA may either be used as soon as it is produced, or
be bound by a ribosome after having moved away from the nucleoid.
In contrast, eukaryotes make mRNA in the cell nucleus and then
translocate it across the nuclear membrane into the cytoplasm, where
protein synthesis then takes place. The rate of protein synthesis is
higher in prokaryotes than eukaryotes and can reach up to 20 amino acids per second.
[]
The process of synthesizing a protein from an mRNA template is known as translation. The mRNA is loaded onto
the ribosome and is read three nucleotides at a time by matching each codon to its base pairing anticodon located on
a transfer RNA molecule, which carries the amino acid corresponding to the codon it recognizes. The enzyme
aminoacyl tRNA synthetase "charges" the tRNA molecules with the correct amino acids. The growing polypeptide is
often termed the nascent chain. Proteins are always biosynthesized from N-terminus to C-terminus.
[3]
The size of a synthesized protein can be measured by the number of amino acids it contains and by its total
molecular mass, which is normally reported in units of daltons (synonymous with atomic mass units), or the
derivative unit kilodalton (kDa). Yeast proteins are on average 466 amino acids long and 53 kDa in mass.
[]
The
largest known proteins are the titins, a component of the muscle sarcomere, with a molecular mass of almost 3,000
kDa and a total length of almost 27,000 amino acids.
[]
Chemical synthesis
Short proteins can also be synthesized chemically by a family of methods known as peptide synthesis, which rely on
organic synthesis techniques such as chemical ligation to produce peptides in high yield.
[]
Chemical synthesis allows
for the introduction of non-natural amino acids into polypeptide chains, such as attachment of fluorescent probes to
amino acid side chains.
[]
These methods are useful in laboratory biochemistry and cell biology, though generally not
for commercial applications. Chemical synthesis is inefficient for polypeptides longer than about 300 amino acids,
and the synthesized proteins may not readily assume their native tertiary structure. Most chemical synthesis methods
proceed from C-terminus to N-terminus, opposite the biological reaction.
[]
Protein
75
Structure
The crystal structure of the chaperonin. Chaperonins assist protein folding.
Three possible representations of the three-dimensional structure of the protein
triose phosphate isomerase. Left: all-atom representation colored by atom type.
Middle: Simplified representation illustrating the backbone conformation, colored
by secondary structure. Right: Solvent-accessible surface representation colored by
residue type (acidic residues red, basic residues blue, polar residues green,
nonpolar residues white)
Mostproteins fold into unique
3-dimensional structures. The shape into
which a protein naturally folds is known as
its native conformation.
[4]
Although many
proteins can fold unassisted, simply through
the chemical properties of their amino acids,
others require the aid of molecular
chaperones to fold into their native states.
[5]
Biochemists often refer to four distinct
aspects of a protein's structure:
[6]
Primary structure: the amino acid
sequence.
Secondary structure: regularly repeating
local structures stabilized by hydrogen
bonds. The most common examples are
the alpha helix, beta sheet and turns.
Because secondary structures are local,
many regions of different secondary
structure can be present in the same
protein molecule.
Tertiary structure: the overall shape of a
single protein molecule; the spatial
relationship of the secondary structures to
one another. Tertiary structure is
generally stabilized by nonlocal interactions, most commonly the formation of a hydrophobic core, but also
through salt bridges, hydrogen bonds, disulfide bonds, and even posttranslational modifications. The term
"tertiary structure" is often used as synonymous with the term fold. The tertiary structure is what controls the
basic function of the protein.
Quaternary structure: the structure formed by several protein molecules (polypeptide chains), usually called
protein subunits in this context, which function as a single protein complex.
Proteins are not entirely rigid molecules. In addition to these levels of structure, proteins may shift between several
related structures while they perform their functions. In the context of these functional rearrangements, these tertiary
or quaternary structures are usually referred to as "conformations", and transitions between them are called
conformational changes. Such changes are often induced by the binding of a substrate molecule to an enzyme's
active site, or the physical region of the protein that participates in chemical catalysis. In solution proteins also
undergo variation in structure through thermal vibration and the collision with other molecules.
[7]
Protein
76
Molecular surface of several proteins showing their comparative sizes. From left to
right are: immunoglobulin G (IgG, an antibody), hemoglobin, insulin (a hormone),
adenylate kinase (an enzyme), and glutamine synthetase (an enzyme).
Proteins can be informally divided into three
main classes, which correlate with typical
tertiary structures: globular proteins, fibrous
proteins, and membrane proteins. Almost all
globular proteins are soluble and many are
enzymes. Fibrous proteins are often
structural, such as collagen, the major
component of connective tissue, or keratin,
the protein component of hair and nails.
Membrane proteins often serve as receptors or provide channels for polar or charged molecules to pass through the
cell membrane.
[8]
A special case of intramolecular hydrogen bonds within proteins, poorly shielded from water attack and hence
promoting their own dehydration, are called dehydrons.
[]
Structure determination
Discovering the tertiary structure of a protein, or the quaternary structure of its complexes, can provide important
clues about how the protein performs its function. Common experimental methods of structure determination include
X-ray crystallography and NMR spectroscopy, both of which can produce information at atomic resolution.
However, NMR experiments are able to provide information from which a subset of distances between pairs of
atoms can be estimated, and the final possible conformations for a protein are determined by solving a distance
geometry problem. Dual polarisation interferometry is a quantitative analytical method for measuring the overall
protein conformation and conformational changes due to interactions or other stimulus. Circular dichroism is another
laboratory technique for determining internal beta sheet/ helical composition of proteins. Cryoelectron microscopy is
used to produce lower-resolution structural information about very large protein complexes, including assembled
viruses;
[9]
a variant known as electron crystallography can also produce high-resolution information in some cases,
especially for two-dimensional crystals of membrane proteins.
[]
Solved structures are usually deposited in the Protein
Data Bank (PDB), a freely available resource from which structural data about thousands of proteins can be obtained
in the form of Cartesian coordinates for each atom in the protein.
[]
Many more gene sequences are known than protein structures. Further, the set of solved structures is biased toward
proteins that can be easily subjected to the conditions required in X-ray crystallography, one of the major structure
determination methods. In particular, globular proteins are comparatively easy to crystallize in preparation for X-ray
crystallography. Membrane proteins, by contrast, are difficult to crystallize and are underrepresented in the PDB.
[]
Structural genomics initiatives have attempted to remedy these deficiencies by systematically solving representative
structures of major fold classes. Protein structure prediction methods attempt to provide a means of generating a
plausible structure for proteins whose structures have not been experimentally determined.
[]
Cellular functions
Proteins are the chief actors within the cell, said to be carrying out the duties specified by the information encoded in
genes.
[]
With the exception of certain types of RNA, most other biological molecules are relatively inert elements
upon which proteins act. Proteins make up half the dry weight of an Escherichia coli cell, whereas other
macromolecules such as DNA and RNA make up only 3% and 20%, respectively.
[10]
The set of proteins expressed
in a particular cell or cell type is known as its proteome.
Protein
77
The enzyme hexokinase is shown as a
conventional ball-and-stick molecular model. To
scale in the top right-hand corner are two of its
substrates, ATP and glucose.
The chief characteristic of proteins that also allows their diverse set of
functions is their ability to bind other molecules specifically and
tightly. The region of the protein responsible for binding another
molecule is known as the binding site and is often a depression or
"pocket" on the molecular surface. This binding ability is mediated by
the tertiary structure of the protein, which defines the binding site
pocket, and by the chemical properties of the surrounding amino acids'
side chains. Protein binding can be extraordinarily tight and specific;
for example, the ribonuclease inhibitor protein binds to human
angiogenin with a sub-femtomolar dissociation constant (<10
15
M)
but does not bind at all to its amphibian homolog onconase (>1 M).
Extremely minor chemical changes such as the addition of a single
methyl group to a binding partner can sometimes suffice to nearly eliminate binding; for example, the aminoacyl
tRNA synthetase specific to the amino acid valine discriminates against the very similar side chain of the amino acid
isoleucine.
[]
Proteins can bind to other proteins as well as to small-molecule substrates. When proteins bind specifically to other
copies of the same molecule, they can oligomerize to form fibrils; this process occurs often in structural proteins that
consist of globular monomers that self-associate to form rigid fibers. Proteinprotein interactions also regulate
enzymatic activity, control progression through the cell cycle, and allow the assembly of large protein complexes
that carry out many closely related reactions with a common biological function. Proteins can also bind to, or even
be integrated into, cell membranes. The ability of binding partners to induce conformational changes in proteins
allows the construction of enormously complex signaling networks.
[11]
Importantly, as interactions between proteins
are reversible, and depend heavily on the availability of different groups of partner proteins to form aggregates that
are capable to carry out discrete sets of function, study of the interactions between specific proteins is a key to
understand important aspects of cellular function, and ultimately the properties that distinguish particular cell
types.
[][]
Enzymes
The best-known role of proteins in the cell is as enzymes, which catalyze chemical reactions. Enzymes are usually
highly specific and accelerate only one or a few chemical reactions. Enzymes carry out most of the reactions
involved in metabolism, as well as manipulating DNA in processes such as DNA replication, DNA repair, and
transcription. Some enzymes act on other proteins to add or remove chemical groups in a process known as
posttranslational modification. About 4,000 reactions are known to be catalyzed by enzymes.
[]
The rate acceleration
conferred by enzymatic catalysis is often enormousas much as 10
17
-fold increase in rate over the uncatalyzed
reaction in the case of orotate decarboxylase (78 million years without the enzyme, 18 milliseconds with the
enzyme).
[]
The molecules bound and acted upon by enzymes are called substrates. Although enzymes can consist of hundreds
of amino acids, it is usually only a small fraction of the residues that come in contact with the substrate, and an even
smaller fractionthree to four residues on averagethat are directly involved in catalysis.
[]
The region of the
enzyme that binds the substrate and contains the catalytic residues is known as the active site.
Dirigent proteins are members of a class of proteins which dictate the stereochemistry of a compound synthesized by
other enzymes.
Protein
78
Cell signaling and ligand binding
Ribbon diagram of a mouse antibody
against cholera that binds a
carbohydrate antigen
Many proteins are involved in the process of cell signaling and signal
transduction. Some proteins, such as insulin, are extracellular proteins that
transmit a signal from the cell in which they were synthesized to other cells in
distant tissues. Others are membrane proteins that act as receptors whose main
function is to bind a signaling molecule and induce a biochemical response in the
cell. Many receptors have a binding site exposed on the cell surface and an
effector domain within the cell, which may have enzymatic activity or may
undergo a conformational change detected by other proteins within the cell.
[12]
Antibodies are protein components of an adaptive immune system whose main
function is to bind antigens, or foreign substances in the body, and target them
for destruction. Antibodies can be secreted into the extracellular environment or
anchored in the membranes of specialized B cells known as plasma cells.
Whereas enzymes are limited in their binding affinity for their substrates by the
necessity of conducting their reaction, antibodies have no such constraints. An
antibody's binding affinity to its target is extraordinarily high.
[13]
Many ligand transport proteins bind particular small biomolecules and transport them to other locations in the body
of a multicellular organism. These proteins must have a high binding affinity when their ligand is present in high
concentrations, but must also release the ligand when it is present at low concentrations in the target tissues. The
canonical example of a ligand-binding protein is haemoglobin, which transports oxygen from the lungs to other
organs and tissues in all vertebrates and has close homologs in every biological kingdom.
[14]
Lectins are
sugar-binding proteins which are highly specific for their sugar moieties. Lectins typically play a role in biological
recognition phenomena involving cells and proteins.
[]
Receptors and hormones are highly specific binding proteins.
Transmembrane proteins can also serve as ligand transport proteins that alter the permeability of the cell membrane
to small molecules and ions. The membrane alone has a hydrophobic core through which polar or charged molecules
cannot diffuse. Membrane proteins contain internal channels that allow such molecules to enter and exit the cell.
Many ion channel proteins are specialized to select for only a particular ion; for example, potassium and sodium
channels often discriminate for only one of the two ions.
[15]
Structural proteins
Structural proteins confer stiffness and rigidity to otherwise-fluid biological components. Most structural proteins are
fibrous proteins; for example, collagen and elastin are critical components of connective tissue such as cartilage, and
keratin is found in hard or filamentous structures such as hair, nails, feathers, hooves, and some animal shells.
[16]
Some globular proteins can also play structural functions, for example, actin and tubulin are globular and soluble as
monomers, but polymerize to form long, stiff fibers that make up the cytoskeleton, which allows the cell to maintain
its shape and size.
Other proteins that serve structural functions are motor proteins such as myosin, kinesin, and dynein, which are
capable of generating mechanical forces. These proteins are crucial for cellular motility of single celled organisms
and the sperm of many multicellular organisms which reproduce sexually. They also generate the forces exerted by
contracting muscles
[17]
and play essential roles in intracellular transport.
Protein
79
Methods of study
As some of the most commonly studied biological molecules, the activities and structures of proteins are examined
both in vitro and in vivo. In vitro studies of purified proteins in controlled environments are useful for learning how a
protein carries out its function: for example, enzyme kinetics studies explore the chemical mechanism of an enzyme's
catalytic activity and its relative affinity for various possible substrate molecules. By contrast, in vivo experiments on
proteins' activities within cells or even within whole organisms can provide complementary information about where
a protein functions and how it is regulated.
Protein purification
To perform in vitro analysis, a protein must be purified away from other cellular components. This process usually
begins with cell lysis, in which a cell's membrane is disrupted and its internal contents released into a solution known
as a crude lysate. The resulting mixture can be purified using ultracentrifugation, which fractionates the various
cellular components into fractions containing soluble proteins; membrane lipids and proteins; cellular organelles, and
nucleic acids. Precipitation by a method known as salting out can concentrate the proteins from this lysate. Various
types of chromatography are then used to isolate the protein or proteins of interest based on properties such as
molecular weight, net charge and binding affinity.
[18]
The level of purification can be monitored using various types
of gel electrophoresis if the desired protein's molecular weight and isoelectric point are known, by spectroscopy if
the protein has distinguishable spectroscopic features, or by enzyme assays if the protein has enzymatic activity.
Additionally, proteins can be isolated according their charge using electrofocusing.
[]
For natural proteins, a series of purification steps may be necessary to obtain protein sufficiently pure for laboratory
applications. To simplify this process, genetic engineering is often used to add chemical features to proteins that
make them easier to purify without affecting their structure or activity. Here, a "tag" consisting of a specific amino
acid sequence, often a series of histidine residues (a "His-tag"), is attached to one terminus of the protein. As a result,
when the lysate is passed over a chromatography column containing nickel, the histidine residues ligate the nickel
and attach to the column while the untagged components of the lysate pass unimpeded. A number of different tags
have been developed to help researchers purify specific proteins from complex mixtures.
[]
Protein
80
Cellular localization
Proteins in different cellular compartments and structures tagged with green
fluorescent protein (here, white)
The study of proteins in vivo is often concerned
with the synthesis and localization of the protein
within the cell. Although many intracellular
proteins are synthesized in the cytoplasm and
membrane-bound or secreted proteins in the
endoplasmic reticulum, the specifics of how
proteins are targeted to specific organelles or
cellular structures is often unclear. A useful
technique for assessing cellular localization uses
genetic engineering to express in a cell a fusion
protein or chimera consisting of the natural
protein of interest linked to a "reporter" such as
green fluorescent protein (GFP).
[]
The fused
protein's position within the cell can be cleanly
and efficiently visualized using microscopy,
[]
as
shown in the figure opposite.
Other methods for elucidating the cellular
location of proteins requires the use of known
compartmental markers for regions such as the
ER, the Golgi, lysosomes or vacuoles,
mitochondria, chloroplasts, plasma membrane,
etc. With the use of fluorescently tagged versions
of these markers or of antibodies to known
markers, it becomes much simpler to identify the
localization of a protein of interest. For example,
indirect immunofluorescence will allow for fluorescence colocalization and demonstration of location. Fluorescent
dyes are used to label cellular compartments for a similar purpose.
[]
Other possibilities exist, as well. For example, immunohistochemistry usually utilizes an antibody to one or more
proteins of interest that are conjugated to enzymes yielding either luminescent or chromogenic signals that can be
compared between samples, allowing for localization information. Another applicable technique is cofractionation in
sucrose (or other material) gradients using isopycnic centrifugation.
[]
While this technique does not prove
colocalization of a compartment of known density and the protein of interest, it does increase the likelihood, and is
more amenable to large-scale studies.
Finally, the gold-standard method of cellular localization is immunoelectron microscopy. This technique also uses an
antibody to the protein of interest, along with classical electron microscopy techniques. The sample is prepared for
normal electron microscopic examination, and then treated with an antibody to the protein of interest that is
conjugated to an extremely electro-dense material, usually gold. This allows for the localization of both
ultrastructural details as well as the protein of interest.
[]
Through another genetic engineering application known as site-directed mutagenesis, researchers can alter the
protein sequence and hence its structure, cellular localization, and susceptibility to regulation. This technique even
allows the incorporation of unnatural amino acids into proteins, using modified tRNAs,
[]
and may allow the rational
design of new proteins with novel properties.
[]
Protein
81
Proteomics and bioinformatics
The total complement of proteins present at a time in a cell or cell type is known as its proteome, and the study of
such large-scale data sets defines the field of proteomics, named by analogy to the related field of genomics. Key
experimental techniques in proteomics include 2D electrophoresis,
[]
which allows the separation of a large number of
proteins, mass spectrometry,
[]
which allows rapid high-throughput identification of proteins and sequencing of
peptides (most often after in-gel digestion), protein microarrays,
[]
which allow the detection of the relative levels of a
large number of proteins present in a cell, and two-hybrid screening, which allows the systematic exploration of
proteinprotein interactions.
[]
The total complement of biologically possible such interactions is known as the
interactome.
[]
A systematic attempt to determine the structures of proteins representing every possible fold is known
as structural genomics.
[]
The large amount of genomic and proteomic data available for a variety of organisms, including the human genome,
allows researchers to efficiently identify homologous proteins in distantly related organisms by sequence alignment.
Sequence profiling tools can perform more specific sequence manipulations such as restriction enzyme maps, open
reading frame analyses for nucleotide sequences, and secondary structure prediction. From this data phylogenetic
trees can be constructed and evolutionary hypotheses developed using special software like ClustalW regarding the
ancestry of modern organisms and the genes they express. The field of bioinformatics seeks to assemble, annotate,
and analyze genomic and proteomic data, applying computational techniques to biological problems such as gene
finding and cladistics.
Structure prediction and simulation
Complementary to the field of structural genomics, protein structure prediction seeks to develop efficient ways to
provide plausible models for proteins whose structures have not yet been determined experimentally.
[]
The most
successful type of structure prediction, known as homology modeling, relies on the existence of a "template"
structure with sequence similarity to the protein being modeled; structural genomics' goal is to provide sufficient
representation in solved structures to model most of those that remain.
[]
Although producing accurate models
remains a challenge when only distantly related template structures are available, it has been suggested that sequence
alignment is the bottleneck in this process, as quite accurate models can be produced if a "perfect" sequence
alignment is known.
[]
Many structure prediction methods have served to inform the emerging field of protein
engineering, in which novel protein folds have already been designed.
[]
A more complex computational problem is
the prediction of intermolecular interactions, such as in molecular docking and proteinprotein interaction
prediction.
[]
The processes of protein folding and binding can be simulated using such technique as molecular mechanics, in
particular, molecular dynamics and Monte Carlo, which increasingly take advantage of parallel and distributed
computing (Folding@home project;
[]
molecular modeling on GPU). The folding of small alpha-helical protein
domains such as the villin headpiece
[]
and the HIV accessory protein
[]
have been successfully simulated in silico, and
hybrid methods that combine standard molecular dynamics with quantum mechanics calculations have allowed
exploration of the electronic states of rhodopsins.
[]
Nutrition
Most microorganisms and plants can biosynthesize all 20 standard amino acids, while animals (including humans)
must obtain some of the amino acids from the diet.
[10]
The amino acids that an organism cannot synthesize on its
own are referred to as essential amino acids. Key enzymes that synthesize certain amino acids are not present in
animals such as aspartokinase, which catalyzes the first step in the synthesis of lysine, methionine, and threonine
from aspartate. If amino acids are present in the environment, microorganisms can conserve energy by taking up the
amino acids from their surroundings and downregulating their biosynthetic pathways.
Protein
82
In animals, amino acids are obtained through the consumption of foods containing protein. Ingested proteins are then
broken down into amino acids through digestion, which typically involves denaturation of the protein through
exposure to acid and hydrolysis by enzymes called proteases. Some ingested amino acids are used for protein
biosynthesis, while others are converted to glucose through gluconeogenesis, or fed into the citric acid cycle. This
use of protein as a fuel is particularly important under starvation conditions as it allows the body's own proteins to be
used to support life, particularly those found in muscle.
[]
Amino acids are also an important dietary source of
nitrogen.
[citation needed]
History and etymology
Proteins were recognized as a distinct class of biological molecules in the eighteenth century by Antoine Fourcroy
and others, distinguished by the molecules' ability to coagulate or flocculate under treatments with heat or acid
[citation
needed]
. Noted examples at the time included albumin from egg whites, blood serum albumin, fibrin, and wheat
gluten.
Proteins were first described by the Dutch chemist Gerardus Johannes Mulder and named by the Swedish chemist
Jns Jacob Berzelius in 1838. Mulder carried out elemental analysis of common proteins and found that nearly all
proteins had the same empirical formula, C
400
H
620
N
100
O
120
P
1
S
1
.
[]
He came to the erroneous conclusion that they
might be composed of a single type of (very large) molecule. The term "protein" to describe these molecules was
proposed by Mulder's associate Berzelius; protein is derived from the Greek word (proteios), meaning
"primary",
[19]
"in the lead", or "standing in front".
[]
Mulder went on to identify the products of protein degradation
such as the amino acid leucine for which he found a (nearly correct) molecular weight of 131 Da.
[]
Early nutritional scientists such as the German Carl von Voit believed that protein was the most important nutrient
for maintaining the structure of the body, because it was generally believed that "flesh makes flesh."
[]
The central
role of proteins as enzymes in living organisms was not fully appreciated until 1926, when James B. Sumner showed
that the enzyme urease was in fact a protein.
[]
The difficulty in purifying proteins in large quantities made them very difficult for early protein biochemists to
study. Hence, early studies focused on proteins that could be purified in large quantities, e.g., those of blood, egg
white, various toxins, and digestive/metabolic enzymes obtained from slaughterhouses. In the 1950s, the Armour
Hot Dog Co. purified 1kg of pure bovine pancreatic ribonuclease A and made it freely available to scientists; this
gesture helped ribonuclease A become a major target for biochemical study for the following decades.
[]
John Kendrew with model of myoglobin in
progress.
Linus Pauling is credited with the successful prediction of regular
protein secondary structures based on hydrogen bonding, an idea first
put forth by William Astbury in 1933.
[]
Later work by Walter
Kauzmann on denaturation,
[][]
based partly on previous studies by Kaj
Linderstrm-Lang,
[]
contributed an understanding of protein folding
and structure mediated by hydrophobic interactions.
The first protein to be sequenced was insulin, by Frederick Sanger, in
1949. Sanger correctly determined the amino acid sequence of insulin,
thus conclusively demonstrating that proteins consisted of linear
polymers of amino acids rather than branched chains, colloids, or
cyclols.
[]
He won the Nobel Prize for this achievement in 1958.
The first protein structures to be solved were hemoglobin and myoglobin, by Max Perutz and Sir John Cowdery
Kendrew, respectively, in 1958.
[][]
The first atomic-resolution structures of proteins were solved by X-ray diffraction
analysis in the 1960s
[citation needed]
(Perutz and Kendrew shared the 1962 Nobel Prize in Chemistry for these
discoveries) and by NMR in the 1980s.
[citation needed]
As of 2009[20], the Protein Data Bank has over 55,000
atomic-resolution structures of proteins.
[]
In more recent times, cryo-electron microscopy of large macromolecular
Protein
83
assemblies
[]
and computational protein structure prediction of small protein domains
[]
are two methods approaching
atomic resolution.
Footnotes
[1] Murray et al., p. 19.
[2] Murray et al., p. 31.
[3] van Holde and Mathews, pp. 100242.
[4] Murray et al., p. 36.
[5] Murray et al., p. 37.
[6] Murray et al., pp. 3034.
[7] van Holde and Mathews, pp. 36875.
[8] van Holde and Mathews, pp. 16585.
[9] Branden and Tooze, pp. 34041.
[10] Voet D, Voet JG. (2004). Biochemistry Vol 1 3rd ed. Wiley: Hoboken, NJ.
[11] van Holde and Mathews, pp. 83049.
[12] Branden and Tooze, pp. 25181.
[13] van Holde and Mathews, pp. 24750.
[14] van Holde and Mathews, pp. 22029.
[15] Branden and Tooze, pp. 23234.
[16] van Holde and Mathews, pp. 17881.
[17] van Holde and Mathews, pp. 25864; 272.
[18] Murray et al., pp. 2124.
[19] [19] New Oxford Dictionary of English
[20] http:/ / en. wikipedia. org/ w/ index. php?title=Protein& action=edit
References
Branden C, Tooze J (1999). Introduction to Protein Structure. New York: Garland Pub. ISBN0-8153-2305-0.
Murray RF, Harper HW, Granner DK, Mayes PA, Rodwell VW (2006). Harper's Illustrated Biochemistry. New
York: Lange Medical Books/McGraw-Hill. ISBN0-07-146197-3.
Van Holde KE, Mathews CK (1996). Biochemistry. Menlo Park, California: Benjamin/Cummings Pub. Co., Inc.
ISBN0-8053-3931-0.
External links
Databases and projects
The Protein Naming Utility (http:/ / www. jcvi. org/ pn-utility)
Human Protein Atlas (http:/ / www. proteinatlas. org/ )
NCBI Entrez Protein database (http:/ / www. ncbi. nlm. nih. gov/ sites/ entrez?db=protein)
NCBI Protein Structure database (http:/ / www. ncbi. nlm. nih. gov/ sites/ entrez?db=structure)
Human Protein Reference Database (http:/ / www. hprd. org/ )
Human Proteinpedia (http:/ / www. humanproteinpedia. org/ )
Folding@Home (Stanford University) (http:/ / folding. stanford. edu/ )
Comparative Toxicogenomics Database (http:/ / ctd. mdibl. org/ ) curates proteinchemical interactions, as well
as gene/proteindisease relationships and chemical-disease relationships.
Bioinformatic Harvester (http:/ / harvester. fzk. de/ ) A Meta search engine (29 databases) for gene and protein
information.
Protein Databank in Europe (http:/ / www. pdbe. org/ ) (see also PDBeQuips (http:/ / www. pdbe. org/ quips),
short articles and tutorials on interesting PDB structures)
Research Collaboratory for Structural Bioinformatics (http:/ / www. rcsb. org/ ) (see also Molecule of the Month
(http:/ / www. rcsb. org/ pdb/ static. do?p=education_discussion/ molecule_of_the_month/ index. html),
Protein
84
presenting short accounts on selected proteins from the PDB)
Proteopedia Life in 3D (http:/ / www. proteopedia. org/ ): rotatable, zoomable 3D model with wiki annotations
for every known protein molecular structure.
UniProt the Universal Protein Resource (http:/ / www. expasy. uniprot. org/ )
neXtProt Exploring the universe of human proteins (http:/ / www. nextprot. org/ ): human-centric protein
knowledge resource
Tutorials and educational websites
"An Introduction to Proteins" (http:/ / hopes. stanford. edu/ basics/ proteins/ p0. html) from HOPES (Huntington's
Disease Outreach Project for Education at Stanford)
Proteins: Biogenesis to Degradation The Virtual Library of Biochemistry and Cell Biology (http:/ / www.
biochemweb. org/ proteins. shtml)
Amino acid
The generic structure of an alpha amino acid in its
un-ionized form
Amino acids (pron.: /mino/, /mano/, or /mno/) are
biologically important organic compounds made from amine
(-NH
2
) and carboxylic acid (-COOH) functional groups, along
with a side-chain specific to each amino acid. The key elements of
an amino acid are carbon, hydrogen, oxygen, and nitrogen, though
other elements are found in the side-chains of certain amino acids.
About 500 amino acids are known
[1]
and can be classified in many
ways. Structurally they can be classified according to the
functional groups' locations as alpha- (-), beta- (-), gamma- (-)
or delta- (-) amino acids; other categories relate to polarity, pH
level, and side chain group type (aliphatic, acyclic, aromatic,
containing hydroxyl or sulphur, et al.). In the form of proteins,
amino acids comprise the second largest component (after water) of human muscles, cells and other tissues.
[2]
Outside proteins, amino acids perform critical roles in processes such as neurotransmitter transport and biosynthesis.
Amino acids having both the amine and carboxylic acid groups attached to the first (alpha-) carbon atom have
particular importance in biochemistry. They are known as 2-, alpha-, or -amino acids (generic formula
H
2
NCHRCOOH in most cases
[3]
where R is an
Amino acid
85
The 21 amino acids found in eukaryotes, grouped according to their
side-chains' pK
a
values and charges carried at physiological pH 7.4
organic substituent known as a "side-chain");
[4]
often the term "amino acid" is used to refer
specifically to these. They include the 23
proteinogenic ("protein-building") amino acids
which combine into peptide chains
("polypeptides") to form the building blocks of
a vast array of proteins.
[]
These are all
L-stereoisomers ("left-handed" isomers)
although a few D-amino acids ("right-handed")
occur in bacterial envelopes and some
antibiotics.
[5][6]
20 of the 23 proteinogenic
amino acids are encoded directly by triplet
codons in the genetic code and are known as
"standard" amino acids. The other three
("non-standard" or "non-canonical") are
pyrrolysine (found in methanogenic organisms
and other eukaryotes), selenocysteine (present
in many noneukaryotes as well as most
eukaryotes), and N-Formylmethionine. For
example, 25 human proteins include
selenocysteine (Sec) in their primary
structure,
[7]
and the structurally characterized
enzymes (selenoenzymes) employ Sec as the
catalytic moiety in their active sites.
[8]
Pyrollysine and selenocysteine are encoded via
variant codons; for example, selenocysteine is encoded by stop codon and SECIS element.
[9][10][]
CodontRNA
combinations not found in nature can also be used to "expand" the genetic code and create novel proteins known as
alloproteins incorporating non-proteinogenic amino acids.
[][][]
Many important proteinogenic and non-proteinogenic amino acids also play critical non-protein roles within the
body. For example: in the human brain, glutamate (standard glutamic acid) and gamma-amino-butyric acid
("GABA", non-standard gamma-amino acid) are respectively the main excitatory and inhibitory neurotransmitters;
[]
hydroxyproline (a major component of the connective tissue collagen) is synthesised from proline; the standard
amino acid glycine is used to synthesise porphyrins used in red blood cells; and the non-standard carnitine is used in
lipid transport.
9 of the 20 standard amino acids are called "essential" for humans because they cannot be created from other
compounds by the human body, and so must be taken in as food. Others may be conditionally essential for certain
ages or medical conditions. Essential amino acids may also differ between species.
[11]
Because of their biological significance, amino acids are important in nutrition and are commonly used in nutritional
supplements, fertilizers, and food technology. Industrial uses include the production of drugs, biodegradable plastics
and chiral catalysts.
Amino acid
86
History
The first few amino acids were discovered in the early 19th century. In 1806, French chemists Louis-Nicolas
Vauquelin and Pierre Jean Robiquet isolated a compound in asparagus that was subsequently named asparagine, the
first amino acid to be discovered.
[12][]
Cystine was discovered in 1810,
[13]
although its monomer, cysteine, remained
undiscovered until 1884.
[][14]
Glycine and leucine were discovered in 1820.
[15]
Usage of the term amino acid in the
English language is from 1898.
[16]
Proteins were found to yield amino acids after enzymatic digestion or acid
hydrolysis. In 1902, Emil Fischer and Franz Hofmeister proposed that proteins are the result of the formation of
bonds between the amino group of one amino acid with the carboxyl group of another, in a linear structure which
Fischer termed peptide.
[17]
General structure
Lysine with the carbon atoms in the side-chain labeled
In the structure shown at the top of the page, R
represents a side-chain specific to each amino acid.
The carbon atom next to the carboxyl group is called
the carbon and amino acids with a side-chain
bonded to this carbon are referred to as alpha amino
acids. These are the most common form found in
nature. In the alpha amino acids, the carbon is a
chiral carbon atom, with the exception of glycine.
[]
In
amino acids that have a carbon chain attached to the
carbon (such as lysine, shown to the right) the
carbons are labeled in order as , , , , and so
on.
[18]
In some amino acids, the amine group is
attached to the or -carbon, and these are therefore
referred to as beta or gamma amino acids.
Amino acids are usually classified by the properties
of their side-chain into four groups. The side-chain
can make an amino acid a weak acid or a weak base, and a hydrophile if the side-chain is polar or a hydrophobe if it
is nonpolar.
[]
The chemical structures of the 22 standard amino acids, along with their chemical properties, are
described more fully in the article on these proteinogenic amino acids.
The phrase "branched-chain amino acids" or BCAA refers to the amino acids having aliphatic side-chains that are
non-linear; these are leucine, isoleucine, and valine. Proline is the only proteinogenic amino acid whose side-group
links to the -amino group and, thus, is also the only proteinogenic amino acid containing a secondary amine at this
position.
[]
In chemical terms, proline is, therefore, an imino acid, since it lacks a primary amino group,
[19]
although it
is still classed as an amino acid in the current biochemical nomenclature,
[20]
and may also be called an "N-alkylated
alpha-amino acid".
[21]
Amino acid
87
The two enantiomers of alanine, D-Alanine and
L-Alanine
Isomerism
Of the standard -amino acids, all but glycine can exist in either of two
enantiomers, called L or D amino acids, which are mirror images of
each other (see also Chirality). While L-amino acids represent all of the
amino acids found in proteins during translation in the ribosome,
D-amino acids are found in some proteins produced by enzyme
posttranslational modifications after translation and translocation to the
endoplasmic reticulum, as in exotic sea-dwelling organisms such as
cone snails.
[22]
They are also abundant components of the
peptidoglycan cell walls of bacteria,
[23]
and D-serine may act as a
neurotransmitter in the brain.
[24]
The L and D convention for amino
acid configuration refers not to the optical activity of the amino acid itself, but rather to the optical activity of the
isomer of glyceraldehyde from which that amino acid can, in theory, be synthesized (D-glyceraldehyde is
dextrorotary; L-glyceraldehyde is levorotatory). In alternative fashion, the (S) and (R) designators are used to indicate
the absolute stereochemistry. Almost all of the amino acids in proteins are (S) at the carbon, with cysteine being
(R) and glycine non-chiral.
[25]
Cysteine is unusual since it has a sulfur atom at the second position in its side-chain,
which has a larger atomic mass than the groups attached to the first carbon, which is attached to the -carbon in the
other standard amino acids, thus the (R) instead of (S).
An amino acid in its (1) un-ionized and (2) zwitterionic forms
Zwitterions
The amine and carboxylic acid
functional groups found in amino acids
allow them to have amphiprotic
properties.
[]
Carboxylic acid groups
(CO
2
H) can be deprotonated to
become negative carboxylates (CO
2

), and -amino groups (NH

2
) can be
protonated to become positive
-ammonium groups (
+
NH
3
). At pH
values greater than the pKa of the
carboxylic acid group (mean for the 20 common amino acids is about 2.2, see the table of amino acid structures
above), the negative carboxylate ion predominates. At pH values lower than the pKa of the -ammonium group
(mean for the 20 common -amino acids is about 9.4), the nitrogen is predominantly protonated as a positively
charged -ammonium group. Thus, at pH between 2.2 and 9.4, the predominant form adopted by -amino acids
contains a negative carboxylate and a positive -ammonium group, as shown in structure (2) on the right, so has net
zero charge. This molecular state is known as a zwitterion, from the German Zwitter meaning hermaphrodite or
hybrid.
[26]
Below pH 2.2, the predominant form will have a neutral carboxylic acid group and a positive
-ammonium ion (net charge +1), and above pH 9.4, a negative carboxylate and neutral -amino group (net charge
1). The fully neutral form (structure (1) on the right) is a very minor species in aqueous solution throughout the pH
range (less than 1 part in 10
7
). Amino acids also exist as zwitterions in the solid phase, and crystallize with salt-like
properties unlike typical organic acids or amines.
Amino acid
88
Isoelectric point
At pH values between the two pKa values, the zwitterion predominates, but coexists in dynamic equilibrium with
small amounts of net negative and net positive ions. At the exact midpoint between the two pKa values, the trace
amount of net negative and trace of net positive ions exactly balance, so that average net charge of all forms present
is zero.
[27]
This pH is known as the isoelectric point pI, so pI = (pKa
1
+ pKa
2
). The individual amino acids all have
slightly different pKa values, so have different isoelectric points. For amino acids with charged side-chains, the pKa
of the side-chain is involved. Thus for Asp, Glu with negative side-chains, pI = (pKa
1
+ pKa
R
), where pKa
R
is the
side-chain pKa. Cysteine also has potentially negative side-chain with pKa
R
= 8.14, so pI should be calculated as for
Asp and Glu, even though the side-chain is not significantly charged at neutral pH. For His, Lys, and Arg with
positive side-chains, pI = (pKa
R
+ pKa
2
). Amino acids have zero mobility in electrophoresis at their isoelectric
point, although this behaviour is more usually exploited for peptides and proteins than single amino acids.
Zwitterions have minimum solubility at their isolectric point and some amino acids (in particular, with non-polar
side-chains) can be isolated by precipitation from water by adjusting the pH to the required isoelectric point.
Occurrence and functions in biochemistry
A polypeptide is an unbranched chain of amino acids.
Standard amino acids
Amino acids are the structural units
(monomers) that make up proteins. They
join together to form short polymer chains
called peptides or longer chains called either
polypeptides or proteins. These polymers
are linear and unbranched, with each amino
acid within the chain attached to two
neighboring amino acids. The process of
making proteins is called translation and
involves the step-by-step addition of amino
acids to a growing protein chain by a
ribozyme that is called a ribosome.
[28]
The
order in which the amino acids are added is read through the genetic code from an mRNA template, which is a RNA
copy of one of the organism's genes.
Twenty-two amino acids are naturally incorporated into polypeptides and are called proteinogenic or natural amino
acids.
[]
Of these, 20 are encoded by the universal genetic code. The remaining 2, selenocysteine and pyrrolysine, are
incorporated into proteins by unique synthetic mechanisms. Selenocysteine is incorporated when the mRNA being
translated includes a SECIS element, which causes the UGA codon to encode selenocysteine instead of a stop
codon.
[29]
Pyrrolysine is used by some methanogenic archaea in enzymes that they use to produce methane. It is
coded for with the codon UAG, which is normally a stop codon in other organisms.
[30]
This UAG codon is followed
by a PYLIS downstream sequence.
[]
Amino acid
89
The amino acid selenocysteine
Non-standard amino acids
Aside from the 22 standard amino acids, there are many
other amino acids that are called non-proteinogenic or
non-standard. Those either are not found in proteins
(for example carnitine, GABA), or are not produced
directly and in isolation by standard cellular machinery
(for example, hydroxyproline and selenomethionine).
Non-standard amino acids that are found in proteins are
formed by post-translational modification, which is
modification after translation during protein synthesis.
These modifications are often essential for the function
or regulation of a protein; for example, the
carboxylation of glutamate allows for better binding of
calcium cations,
[31]
and the hydroxylation of proline is
critical for maintaining connective tissues.
[32]
Another
example is the formation of hypusine in the translation
initiation factor EIF5A, through modification of a
lysine residue.
[33]
Such modifications can also
determine the localization of the protein, e.g., the
addition of long hydrophobic groups can cause a
protein to bind to a phospholipid membrane.
[34]
-alanine and its -alanine isomer
Some nonstandard amino acids are not found in
proteins. Examples include lanthionine,
2-aminoisobutyric acid, dehydroalanine, and the
neurotransmitter gamma-aminobutyric acid.
Nonstandard amino acids often occur as intermediates
in the metabolic pathways for standard amino acids
for example, ornithine and citrulline occur in the urea
cycle, part of amino acid catabolism (see below).
[35]
A
rare exception to the dominance of -amino acids in
biology is the -amino acid beta alanine
(3-aminopropanoic acid), which is used in plants and microorganisms in the synthesis of pantothenic acid (vitamin
B
5
), a component of coenzyme A.
[36]
In human nutrition
When taken up into the human body from the diet, the 22 standard amino acids either are used to synthesize proteins
and other biomolecules or are oxidized to urea and carbon dioxide as a source of energy.
[37]
The oxidation pathway
starts with the removal of the amino group by a transaminase, the amino group is then fed into the urea cycle. The
other product of transamidation is a keto acid that enters the citric acid cycle.
[38]
Glucogenic amino acids can also be
converted into glucose, through gluconeogenesis.
[39]
Amino acid
90
Pyrrolysine trait is restricted to several microbes, and only one organism has both Pyl and Sec. Of the 22 standard
amino acids, 9 are called essential amino acids because the human body cannot synthesize them from other
compounds at the level needed for normal growth, so they must be obtained from food.
[40]
In addition, cysteine,
taurine, tyrosine, and arginine are considered semiessential amino-acids in children (though taurine is not technically
an amino acid), because the metabolic pathways that synthesize these amino acids are not fully developed.
[41][42]
The amounts required also depend on the age and health of the individual, so it is hard to make general statements
about the dietary requirement for some amino acids.
Essential Nonessential
Histidine Alanine
Isoleucine Arginine*
Leucine Asparagine
Lysine Aspartic acid
Methionine Cysteine*
Phenylalanine Glutamic acid
Threonine Glutamine*
Tryptophan Glycine
Valine Ornithine*
Proline*
Serine*
Tyrosine*
(*) Essential only in certain cases.
[43][44]
Classification of Amino Acids
Although there are many ways to classify amino acids, these molecules can be assorted into six main groups, on the
basis of their structure and the general chemical characteristics of their R groups.
Class Name of the amino acids
Aliphatic Glycine, Alanine, Valine, Leucine, Isoleucine
Hydroxyl or Sulfur-containing Serine, Cysteine, Threonine, Methionine
Cyclic Proline
Aromatic Phenylalanine, Tyrosine, Tryptophan
Basic Histidine, Lysine, Arginine
Acidic and their Amide Aspartate, Glutamate, Asparagine, Glutamine
Amino acid
91
Non-protein functions
In humans, non-protein amino acids also have important roles as metabolic intermediates, such as in the biosynthesis
of the neurotransmitter gamma-aminobutyric acid. Many amino acids are used to synthesize other molecules, for
example:
Tryptophan is a precursor of the neurotransmitter serotonin.
[45]
Tyrosine (and its precursor phenylalanine) are precursors of the catecholamine neurotransmitters dopamine,
epinephrine and norepinephrine.
Glycine is a precursor of porphyrins such as heme.
[46]
Arginine is a precursor of nitric oxide.
[47]
Ornithine and S-adenosylmethionine are precursors of polyamines.
[48]
Aspartate, glycine, and glutamine are precursors of nucleotides.
[49]
Phenylalanine is a precursor of various phenylpropanoids, which are important in plant metabolism.
However, not all of the functions of other abundant non-standard amino acids are known.
Some non-standard amino acids are used as defenses against herbivores in plants.
[]
For example canavanine is an
analogue of arginine that is found in many legumes,
[]
and in particularly large amounts in Canavalia gladiata (sword
bean).
[50]
This amino acid protects the plants from predators such as insects and can cause illness in people if some
types of legumes are eaten without processing.
[51]
The non-protein amino acid mimosine is found in other species of
legume, particularly Leucaena leucocephala.
[52]
This compound is an analogue of tyrosine and can poison animals
that graze on these plants.
Uses in technology
Amino acids are used for a variety of applications in industry, but their main use is as additives to animal feed. This
is necessary, since many of the bulk components of these feeds, such as soybeans, either have low levels or lack
some of the essential amino acids: Lysine, methionine, threonine, and tryptophan are most important in the
production of these feeds.
[]
In this industry, amino acids are also used to chelate metal cations in order to improve the
absorption of minerals from supplements, which may be required to improve the health or production of these
animals.
[53]
The food industry is also a major consumer of amino acids, in particular, glutamic acid, which is used as a flavor
enhancer,
[]
and Aspartame (aspartyl-phenylalanine-1-methyl ester) as a low-calorie artificial sweetener.
[54]
Similar
technology to that used for animal nutrition is employed in the human nutrition industry to alleviate symptoms of
mineral deficiencies, such as anemia, by improving mineral absorption and reducing negative side effects from
inorganic mineral supplementation.
[55]
The chelating ability of amino acids has been used in fertilizers for agriculture to facilitate the delivery of minerals to
plants in order to correct mineral deficiencies, such as iron chlorosis. These fertilizers are also used to prevent
deficiencies from occurring and improving the overall health of the plants.
[56]
The remaining production of amino
acids is used in the synthesis of drugs and cosmetics.
[]
Amino acid
92
Amino acid derivative Pharmaceutical application
5-HTP (5-hydroxytryptophan)
Experimental treatment for depression.
[57]
L-DOPA (L-dihydroxyphenylalanine)
Treatment for Parkinsonism.
[58]
Eflornithine
Drug that inhibits ornithine decarboxylase and is used in the treatment of sleeping sickness.
[59]
Expanded genetic code
Since 2001, 40 non-natural amino acids have been added into protein by creating a unique codon (recoding) and a
corresponding transfer-RNA:aminoacyl tRNA-synthetase pair to encode it with diverse physicochemical and
biological properties in order to be used as a tool to exploring protein structure and function or to create novel or
enhanced proteins.
[][]
Chemical building blocks
Amino acids are important as low-cost feedstocks. These compounds are used in chiral pool synthesis as
enantiomerically pure building blocks.
[]
Amino acids have been investigated as precursors chiral catalysts, e.g., for asymmetric hydrogenation reactions,
although no commercial applications exist.
[]
Biodegradable plastics
Amino acids are under development as components of a range of biodegradable polymers. These materials have
applications as environmentally friendly packaging and in medicine in drug delivery and the construction of
prosthetic implants. These polymers include polypeptides, polyamides, polyesters, polysulfides, and polyurethanes
with amino acids either forming part of their main chains or bonded as side-chains. These modifications alter the
physical properties and reactivities of the polymers.
[]
An interesting example of such materials is polyaspartate, a
water-soluble biodegradable polymer that may have applications in disposable diapers and agriculture.
[]
Due to its
solubility and ability to chelate metal ions, polyaspartate is also being used as a biodegradeable anti-scaling agent
and a corrosion inhibitor.
[60][]
In addition, the aromatic amino acid tyrosine is being developed as a possible
replacement for toxic phenols such as bisphenol A in the manufacture of polycarbonates.
[]
Reactions
As amino acids have both a primary amine group and a primary carboxyl group, these chemicals can undergo most
of the reactions associated with these functional groups. These include nucleophilic addition, amide bond formation
and imine formation for the amine group and esterification, amide bond formation and decarboxylation for the
carboxylic acid group.
[61]
The combination of these functional groups allow amino acids to be effective polydentate
ligands for metal-amino acid chelates.
[62]
The multiple side-chains of amino acids can also undergo chemical
reactions.
[63]
The types of these reactions are determined by the groups on these side-chains and are, therefore,
different between the various types of amino acid.
Amino acid
93
The Strecker amino acid synthesis
Chemical synthesis
Several methods exist to synthesize
amino acids. One of the oldest methods
begins with the bromination at the
-carbon of a carboxylic acid.
Nucleophilic substitution with
ammonia then converts the alkyl bromide to the amino acid.
[64]
In alternative fashion, the Strecker amino acid
synthesis involves the treatment of an aldehyde with potassium cyanide and ammonia, this produces an -amino
nitrile as an intermediate. Hydrolysis of the nitrile in acid then yields a -amino acid.
[65]
Using ammonia or
ammonium salts in this reaction gives unsubstituted amino acids, while substituting primary and secondary amines
will yield substituted amino acids.
[66]
Likewise, using ketones, instead of aldehydes, gives ,-disubstituted amino
acids.
[67]
The classical synthesis gives racemic mixtures of -amino acids as products, but several alternative
procedures using asymmetric auxiliaries
[68]
or asymmetric catalysts
[69][70]
have been developed.
[71]
At the current time, the most-adopted method is an automated synthesis on a solid support (e.g., polystyrene beads),
using protecting groups (e.g., Fmoc and t-Boc) and activating groups (e.g., DCC and DIC).
Peptide bond formation
The condensation of two amino acids to form a dipeptide through a peptide bond
As both the amine and carboxylic acid
groups of amino acids can react to
form amide bonds, one amino acid
molecule can react with another and
become joined through an amide
linkage. This polymerization of amino
acids is what creates proteins. This
condensation reaction yields the newly
formed peptide bond and a molecule of
water. In cells, this reaction does not
occur directly; instead the amino acid
is first activated by attachment to a
transfer RNA molecule through an
ester bond. This aminoacyl-tRNA is
produced in an ATP-dependent
reaction carried out by an aminoacyl
tRNA synthetase.
[72]
This
aminoacyl-tRNA is then a substrate for
the ribosome, which catalyzes the
attack of the amino group of the elongating protein chain on the ester bond.
[73]
As a result of this mechanism, all
proteins made by ribosomes are synthesized starting at their N-terminus and moving towards their C-terminus.
However, not all peptide bonds are formed in this way. In a few cases, peptides are synthesized by specific enzymes.
For example, the tripeptide glutathione is an essential part of the defenses of cells against oxidative stress. This
peptide is synthesized in two steps from free amino acids.
[74]
In the first step gamma-glutamylcysteine synthetase
condenses cysteine and glutamic acid through a peptide bond formed between the side-chain carboxyl of the
glutamate (the gamma carbon of this side-chain) and the amino group of the cysteine. This dipeptide is then
condensed with glycine by glutathione synthetase to form glutathione.
[75]
Amino acid
94
In chemistry, peptides are synthesized by a variety of reactions. One of the most-used in solid-phase peptide
synthesis uses the aromatic oxime derivatives of amino acids as activated units. These are added in sequence onto the
growing peptide chain, which is attached to a solid resin support.
[76]
The ability to easily synthesize vast numbers of
different peptides by varying the types and order of amino acids (using combinatorial chemistry) has made peptide
synthesis particularly important in creating libraries of peptides for use in drug discovery through high-throughput
screening.
[77]
Biosynthesis
In plants, nitrogen is first assimilated into organic compounds in the form of glutamate, formed from
alpha-ketoglutarate and ammonia in the mitochondrion. In order to form other amino acids, the plant uses
transaminases to move the amino group to another alpha-keto carboxylic acid. For example, aspartate
aminotransferase converts glutamate and oxaloacetate to alpha-ketoglutarate and aspartate.
[78]
Other organisms use
transaminases for amino acid synthesis, too.
Nonstandard amino acids are usually formed through modifications to standard amino acids. For example,
homocysteine is formed through the transsulfuration pathway or by the demethylation of methionine via the
intermediate metabolite S-adenosyl methionine,
[]
while hydroxyproline is made by a posttranslational modification
of proline.
[79]
Microorganisms and plants can synthesize many uncommon amino acids. For example, some microbes make
2-aminoisobutyric acid and lanthionine, which is a sulfide-bridged derivative of alanine. Both of these amino acids
are found in peptidic lantibiotics such as alamethicin.
[80]
While in plants, 1-aminocyclopropane-1-carboxylic acid is
a small disubstituted cyclic amino acid that is a key intermediate in the production of the plant hormone ethylene.
[81]
Catabolism
Catabolism of proteinogenic amino acids. Amino acids can be classified according
to the properties of their main products as either of the following:
[82]
*
Glucogenic, with the products having the ability to form glucose by
gluconeogenesis* Ketogenic, with the products not having the ability to form
glucose. These products may still be used for ketogenesis or lipid synthesis.*
Amino acids catabolized into both glucogenic and ketogenic products.
Degradation of an amino acid often involves
deamination by moving its amino group to
alpha-ketoglutarate, forming glutamate. This
process involves transaminases, often the
same as those used in amination during
synthesis. In many vertebrates, the amino
group is then removed through the urea
cycle and is excreted in the form of urea.
However, amino acid degradation can
produce uric acid or ammonia instead. For
example, serine dehydratase converts serine
to pyruvate and ammonia.
[83]
After removal
of one or more amino groups, the remainder
of the molecule can sometimes be used to
synthesize new amino acids, or it can be
used for energy by entering glycolysis or the
citric acid cycle, as detailed in image at
right.
Physicochemical properties of amino acids
Amino acid
95
The 20 amino acids encoded directly by the genetic code can be divided into several groups based on their
properties. Important factors are charge, hydrophilicity or hydrophobicity, size, and functional groups.
[]
These
properties are important for protein structure and proteinprotein interactions. The water-soluble proteins tend to
have their hydrophobic residues (Leu, Ile, Val, Phe, and Trp) buried in the middle of the protein, whereas
hydrophilic side-chains are exposed to the aqueous solvent. The integral membrane proteins tend to have outer rings
of exposed hydrophobic amino acids that anchor them into the lipid bilayer. In the case part-way between these two
extremes, some peripheral membrane proteins have a patch of hydrophobic amino acids on their surface that locks
onto the membrane. In similar fashion, proteins that have to bind to positively charged molecules have surfaces rich
with negatively charged amino acids like glutamate and aspartate, while proteins binding to negatively charged
molecules have surfaces rich with positively charged chains like lysine and arginine. There are different
hydrophobicity scales of amino acid residues.
[84]
Some amino acids have special properties such as cysteine, that can form covalent disulfide bonds to other cysteine
residues, proline that forms a cycle to the polypeptide backbone, and glycine that is more flexible than other amino
acids.
Many proteins undergo a range of posttranslational modifications, when additional chemical groups are attached to
the amino acids in proteins. Some modifications can produce hydrophobic lipoproteins,
[85]
or hydrophilic
glycoproteins.
[86]
These type of modification allow the reversible targeting of a protein to a membrane. For example,
the addition and removal of the fatty acid palmitic acid to cysteine residues in some signaling proteins causes the
proteins to attach and then detach from cell membranes.
[87]
Table of standard amino acid abbreviations and properties
Amino Acid
3-Letter
[]
1-Letter
[]
Side-chain
polarity
[]
Side-chain charge
(pH 7.4)
[]
Hydropathy
index
[88]
Absorbance

max
(nm)
[]
at
max
(x10
3
M
1
cm
1
)
[]
Alanine Ala A nonpolar neutral 1.8
Arginine Arg R Basic polar positive 4.5
Asparagine Asn N polar neutral 3.5
Aspartic acid Asp D acidic polar negative 3.5
Cysteine Cys C nonpolar neutral 2.5 250 0.3
Glutamic acid Glu E acidic polar negative 3.5
Glutamine Gln Q polar neutral 3.5
Glycine Gly G nonpolar neutral 0.4
Histidine His H Basic polar positive(10%)
neutral(90%)
3.2 211 5.9
Isoleucine Ile I nonpolar neutral 4.5
Leucine Leu L nonpolar neutral 3.8
Lysine Lys K Basic polar positive 3.9
Methionine Met M nonpolar neutral 1.9
Phenylalanine Phe F nonpolar neutral 2.8 257, 206, 188 0.2, 9.3, 60.0
Proline Pro P nonpolar neutral 1.6
Serine Ser S polar neutral 0.8
Threonine Thr T polar neutral 0.7
Tryptophan Trp W nonpolar neutral 0.9 280, 219 5.6, 47.0
Amino acid
96
Tyrosine Tyr Y polar neutral 1.3 274, 222, 193 1.4, 8.0, 48.0
Valine Val V nonpolar neutral 4.2
Two additional amino acids are in some species coded for by codons that are usually interpreted as stop codons:
21st and 22nd amino acids 3-Letter 1-Letter
Selenocysteine Sec U
Pyrrolysine Pyl O
In addition to the specific amino acid codes, placeholders are used in cases where chemical or crystallographic
analysis of a peptide or protein cannot conclusively determine the identity of a residue.
Ambiguous Amino Acids 3-Letter 1-Letter
Asparagine or aspartic acid Asx B
Glutamine or glutamic acid Glx Z
Leucine or Isoleucine Xle J
Unspecified or unknown amino acid Xaa X
Unk is sometimes used instead of Xaa, but is less standard.
In addition, many non-standard amino acids have a specific code. For example, several peptide drugs, such as
Bortezomib and MG132, are artificially synthesized and retain their protecting groups, which have specific codes.
Bortezomib is Pyz-Phe-boroLeu, and MG132 is Z-Leu-Leu-Leu-al. To aid in the analysis of protein structure,
photocrosslinking amino acid analogues are available. These include photoleucine (pLeu) and photomethionine
(pMet).
[89]
References and notes
[2] Human nutrition in the developing world (http:/ / www. fao. org/ docrep/ W0073E/ w0073e04. htm#P1625_217364) United Nations Food
and Agriculture Organization, ch.8
[3] Proline is an exception to this general formula. It lacks the NH
2
group because of the cyclization of the side-chain and is known as an imino
acid; it falls under the category of special structured amino acids.
[4] INTRODUCING AMINO ACIDS (http:/ / www. chemguide. co. uk/ organicprops/ aminoacids/ background. html)
[5] "Biochemical pathways: an atlas of biochemistry and molecular biology" Michal, p.5
[7] Kryukov GV, Castellano S, Novoselov SV, Lobanov AV, Zehtab O, Guigo R, et al. Characterization of mammalian selenoproteomes.
Science. 2003;300:14391443.
[8] [8] Gromer, S., Urig, S., Becker, K. (2004) The Thioredoxin System - From Science to Clinic. Medicinal Research Reviews. 24(1):40-89.
[9] Modeling Electrostatic Contributions to Protein Folding and Binding (http:/ / books. google. com/ books?id=BDn-AI_YBlMC& pg=PA1&
lpg=PA1& ots=WSsFhHJwDy& sig=jkSLFr7AK8iu6OhdX7KOc10eKRY& hl=en& sa=X& ei=gshLUOWZLIin0AXRm4GoBg) Tjong,
p.1 footnote
[10] Frontiers in Drug Design and Discovery (http:/ / books. google. com/ books?id=VoJw6fIISSkC& pg=PA299& lpg=PA299&
ots=C20L115r05& sig=4cix7yKNlod3xbzy2TWiOzEe6As& hl=en& sa=X& ei=H81LUL6MOfC10QX4wYG4Cw& ved=0CIcBEOgBMA8)
ed. Atta-Ur-Rahman & others, p.299
[11] For example, ruminants such as cows obtain a number of amino acids via microbes in the first two stomach chambers.
[82] Stipanuk, M. H. (2006). Biochemical, physiological, & molecular aspects of human nutrition (2 ed.): Saunders Elsevier.
Amino acid
97
Further reading
Tymoczko, John L. (2012). "Protein Composition and Structure". Biochemistry. New York: W. H. Freeman and
company. pp.2831. ISBN9781429229364.
Doolittle, Russell F. (1989). "Redundancies in protein sequences". In Fasman, G.D.. Predictions of Protein
Structure and the Principles of Protein Conformation. New York: Plenum Press. pp.599623.
ISBN978-0-306-43131-9. LCCN 89008555 (http:/ / lccn. loc. gov/ 89008555).
Nelson, David L.; Cox, Michael M. (2000). Lehninger Principles of Biochemistry (3rd ed.). Worth Publishers.
ISBN978-1-57259-153-0. LCCN 99049137 (http:/ / lccn. loc. gov/ 99049137).
Meierhenrich, Uwe (2008). Amino acids and the asymmetry of life (http:/ / rogov. zwz. ru/ Macroevolution/
amino. pdf) (PDF, 11.2 MB). Berlin: Springer Verlag. ISBN978-3-540-76885-2. LCCN 2008930865 (http:/ /
lccn. loc. gov/ 2008930865).
Morelli, Robert J. (1952). Studies of amino acid absorption from the small intestine. San Francisco.
External links
The origin of the single-letter code for the amino acids (http:/ / www. biology. arizona. edu/ biochemistry/
problem_sets/ aa/ Dayhoff. html)
Properties of the twenty amino acids
Proteinogenic amino acids are amino acids that are precursors to proteins, and are produced by cellular machinery
coded for in the genetic code
[1]
of any organism. There are 22 standard amino acids, but only 21 are found in
eukaryotes. Of the 22, selenocysteine and pyrrolysine are incorporated into proteins by distinctive biosynthetic
mechanisms. The other 20 are directly encoded by the universal genetic code. Humans can synthesize 11 of these 20
from each other or from other molecules of intermediary metabolism. The other 9 must be consumed (usually as
their protein derivatives) in the diet and so are thus called essential amino acids. The essential amino acids are
histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine.
The word proteinogenic means "protein building". Proteinogenic amino acids can be condensed into a polypeptide
(the subunit of a protein) through a process called translation (the second stage of protein biosynthesis, part of the
overall process of gene expression).
In contrast, non-proteinogenic amino acids are either not incorporated in proteins (like GABA, L-DOPA, or
triiodothyronine), or are not produced directly and in isolation by standard cellular machinery (like hydroxyproline
and selenomethionine). The latter often results from posttranslational modification of proteins.
The proteinogenic amino acids have been found to be related to the set of amino acids that can be recognized by
ribozyme auto-aminoacylation systems.
[2]
Thus, non-proteinogenic amino acids would have been excluded by the
contingent evolutionary success of nucleotide-based life forms. Other reasons have been offered to explain why
certain specific non-proteinogenic amino acids turns into proteins: for example, ornithine and homoserine cyclize
against the peptide backbone and fragment the protein with relatively short half-lives, while others are toxic because
they can be mistakenly incorporated into proteins, such as the arginine analog canavanine.
Non-proteinogenic amino acids are incorporated in nonribosomal peptides, which are not produced by the ribosome
during translation.
Properties of the twenty amino acids
98
Structures
The following illustrates the structures and abbreviations of the 21 amino acids that are directly encoded for protein
synthesis by the genetic code of eukaryotes. The structures given below are standard chemical structures, not the
typical zwitterion forms that exist in aqueous solutions.
Grouped table of 21 amino acids' structures, nomenclature, and their side
groups' pKa's.
L-Alanine
(Ala/A)
L-Arginine
(Arg/R)
L-Asparagine
(Asn/N)
L-Aspartic acid
(Asp/D)
L-Cysteine
(Cys/C)
L-Glutamic acid
(Glu/E)
L-Glutamine
(Gln/Q)
Glycine
(Gly/G)
Properties of the twenty amino acids
99
L-Histidine
(His/H)
L-Isoleucine
(Ile/I)
L-Leucine
(Leu/L)
L-Lysine
(Lys/K)
L-Methionine
(Met/M)
L-Phenylalanine
(Phe/F)
L-Proline
(Pro/P)
L-Serine
(Ser/S)
L-Threonine
(Thr/T)
L-Tryptophan
(Trp/W)
L-Tyrosine
(Tyr/Y)
L-Valine
(Val/V)
IUPAC/IUBMB now also recommends standard abbreviations for the one amino acid :
L-Pyrrolysine
(Pyl/O)
Properties of the twenty amino acids
100
Non-specific abbreviations
Sometimes the specific identity of an amino acid cannot be determined unambiguously. Certain protein sequencing
techniques do not distinguish among certain pairs. Thus, the following codes are used:
Asx (B) is "asparagine or aspartic acid"
Glx (Z) is "glutamic acid or glutamine"
Xle (J) is "leucine or isoleucine"
In addition, the symbol X is used to indicate an amino acid that is completely unidentified.
Chemical properties
Following is a table listing the one-letter symbols, the three-letter symbols, and the chemical properties of the
side-chains of the standard amino acids. The masses listed are based on weighted averages of the elemental isotopes
at their natural abundances. Note that forming a peptide bond results in elimination of a molecule of water, so the
mass of an amino acid unit within a protein chain is reduced by 18.01524 Da.
General chemical properties
Amino Acid Short Abbrev. Avg. Mass (Da) pI pK
1
(-COOH)
pK
2
(-
+
NH
3
)
Alanine A Ala 89.09404 6.01 2.35 9.87
Cysteine C Cys 121.15404 5.05 1.92 10.70
Aspartic acid D Asp 133.10384 2.85 1.99 9.90
Glutamic acid E Glu 147.13074 3.15 2.10 9.47
Phenylalanine F Phe 165.19184 5.49 2.20 9.31
Glycine G Gly 75.06714 6.06 2.35 9.78
Histidine H His 155.15634 7.60 1.80 9.33
Isoleucine I Ile 131.17464 6.05 2.32 9.76
Lysine K Lys 146.18934 9.60 2.16 9.06
Leucine L Leu 131.17464 6.01 2.33 9.74
Methionine M Met 149.20784 5.74 2.13 9.28
Asparagine N Asn 132.11904 5.41 2.14 8.72
Pyrrolysine O Pyl
Proline P Pro 115.13194 6.30 1.95 10.64
Glutamine Q Gln 146.14594 5.65 2.17 9.13
Arginine R Arg 174.20274 10.76 1.82 8.99
Serine S Ser 105.09344 5.68 2.19 9.21
Threonine T Thr 119.12034 5.60 2.09
Valine V Val 117.14784 6.00 2.39 9.74
Tryptophan W Trp 204.22844 5.89 2.46 9.41
Tyrosine Y Tyr 181.19124 5.64 2.20 9.21
Properties of the twenty amino acids
101
Side chain properties
Amino Acid Short Abbrev. Side chain Hydro-
phobic
pKa Polar pH Small Tiny Aromatic
or Aliphatic
van der
Waals
volume
Alanine A Ala -CH
3
X - - - X X - 67
Cysteine C Cys -CH
2
SH - 8.18 - acidic X X - 86
Aspartic acid D Asp -CH
2
COOH - 3.90 X acidic X - - 91
Glutamic acid E Glu -CH
2
CH
2
COOH - 4.07 X acidic - - - 109
Phenylalanine F Phe -CH
2
C
6
H
5
X - - - - - Aromatic 135
Glycine G Gly -H X - - - X X - 48
Histidine H His -CH
2
-C
3
H
3
N
2
- 6.04 X weak basic - - Aromatic 118
Isoleucine I Ile -CH(CH
3
)CH
2
CH
3
X - - - - - Aliphatic 124
Lysine K Lys -(CH
2
)
4
NH
2
- 10.54 X basic - - - 135
Leucine L Leu -CH
2
CH(CH
3
)
2
X - - - - - Aliphatic 124
Methionine M Met -CH
2
CH
2
SCH
3
X - - - - - - 124
Asparagine N Asn -CH
2
CONH
2
- 5.41 X weak basic X - - 96
Pyrrolysine O Pyl -(CH
2
)
4
NHCOC
4
H
5
NCH
3
- - X weak basic - - -
Proline P Pro -CH
2
CH
2
CH
2
- X - - - X - - 90
Glutamine Q Gln -CH
2
CH
2
CONH
2
- - X weak basic - - - 114
Arginine R Arg -(CH
2
)
3
NH-C(NH)NH
2
- 12.48 X strongly basic - - - 148
Serine S Ser -CH
2
OH - 5.68 X weak acidic X X - 73
Threonine T Thr -CH(OH)CH
3
- 5.53 - weak acidic X - - 93
Valine V Val -CH(CH
3
)
2
X - - - X - Aliphatic 105
Tryptophan W Trp -CH
2
C
8
H
6
N - 5.885 X weak basic - - Aromatic 163
Tyrosine Y Tyr -CH
2
-C
6
H
4
OH - 10.46 X weak acidic - - Aromatic 141
Note: The pKa values of amino acids are typically slightly different when the amino acid is inside a protein. Protein
pKa calculations are sometimes used to calculate the change in the pKa value of an amino acid in this situation.
Gene expression and biochemistry
Amino Acid Short Abbrev. Codon(s) Occurrence
in human
proteins
(%)
Essential in humans
Alanine A Ala GCU, GCC, GCA, GCG 7.8 No
Cysteine C Cys UGU, UGC 1.9 Conditionally
Aspartic acid D Asp GAU, GAC 5.3 No
Glutamic acid E Glu GAA, GAG 6.3 Conditionally
Phenylalanine F Phe UUU, UUC 3.9 Yes
Glycine G Gly GGU, GGC, GGA, GGG 7.2 Conditionally
Histidine H His CAU, CAC 2.3 Yes
Properties of the twenty amino acids
102
Isoleucine I Ile AUU, AUC, AUA 5.3 Yes
Lysine K Lys AAA, AAG 5.9 Yes
Leucine L Leu UUA, UUG, CUU, CUC, CUA, CUG 9.1 Yes
Methionine M Met AUG 2.3 Yes
Asparagine N Asn AAU, AAC 4.3 No
Pyrrolysine O Pyl UAG* 0 No
Proline P Pro CCU, CCC, CCA, CCG 5.2 No
Glutamine Q Gln CAA, CAG 4.2 No
Arginine R Arg CGU, CGC, CGA, CGG, AGA, AGG 5.1 Conditionally
Serine S Ser UCU, UCC, UCA, UCG, AGU, AGC 6.8 No
Threonine T Thr ACU, ACC, ACA, ACG 5.9 Yes
Valine V Val GUU, GUC, GUA, GUG 6.6 Yes
Tryptophan W Trp UGG 1.4 Yes
Tyrosine Y Tyr UAU, UAC 3.2 Conditionally
Stop codon - Term UAA, UAG, UGA - -
* UAG is normally the amber stop codon, but encodes pyrrolysine if a PYLIS element is present.
** UGA is normally the opal (or umber) stop codon, but encodes selenocysteine if a SECIS element is present.
The stop codon is not an amino acid, but is included for completeness.
UAG and UGA do not always act as stop codons (see above).
An essential amino acid cannot be synthesized in humans and must, therefore, be supplied in the diet.
Conditionally essential amino acids are not normally required in the diet, but must be supplied exogenously to
specific populations that do not synthesize it in adequate amounts.
Mass spectrometry
In mass spectrometry of peptides and proteins, it is useful to know the masses of the residues. The mass of the
peptide or protein is the sum of the residue masses plus the mass of water.
[3]
Amino Acid Short Abbrev. Formula Mon. Mass (Da) Avg. Mass (Da)
Alanine A Ala C
3
H
5
NO 71.03711 71.0788
Cysteine C Cys C
3
H
5
NOS 103.00919 103.1388
Aspartic acid D Asp C
4
H
5
NO
3
115.02694 115.0886
Glutamic acid E Glu C
5
H
7
NO
3
129.04259 129.1155
Phenylalanine F Phe C
9
H
9
NO 147.06841 147.1766
Glycine G Gly C
2
H
3
NO 57.02146 57.0519
Histidine H His C
6
H
7
N
3
O 137.05891 137.1411
Isoleucine I Ile C
6
H
11
NO 113.08406 113.1594
Lysine K Lys C
6
H
12
N
2
O 128.09496 128.1741
Leucine L Leu C
6
H
11
NO 113.08406 113.1594
Methionine M Met C
5
H
9
NOS 131.04049 131.1986
Asparagine N Asn C
4
H
6
N
2
O
2
114.04293 114.1039
Pyrrolysine O Pyl C
12
H
21
N
3
O
3
255.15829 255.3172
Properties of the twenty amino acids
103
Proline P Pro C
5
H
7
NO 97.05276 97.1167
Glutamine Q Gln C
5
H
8
N
2
O
2
128.05858 128.1307
Arginine R Arg C
6
H
12
N
4
O 156.10111 156.1875
Serine S Ser C
3
H
5
NO
2
87.03203 87.0782
Threonine T Thr C
4
H
7
NO
2
101.04768 101.1051
Valine V Val C
5
H
9
NO 99.06841 99.1326
Tryptophan W Trp C
11
H
10
N
2
O 186.07931 186.2132
Tyrosine Y Tyr C
9
H
9
NO
2
163.06333 163.1760
Monoisotopic mass
Stoichiometry and metabolic cost in cell
Following table lists the abundance of amino acids in E.coli cell and the metabolic cost (ATP) for synthesis the
amino acids. Negative numbers indicate the metabolic processes are energy favorable and do not cost net ATP of the
cell.
[4]
Note that the abundance of amino acids include amino acids in free-form and in polymerization form
(proteins).
Amino acid
Abundance
(# of molecules
(10
8
)
per E. coli cell)
ATP cost in
synthesis
under aerobic
condition
ATP cost in
synthesis
under anaerobic
condition
Alanine 2.9 -1 1
Cysteine 0.52 11 15
Aspartic acid 1.4 0 2
Glutamic acid 1.5 -7 -1
Phenylalanine 1.1 -6 2
Glycine 3.5 -2 2
Histidine 0.54 1 7
Isoleucine 1.7 7 11
Lysine 2.0 5 9
Leucine 2.6 -9 1
Methionine 0.88 21 23
Asparagine 1.4 3 5
Proline 1.3 -2 4
Glutamine 1.5 -6 0
Arginine 1.7 5 13
Serine 1.2 -2 2
Threonine 1.5 6 8
Tryptophan 0.33 -7 7
Tyrosine 0.79 -8 2
Valine 2.4 -2 2
Properties of the twenty amino acids
104
Remarks
Amino Acid Abbrev. Remarks
Alanine A Ala Very abundant, very versatile. More stiff than glycine, but small enough to pose only small steric limits for the
protein conformation. It behaves fairly neutrally, and can be located in both hydrophilic regions on the protein
outside and the hydrophobic areas inside.
Asparagine or
aspartic acid
B Asx A placeholder when either amino acid may occupy a position.
Cysteine C Cys The sulfur atom bonds readily to heavy metal ions. Under oxidizing conditions, two cysteines can join together in a
disulfide bond to form the amino acid cystine. When cystines are part of a protein, insulin for example, the tertiary
structure is stabilized, which makes the protein more resistant to denaturation; therefore, disulfide bonds are common
in proteins that have to function in harsh environments including digestive enzymes (e.g., pepsin and chymotrypsin)
and structural proteins (e.g., keratin). Disulfides are also found in peptides too small to hold a stable shape on their
own (e.g. insulin).
Aspartic acid D Asp Behaves similarly to glutamic acid. Carries a hydrophilic acidic group with strong negative charge. Usually is located
on the outer surface of the protein, making it water-soluble. Binds to positively-charged molecules and ions, often
used in enzymes to fix the metal ion. When located inside of the protein, aspartate and glutamate are usually paired
with arginine and lysine.
Glutamic acid E Glu Behaves similarly to aspartic acid. Has longer, slightly more flexible side chain.
Phenylalanine F Phe Essential for humans. Phenylalanine, tyrosine, and tryptophan contain large rigid aromatic group on the side-chain.
These are the biggest amino acids. Like isoleucine, leucine and valine, these are hydrophobic and tend to orient
towards the interior of the folded protein molecule. Phenylalanine can be converted into Tyrosine.
Glycine G Gly Because of the two hydrogen atoms at the carbon, glycine is not optically active. It is the smallest amino acid,
rotates easily, adds flexibility to the protein chain. It is able to fit into the tightest spaces, e.g., the triple helix of
collagen. As too much flexibility is usually not desired, as a structural component it is less common than alanine.
Histidine H His In even slightly acidic conditions protonation of the nitrogen occurs, changing the properties of histidine and the
polypeptide as a whole. It is used by many proteins as a regulatory mechanism, changing the conformation and
behavior of the polypeptide in acidic regions such as the late endosome or lysosome, enforcing conformation change
in enzymes. However only a few histidines are needed for this, so it is comparatively scarce.
Isoleucine I Ile Essential for humans. Isoleucine, leucine and valine have large aliphatic hydrophobic side chains. Their molecules
are rigid, and their mutual hydrophobic interactions are important for the correct folding of proteins, as these chains
tend to be located inside of the protein molecule.
Leucine or
isoleucine
J Xle A placeholder when either amino acid may occupy a position
Lysine K Lys Essential for humans. Behaves similarly to arginine. Contains a long flexible side-chain with a positively-charged
end. The flexibility of the chain makes lysine and arginine suitable for binding to molecules with many negative
charges on their surfaces. E.g., DNA-binding proteins have their active regions rich with arginine and lysine. The
strong charge makes these two amino acids prone to be located on the outer hydrophilic surfaces of the proteins;
when they are found inside, they are usually paired with a corresponding negatively-charged amino acid, e.g.,
aspartate or glutamate.
Leucine L Leu Essential for humans. Behaves similar to isoleucine and valine. See isoleucine.
Methionine M Met Essential for humans. Always the first amino acid to be incorporated into a protein; sometimes removed after
translation. Like cysteine, contains sulfur, but with a methyl group instead of hydrogen. This methyl group can be
activated, and is used in many reactions where a new carbon atom is being added to another molecule.
Asparagine N Asn Similar to aspartic acid. Asn contains an amide group where Asp has a carboxyl.
Pyrrolysine O Pyl Similar to lysine, with a pyrroline ring attached.
Proline P Pro Contains an unusual ring to the N-end amine group, which forces the CO-NH amide sequence into a fixed
conformation. Can disrupt protein folding structures like helix or sheet, forcing the desired kink in the protein
chain. Common in collagen, where it often undergoes a posttranslational modification to hydroxyproline.
Properties of the twenty amino acids
105
Glutamine Q Gln Similar to glutamic acid. Gln contains an amide group where Glu has a carboxyl. Used in proteins and as a storage
for ammonia. The most abundant Amino Acid in the body.
Arginine R Arg Functionally similar to lysine.
Serine S Ser Serine and threonine have a short group ended with a hydroxyl group. Its hydrogen is easy to remove, so serine and
threonine often act as hydrogen donors in enzymes. Both are very hydrophilic, therefore the outer regions of soluble
proteins tend to be rich with them.
Threonine T Thr Essential for humans. Behaves similarly to serine.
Valine V Val Essential for humans. Behaves similarly to isoleucine and leucine. See isoleucine.
Tryptophan W Trp Essential for humans. Behaves similarly to phenylalanine and tyrosine (see phenylalanine). Precursor of serotonin.
Naturally fluorescent.
Unknown X Xaa Placeholder when the amino acid is unknown or unimportant.
Tyrosine Y Tyr Behaves similarly to phenylalanine (precursor to Tyrosine) and tryptophan (see phenylalanine). Precursor of melanin,
epinephrine, and thyroid hormones. Naturally fluorescent, although fluorescence is usually quenched by energy
transfer to tryptophans.
Glutamic acid
or glutamine
Z Glx A placeholder when either amino acid may occupy a position.
Catabolism
Amino acids can be classified according to the properties of their main products as either of the
following:
[5]
Glucogenic, with the products having the ability to form glucose by gluconeogenesisKetogenic,
with the products not having the ability to form glucose. These products may still be used for ketogenesis or
lipid synthesis.Amino acids catabolized into both glucogenic and ketogenic products.
Properties of the twenty amino acids
106
References
[4] Physical Biology of the Cell (Garland Science) p. 178
[5] [5] Chapter 20 (Amino Acid Degradation and Synthesis) in:
Nelson, David L.; Cox, Michael M. (2000). Lehninger Principles of Biochemistry (3rd ed.). Worth Publishers.
ISBN1-57259-153-6.
Kyte, J.; Doolittle, R. F. (1982). "A simple method for displaying the hydropathic character of a protein". J. Mol.
Biol. 157 (1): 105132. doi: 10.1016/0022-2836(82)90515-0 (http:/ / dx. doi. org/ 10. 1016/
0022-2836(82)90515-0). PMID 7108955 (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 7108955).
Meierhenrich, Uwe J. (2008). Amino acids and the asymmetry of life (1st ed.). Springer.
ISBN978-3-540-76885-2.
Myoglobin
107
Myoglobin
Myoglobin
Model of helical domains in myoglobin.
[1]
Available structures
PDB
Ortholog search: PDBe
[2]
, RCSB
[3]
List of PDB id codes
3RGK
[4]
Identifiers
Symbols
MB
[5]
; PVALB
External IDs
OMIM: 160000
[6]
MGI: 96922
[7]
HomoloGene: 3916
[8]
GeneCards: MB Gene
[9]
Gene Ontology
Molecular function
oxygen transporter activity
[10]
iron ion binding
[11]
oxygen binding
[12]
heme binding
[13]
Biological process
response to hypoxia
[14]
heart development
[15]
response to hormone stimulus
[16]
slow-twitch skeletal muscle fiber contraction
[17]
response to hydrogen peroxide
[18]
enucleate erythrocyte differentiation
[19]
brown fat cell differentiation
[20]
Sources: Amigo
[21]
/ QuickGO
[22]
RNA expression pattern
Myoglobin
108
More reference expression data
[23]
Orthologs
Species Human Mouse
Entrez
4151
[24]
17189
[25]
Ensembl
ENSG00000198125
[26]
ENSMUSG00000018893
[27]
UniProt
P02144
[28]
P04247
[29]
RefSeq (mRNA)
NM_005368
[30]
NM_001164047
[31]
RefSeq (protein)
NP_005359
[32]
NP_001157519
[33]
Location (UCSC)
Chr 22:
36 36.03 Mb
[34]
Chr 15:
77.02 77.05 Mb
[35]
PubMed search
[36] [37]
Myoglobin is an iron- and oxygen-binding protein found in the muscle tissue of vertebrates in general and in almost
all mammals. It is related to hemoglobin, which is the iron- and oxygen-binding protein in blood, specifically in the
red blood cells. The only time myoglobin is found in the bloodstream is when it is released following muscle injury.
It is an abnormal finding, and can be diagnostically relevant when found in blood.
[]
Myoglobin (abbreviated Mb) is a single-chain globular protein of 153
[]
or 154
[]
amino acids, containing a heme
(iron-containing porphyrin) prosthetic group in the center around which the remaining apoprotein folds. It has eight
alpha helices and a hydrophobic core. It has a molecular weight of 17,699 daltons (with heme), and is the primary
oxygen-carrying pigment of muscle tissues.
[]
Unlike the blood-borne hemoglobin, to which it is structurally related,
[]
this protein does not exhibit cooperative binding of oxygen, since positive cooperativity is a property of
multimeric/oligomeric proteins only. High concentrations of myoglobin in muscle cells allow organisms to hold their
breaths longer. Diving mammals such as whales and seals have muscles with particularly high myoglobin
abundance.
[]
Myoglobin was the first protein to have its three-dimensional structure revealed.
[38]
In 1958, John Kendrew and
associates successfully determined the structure of myoglobin by high-resolution X-ray crystallography.
[]
For this
discovery, John Kendrew shared the 1962 Nobel Prize in chemistry with Max Perutz.
[39]
Despite being one of the
most studied proteins in biology, its true physiological function is not yet conclusively established: mice genetically
engineered to lack myoglobin are viable, but showed a 30% reduction in cardiac systolic output. They adapted to this
deficiency through hypoxic genetic mechanisms and increased vasodilation.
[]
In humans myoglobin is encoded by
the MB gene.
[]
Myoglobin
109
Meat color
Myoglobin forms pigments responsible for making meat red. The color that meat takes is partly determined by the
oxidation states of the iron atom in myoglobin and the oxygen species attached to it. When meat is in its raw state,
the iron atom is in the +2 oxidation state, and is bound to a dioxygen molecule (O
2
). Meat cooked well done is
brown because the iron atom is now in the +3 oxidation state, having lost an electron, and is now coordinated by a
water molecule. Under some conditions, meat can also remain pink all through cooking, despite being heated to high
temperatures. If meat has been exposed to nitrites, it will remain pink because the iron atom is bound to NO, nitric
oxide (true of, e.g., corned beef or cured hams). Grilled meats can also take on a pink "smoke ring" that comes from
the iron binding to a molecule of carbon monoxide.
[40]
Raw meat packed in a carbon monoxide atmosphere also
shows this same pink "smoke ring" due to the same coordination chemistry. Notably, the surface of this raw meat
also displays the pink color, which is usually associated in consumers' minds with fresh meat. This artificially
induced pink color can persist in the meat for a very long time, reportedly up to one year.
[]
Hormel and Cargill are
both reported to use this meat-packing process, and meat treated this way has been in the consumer market since
2003.
[]
Myoglobin is found in Type I muscle, Type II A and Type II B, but most texts consider myoglobin not to be
found in smooth muscle.
Role in disease
Myoglobin is released from damaged muscle tissue (rhabdomyolysis), which has very high concentrations of
myoglobin. The released myoglobin is filtered by the kidneys but is toxic to the renal tubular epithelium and so may
cause acute renal failure.
[]
It is not the myoglobin itself that is toxic (it is a protoxin) but the ferrihemate portion that
is dissociated from myoglobin in acidic environments (e.g., acidic urine, lysosomes).
Myoglobin is a sensitive marker for muscle injury, making it a potential marker for heart attack in patients with chest
pain.
[]
However, elevated myoglobin has low specificity for acute myocardial infarction (AMI) and thus CK-MB,
cTnT, ECG, and clinical signs should be taken into account to make the diagnosis.
Structure, bonding and solubility
Myoglobin contains a porphyrin ring with an iron center. There is a proximal histidine group attached directly to the
iron center, and a distal histidine group on the opposite face, not bonded to the iron.
Many functional models of myoglobin have been studied. One of the most important is that of picket fence porphyrin
by James P. Collman. This model was used to show the importance of the distal prosthetic group. It serves three
functions:
1. To form hydrogen bonds with the dioxygen moiety, increasing the O
2
binding constant
2. To prevent the binding of carbon monoxide, whether from within or without the body. Carbon monoxide binds to
iron in an end-on fashion, and is hindered by the presence of the distal histidine, which forces it into a bent
conformation. CO binds to hemeWikipedia:Avoid weasel words 23,000 times better than O
2
, but only 200 times
better in hemoglobin and myoglobin. Oxygen binds in a bent fashion, which can fit with the distal histidine.
[]
3. To prevent irreversible dimerization of the oxymyoglobin with another deoxymyoglobin species
In chemistry studies, which mostly deal with organic compounds, myoglobin can be dissolved in protic solvents by
taking advantage of its structural and bonding characteristics. Dr. Katia C. S. Figueiredo and colleagues have studied
myoglobin's structural stability in organic media. In this study they studied the effect of pH, organic solvents, and
hydrophobic ion pairing on myoglobin's stability. This study has proved that the structure of myoglobin is least
altered at range of pH=5 to pH=7. Study of different solvents effect on myoglobin's structure demonstrated that
protic compounds have better performance as myoglobin solvents compared to aprotic ones. Dr. Figueiredo studied
three main organic functional groups of protic solvent including alcohols, glycols, and amide. The behavior of
myoglobin's solution in alcohols demonstrated a direct proportionality between chain branching and an inverse
Myoglobin
110
proportionality to the hydrocarbonic content. This study also showed that alcohols dissolve myoglobin with minor
modifications in the heme environment. Ethylene glycol and glycerol were the best solvents when making 50% of
the volume of an aqueous solution. Study of aprotic solvents demonstrated that high polar compounds such as
N-methylpyrrolidone and dimethyl sulfoxide dissolved myoglobin. However, they damaged the secondary structure
of myoglobin. The hydrophobic ion pairing technique showed that the superficial moiety of the protein can be
altered by adding very low amounts of SDS, or sodium dodecyl sulfate, which increased the solubility of myoglobin
in hexane.
[]
References
[1] [1] ;
[2] http:/ / www. ebi. ac. uk/ pdbe/ searchResults.html?display=both&
term=P02144%20or%20P02192%20or%20P04247%20or%20Q3UVB1%20or%20Q9QZ76%20or%20B8JLC7%20or%20Q6VN46
[3] http:/ / www. rcsb.org/ pdb/ search/ smartSubquery. do?smartSearchSubtype=UpAccessionIdQuery&
accessionIdList=P02144,P02192,P04247,Q3UVB1,Q9QZ76,B8JLC7,Q6VN46
[4] http:/ / www. rcsb.org/ pdb/ cgi/ explore.cgi?pdbId=3RGK
[5] http:/ / www. genenames.org/ data/ hgnc_data.php?hgnc_id=6915
[6] http:/ / omim. org/ entry/ 160000
[7] http:/ / www. informatics. jax. org/ searches/ accession_report. cgi?id=MGI:96922
[8] http:/ / www. ncbi. nlm.nih. gov/ entrez/ query.fcgi?cmd=Retrieve& db=homologene& dopt=HomoloGene& list_uids=3916
[9] http:/ / www. genecards.org/ cgi-bin/ carddisp.pl?id_type=entrezgene& id=4151
[10] http:/ / amigo. geneontology.org/ cgi-bin/ amigo/ go.cgi?view=details& search_constraint=terms& depth=0& query=GO:0005344
[11] http:/ / amigo. geneontology.org/ cgi-bin/ amigo/ go.cgi?view=details& search_constraint=terms& depth=0& query=GO:0005506
[12] http:/ / amigo. geneontology.org/ cgi-bin/ amigo/ go.cgi?view=details& search_constraint=terms& depth=0& query=GO:0019825
[13] http:/ / amigo. geneontology.org/ cgi-bin/ amigo/ go.cgi?view=details& search_constraint=terms& depth=0& query=GO:0020037
[14] http:/ / amigo. geneontology.org/ cgi-bin/ amigo/ go.cgi?view=details& search_constraint=terms& depth=0& query=GO:0001666
[15] http:/ / amigo. geneontology.org/ cgi-bin/ amigo/ go.cgi?view=details& search_constraint=terms& depth=0& query=GO:0007507
[16] http:/ / amigo. geneontology.org/ cgi-bin/ amigo/ go.cgi?view=details& search_constraint=terms& depth=0& query=GO:0009725
[17] http:/ / amigo. geneontology.org/ cgi-bin/ amigo/ go.cgi?view=details& search_constraint=terms& depth=0& query=GO:0031444
[18] http:/ / amigo. geneontology.org/ cgi-bin/ amigo/ go.cgi?view=details& search_constraint=terms& depth=0& query=GO:0042542
[19] http:/ / amigo. geneontology.org/ cgi-bin/ amigo/ go.cgi?view=details& search_constraint=terms& depth=0& query=GO:0043353
[20] http:/ / amigo. geneontology.org/ cgi-bin/ amigo/ go.cgi?view=details& search_constraint=terms& depth=0& query=GO:0050873
[21] http:/ / amigo. geneontology.org/ cgi-bin/ amigo/ gp-assoc. cgi?gp=UniProtKB:P02144
[22] http:/ / www.ebi. ac.uk/ QuickGO/ GProtein?ac=P02144
[23] http:/ / biogps. org/ gene/ 4151/
[24] http:/ / www.ncbi. nlm.nih. gov/ entrez/ query. fcgi?db=gene& cmd=retrieve& dopt=default& list_uids=4151& rn=1
[25] http:/ / www.ncbi. nlm.nih. gov/ entrez/ query. fcgi?db=gene& cmd=retrieve& dopt=default& list_uids=17189& rn=1
[26] http:/ / www.ensembl. org/ Homo_sapiens/ geneview?gene=ENSG00000198125;db=core
[27] http:/ / www.ensembl. org/ Mus_musculus/ geneview?gene=ENSMUSG00000018893;db=core
[28] http:/ / www.uniprot. org/ uniprot/ P02144
[29] http:/ / www.uniprot. org/ uniprot/ P04247
[30] http:/ / www.ncbi. nlm.nih. gov/ entrez/ viewer. fcgi?val=NM_005368
[31] http:/ / www.ncbi. nlm.nih. gov/ entrez/ viewer. fcgi?val=NM_001164047
[32] http:/ / www.ncbi. nlm.nih. gov/ entrez/ viewer. fcgi?val=NP_005359
[33] http:/ / www.ncbi. nlm.nih. gov/ entrez/ viewer. fcgi?val=NP_001157519
[34] http:/ / genome. ucsc.edu/ cgi-bin/ hgTracks?org=Human& db=hg19& position=chr22:36002811-36033998
[35] http:/ / genome. ucsc.edu/ cgi-bin/ hgTracks?org=Mouse& db=mm9& position=chr15:77015489-77050670
[36] http:/ / www.ncbi. nlm.nih. gov/ sites/ entrez?db=gene& cmd=Link& LinkName=gene_pubmed& from_uid=4151
[37] http:/ / www.ncbi. nlm.nih. gov/ sites/ entrez?db=gene& cmd=Link& LinkName=gene_pubmed& from_uid=17189
[38] (U.S.) National Science Foundation: Protein Data Bank Chronology (Jan. 21, 2004) (http:/ / www. nsf. gov/ news/ news_summ.
jsp?cntn_id=100689). Retrieved 3.17.2010
[39] The Nobel Prize in Chemistry 1962 (http:/ / nobelprize. org/ chemistry/ laureates/ 1962/ index. html)
Myoglobin
111
Further reading
Collman JP, Boulatov R, Sunderland CJ, Fu L (February 2004). "Functional analogues of cytochrome c oxidase,
myoglobin, and hemoglobin". Chem. Rev. 104 (2): 56188. doi: 10.1021/cr0206059 (http:/ / dx. doi. org/ 10.
1021/ cr0206059). PMID 14871135 (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 14871135).
Reeder BJ, Svistunenko DA, Cooper CE, Wilson MT (December 2004). "The radical and redox chemistry of
myoglobin and hemoglobin: from in vitro studies to human pathology". Antioxid. Redox Signal. 6 (6): 95466.
doi: 10.1089/ars.2004.6.954 (http:/ / dx. doi. org/ 10. 1089/ ars. 2004. 6. 954). PMID 15548893 (http:/ / www.
ncbi. nlm. nih. gov/ pubmed/ 15548893).
Schlieper G, Kim JH, Molojavyi A, Jacoby C, Laussmann T, Flgel U, Gdecke A, Schrader J (April 2004).
"Adaptation of the myoglobin knockout mouse to hypoxic stress". Am. J. Physiol. Regul. Integr. Comp. Physiol.
286 (4): R78692. doi: 10.1152/ajpregu.00043.2003 (http:/ / dx. doi. org/ 10. 1152/ ajpregu. 00043. 2003). PMID
14656764 (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 14656764).
Takano, T (1977). "Structure of myoglobin refined at 2-0 A resolution. II. Structure of deoxymyoglobin from
sperm whale". J. Mol. Biol. 110 (3): 569584. doi: 10.1016/S0022-2836(77)80112-5 (http:/ / dx. doi. org/ 10.
1016/ S0022-2836(77)80112-5). PMID 845960 (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 845960).
Roy A, Sen S, Chakraborti AS (February 2004). "In vitro nonenzymatic glycation enhances the role of myoglobin
as a source of oxidative stress". Free Radic. Res. 38 (2): 13946. doi: 10.1080/10715160310001638038 (http:/ /
dx. doi. org/ 10. 1080/ 10715160310001638038). PMID 15104207 (http:/ / www. ncbi. nlm. nih. gov/ pubmed/
15104207).
Stewart JM, Blakely JA, Karpowicz PA, Kalanxhi E, Thatcher BJ, Martin BM (March 2004). "Unusually weak
oxygen binding, physical properties, partial sequence, autoxidation rate and a potential phosphorylation site of
beluga whale (Delphinapterus leucas) myoglobin". Comp. Biochem. Physiol. B, Biochem. Mol. Biol. 137 (3):
40112. doi: 10.1016/j.cbpc.2004.01.007 (http:/ / dx. doi. org/ 10. 1016/ j. cbpc. 2004. 01. 007). PMID 15050527
(http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 15050527).
Wu G, Wainwright LM, Poole RK (2003). "Microbial globins". Adv. Microb. Physiol. 47: 255310. doi:
10.1016/S0065-2911(03)47005-7 (http:/ / dx. doi. org/ 10. 1016/ S0065-2911(03)47005-7). PMID 14560666
(http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 14560666).
External links
The Myoglobin Protein (http:/ / macromoleculeinsights. com/ myoglobin. php)
Protein Database featured molecule (http:/ / pdbdev. sdsc. edu:48346/ pdb/ molecules/ mb1. html)
Online 'Mendelian Inheritance in Man' (OMIM) 160000 (http:/ / omim. org/ entry/ 160000) human genetics
Which Cut Is Older? (It's a Trick Question) (http:/ / www. nytimes. com/ 2006/ 02/ 21/ national/ 21meat. html)
New York Times, February 21, 2006 article regarding meat industry use of carbon monoxide to keep meat
looking red.
Stores React to Meat Reports (http:/ / www. nytimes. com/ 2006/ 03/ 01/ dining/ 01meat. html) New York Times,
March 1, 2006 article on the use of carbon monoxide to make meat appear fresh.
Hemoglobin
112
Hemoglobin
Haemoglobin, human, adult
(heterotetramer, ()
2
)
Structure of human hemoglobin. The proteins' and subunits are in red and blue, and the iron-containing heme groups in green. From PDB

1GZX

[1]
Proteopedia

Hemoglobin

[2]
-
Protein type metalloprotein, globulin
Function oxygen-transport
Cofactor(s) heme (4)
-
Subunit
Name
Gene Chromosomal
Locus
Hb-1 HBA1
Chr. 16 p13.3
[3]
Hb-2 HBA2
Chr. 16 p13.3
[3]
Hb- HBB
Chr. 11 p15.5
[4]
Hemoglobin (pron.: /himlobn/; also spelled haemoglobin and abbreviated Hb or Hgb) is the iron-containing
oxygen-transport metalloprotein in the red blood cells of all vertebrates
[5]
(with the exception of the fish family
Channichthyidae
[6]
) as well as the tissues of some invertebrates. Hemoglobin in the blood carries oxygen from the
respiratory organs (lungs or gills) to the rest of the body (i.e. the tissues) where it releases the oxygen to burn
nutrients to provide energy to power the functions of the organism, and collects the resultant carbon dioxide to bring
it back to the respiratory organs to be dispensed from the organism.
In mammals, the protein makes up about 97% of the red blood cells' dry content, and around 35% of the total content
(including water).
[7]
Hemoglobin has an oxygen binding capacity of 1.34 mL O
2
per gram of hemoglobin,
[8]
which
increases the total blood oxygen capacity seventy-fold compared to dissolved oxygen in blood. The mammalian
hemoglobin molecule can bind (carry) up to four oxygen molecules.
[]
Hemoglobin is involved in the transport of other gases: it carries some of the body's respiratory carbon dioxide
(about 10% of the total) as carbaminohemoglobin, in which CO
2
is bound to the globin protein. The molecule also
carries the important regulatory molecule nitric oxide bound to a globin protein thiol group, releasing it at the same
time as oxygen.
[9]
Hemoglobin is also found outside red blood cells and their progenitor lines. Other cells that contain hemoglobin
include the A9 dopaminergic neurons in the substantia nigra, macrophages, alveolar cells, and mesangial cells in the
kidney. In these tissues, hemoglobin has a non-oxygen-carrying function as an antioxidant and a regulator of iron
Hemoglobin
113
metabolism.
[]
Hemoglobin and hemoglobin-like molecules are also found in many invertebrates, fungi, and plants. In these
organisms, hemoglobins may carry oxygen, or they may act to transport and regulate other things such as carbon
dioxide, nitric oxide, hydrogen sulfide and sulfide. A variant of the molecule, called leghemoglobin, is used to
scavenge oxygen away from anaerobic systems, such as the nitrogen-fixing nodules of leguminous plants, before the
oxygen can poison the system.
Research history
In 1825 J.F. Engelhard
[10]
discovered that the ratio of iron to protein is identical in the hemoglobins of several
species. From the known atomic mass of iron he calculated the molecular mass of hemoglobin to n 16000 (n =
number of irons per hemoglobin, now known to be 4), the first determination of a protein's molecular mass. This
"hasty conclusion" drew a lot of ridicule at the time from scientists who could not believe that any molecule could be
that big. Adair confirmed Engelhard's results in 1925 by measuring the osmotic pressure of hemoglobin solutions.
[11]
The oxygen-carrying protein hemoglobin was discovered by Hnefeld in 1840.
[]
In 1851,
[]
Otto Funke published a
series of articles in which he described growing hemoglobin crystals by successively diluting red blood cells with a
solvent such as pure water, alcohol or ether, followed by slow evaporation of the solvent from the resulting protein
solution.
[]
Hemoglobin's reversible oxygenation was described a few years later by Felix Hoppe-Seyler.
[]
In 1959 Max Perutz determined the molecular structure of myoglobin(similar to hemoglobin) by X-ray
crystallography.
[][]
This work resulted in his sharing with John Kendrew the 1962 Nobel Prize in Chemistry.
The role of hemoglobin in the blood was elucidated by physiologist Claude Bernard. The name hemoglobin is
derived from the words heme and globin, reflecting the fact that each subunit of hemoglobin is a globular protein
with an embedded heme group. Each heme group contains one iron atom, that can bind one oxygen molecule
through ion-induced dipole forces. The most common type of hemoglobin in mammals contains four such subunits.
Genetics
Hemoglobin consists mostly of protein subunits (the "globin" chains), and these proteins, in turn, are folded chains of
a large number of different amino acids called polypeptides. The amino acid sequence of any polypeptide created by
a cell is in turn determined by the stretches of DNA called genes. In all proteins, it is the amino acid sequence which
determines the protein's chemical properties and function.
There is more than one hemoglobin gene. The amino acid sequences of the globin proteins in hemoglobins usually
differ between species. These differences grow with evolutionary distance between species. For example, the most
common hemoglobin sequences in humans and chimpanzees are nearly identical, differing by only one amino acid in
both the alpha and the beta globin protein chains. These differences grow larger between less closely related species.
Even within a species, different variants of hemoglobin always exist, although one sequence is usually a "most
common" one in each species. Mutations in the genes for the hemoglobin protein in a species result in hemoglobin
variants.
[12][13]
Many of these mutant forms of hemoglobin cause no disease. Some of these mutant forms of
hemoglobin, however, cause a group of hereditary diseases termed the hemoglobinopathies. The best known
hemoglobinopathy is sickle-cell disease, which was the first human disease whose mechanism was understood at the
molecular level. A (mostly) separate set of diseases called thalassemias involves underproduction of normal and
sometimes abnormal hemoglobins, through problems and mutations in globin gene regulation. All these diseases
produce anemia.
[14]
Variations in hemoglobin amino acid sequences, as with other proteins, may be adaptive. For example, recent studies
have suggested genetic variants in deer mice that help explain how deer mice that live in the mountains are able to
survive in the thin air that accompanies high altitudes. A researcher from the University of Nebraska-Lincoln found
mutations in four different genes that can account for differences between deer mice that live in lowland prairies
Hemoglobin
114
versus the mountains. After examining wild mice captured from both highlands and lowlands, it was found that: the
genes of the two breeds are "virtually identicalexcept for those that govern the oxygen-carrying capacity of their
hemoglobin". "The genetic difference enables highland mice to make more efficient use of their oxygen", since less
is available at higher altitudes, such as those in the mountains.
[15]
Mammoth hemoglobin featured mutations that
allowed for oxygen delivery at lower temperatures, thus enabling mammoths to migrate to higher latitudes during the
Pleistocene.
[16]
Synthesis
Hemoglobin (Hb) is synthesized in a complex series of steps. The heme part is synthesized in a series of steps in the
mitochondria and the cytosol of immature red blood cells, while the globin protein parts are synthesized by
ribosomes in the cytosol.
[17]
Production of Hb continues in the cell throughout its early development from the
proerythroblast to the reticulocyte in the bone marrow. At this point, the nucleus is lost in mammalian red blood
cells, but not in birds and many other species. Even after the loss of the nucleus in mammals, residual ribosomal
RNA allows further synthesis of Hb until the reticulocyte loses its RNA soon after entering the vasculature (this
hemoglobin-synthetic RNA in fact gives the reticulocyte its reticulated appearance and name).
Structure
Heme b group
Hemoglobin has a quaternary structure characteristic of many
multi-subunit globular proteins.
[18]
Most of the amino acids in
hemoglobin form alpha helices, connected by short non-helical
segments. Hydrogen bonds stabilize the helical sections inside this
protein, causing attractions within the molecule, folding each
polypeptide chain into a specific shape.
[19]
Hemoglobin's quaternary
structure comes from its four subunits in roughly a tetrahedral
arrangement.
[18]
In most vertebrates, the hemoglobin molecule is an assembly of four
globular protein subunits. Each subunit is composed of a protein chain
tightly associated with a non-protein heme group. Each protein chain
arranges into a set of alpha-helix structural segments connected
together in a globin fold arrangement, so called because this
arrangement is the same folding motif used in other heme/globin
proteins such as myoglobin.
[][]
This folding pattern contains a pocket that strongly binds the heme group.
A heme group consists of an iron (Fe) ion (charged atom) held in a heterocyclic ring, known as a porphyrin. This
porphyrin ring consists of four pyrrole molecules cyclically linked together (by methine bridges) with the iron ion
bound in the center.
[20]
The iron ion, which is the site of oxygen binding, coordinates with the four nitrogens in the
center of the ring, which all lie in one plane. The iron is bound strongly (covalently) to the globular protein via the
imidazole ring of F8 histidine residue (also known as the proximal histidine) below the porphyrin ring. A sixth
position can reversibly bind oxygen by a coordinate covalent bond,
[21]
completing the octahedral group of six
ligands. Oxygen binds in an "end-on bent" geometry where one oxygen atom binds Fe and the other protrudes at an
angle. When oxygen is not bound, a very weakly bonded water molecule fills the site, forming a distorted
octahedron.
Even though carbon dioxide is carried by hemoglobin, it does not compete with oxygen for the iron-binding
positions, but is actually bound to the protein chains of the structure.
The iron ion may be either in the Fe
2+
or in the Fe
3+
state, but ferrihemoglobin (methemoglobin) (Fe
3+
) cannot bind
oxygen.
[22]
In binding, oxygen temporarily and reversibly oxidizes (Fe
2+
) to (Fe
3+
) while oxygen temporarily turns
Hemoglobin
115
into superoxide, thus iron must exist in the +2 oxidation state to bind oxygen. If superoxide ion associated to Fe
3+
is
protonated the hemoglobin iron will remain oxidized and incapable of binding oxygen. In such cases, the enzyme
methemoglobin reductase will be able to eventually reactivate methemoglobin by reducing the iron center.
In adult humans, the most common hemoglobin type is a tetramer (which contains 4 subunit proteins) called
hemoglobin A, consisting of two and two subunits non-covalently bound, each made of 141 and 146 amino acid
residues, respectively. This is denoted as
2

2
. The subunits are structurally similar and about the same size. Each
subunit has a molecular weight of about 16,000daltons,
[23]
for a total molecular weight of the tetramer of about
64,000daltons (64,458 g/mol).
[]
Thus, 1 g/dL = 0.1551mmol/L. Hemoglobin A is the most intensively studied of the
hemoglobin molecules.
In human infants, the hemoglobin molecule is made up of 2 chains and 2 chains. The gamma chains are
gradually replaced by chains as the infant grows.
[24]
The four polypeptide chains are bound to each other by salt bridges, hydrogen bonds, and the hydrophobic effect.
Oxygen saturation
In general, hemoglobin can be saturated with oxygen molecules (oxyhemoglobin), or desaturated with oxygen
molecules (deoxyhemoglobin).
[25]
Oxyhemoglobin
Oxyhemoglobin is formed during physiological respiration when oxygen binds to the heme component of the protein
hemoglobin in red blood cells. This process occurs in the pulmonary capillaries adjacent to the alveoli of the lungs.
The oxygen then travels through the blood stream to be dropped off at cells where it is utilized as a terminal electron
acceptor in the production of ATP by the process of oxidative phosphorylation. It does not, however, help to
counteract a decrease in blood pH. Ventilation, or breathing, may reverse this condition by removal of carbon
dioxide, thus causing a shift up in pH.
[]
Hemoglobin exists in two forms, a taut (tense) form (T) and a relaxed form (R). Various factors such as low pH, high
CO
2
and high 2,3 BPG at the level of the tissues favor the taut form, which has low oxygen affinity and releases
oxygen in the tissues. Conversely, a high pH, low CO
2
, or low 2,3 BPG favors the relaxed form which can better
bind oxygen.
[]
The partial pressure of the system also affects O
2
affinity where, at high partial pressures of oxygen
(such as those present in the alveoli), the relaxed (high affinity, R) state is favoured. Inversely, at low partial
pressures (such as those present in respiring tissues), the (low affinity, T) tense state is favoured.
[26]
Additionally, the
binding of oxygen to the Iron-II heme pulls the iron into the plane of the porphryn ring, causing a slight
conformational shift. The shift encourages oxygen to bind to the three remaining hemes within hemoglobin (thus,
oxygen binding is cooperative).
Hemoglobin
116
Deoxygenated hemoglobin
Deoxygenated hemoglobin is the form of hemoglobin without the bound oxygen. The absorption spectra of
oxyhemoglobin and deoxyhemoglobin differ. The oxyhemoglobin has significantly lower absorption of the 660nm
wavelength than deoxyhemoglobin, while at 940nm its absorption is slightly higher. This difference is used for
measurement of the amount of oxygen in patient's blood by an instrument called pulse oximeter. This difference also
accounts for the presentation of cyanosis, the blue to purplish color that tissues develop during hypoxia.
Iron's oxidation state in oxyhemoglobin
Assigning oxygenated hemoglobin's oxidation state is difficult because oxyhemoglobin (Hb-O
2
), by experimental
measurement, is diamagnetic (no net unpaired electrons), yet the low-energy electron configurations in both oxygen
and iron are paramagnetic (suggesting at least one unpaired electron in the complex). The lowest-energy form of
oxygen, and the lowest energy forms of the relevant oxidation states of iron, are these:
Triplet oxygen, the lowest energy molecular oxygen species, has two unpaired electrons in antibonding *
molecular orbitals.
Iron(II) tends to exist in a high-spin configuration where unpaired electrons exist in E
g
antibonding orbitals.
Iron(III) has an odd number of electrons, and thus must have one or more unpaired electrons, in any energy state.
All of these structures are paramagnetic (have unpaired electrons), not diamagnetic. Thus, a non-intuitive (e.g., a
higher-energy for at least one species) distribution of electrons in the combination of iron and oxygen must exist, in
order to explain the observed diamagnetism and no unpaired electrons.
The three logical possibilities to produce diamagnetic (no net spin) Hb-O
2
are:
1. Low-spin Fe
2+
binds to singlet oxygen. Both low-spin iron and singlet oxygen are diamagnetic. However, the
singlet form of oxygen is the higher-energy form of the molecule.
2. Low-spin Fe
3+
binds to .O
2
-
(the superoxide ion) and the two unpaired electrons couple antiferromagnetically,
giving diamagnetic properties.
3. Low-spin Fe
4+
binds to peroxide, O
2
2-
. Both are diamagnetic.
Direct experimental data:
X-ray photoelectron spectroscopy suggests iron has an oxidation state of approximately 3.2
infrared stretching frequencies of the O-O bond suggests a bond length fitting with superoxide (a bond order of
about 1.6, with superoxide being 1.5).
X-ray Absorption Near Edge Structures at the iron K-edge. The energy shift of 5 eV between Deoxyhemoglobin
and Oxyhemoglobin, as for all the Methemoglobin species, strongly suggests an actual local charge closer to Fe
3+
Hemoglobin
117
than Fe
2+
.
[27][28][29]
Thus, the nearest formal oxidation state of iron in Hb-O
2
is the +3 state, with oxygen in the -1 state (as superoxide
.O
2
-
). The diamagnetism in this configuration arises from the single unpaired electron on superoxide aligning
antiferromagnetically from the single unpaired electron on iron, to give no net spin to the entire configuration, in
accordance with diamagnetic oxyhemoglobin from experiment.
[][]
The second choice of the three logical possibilities above for diamagnetic oxyhemoglobin being found correct by
experiment, is not surprising: singlet oxygen (possibility #1) and large separations of charge (possibility #3) are both
unfavorably high-energy states. Iron's shift to a higher oxidation state in Hb-O
2
decreases the atom's size, and allows
it into the plane of the porphyrin ring, pulling on the coordinated histidine residue and initiating the allosteric
changes seen in the globulins.
Early postulates by bio-inorganic chemists claimed that possibility #1 (above) was correct and that iron should exist
in oxidation state II. This seemed particularly likely since the iron oxidation state III as methemoglobin, when not
accompanied by superoxide .O
2
-
to "hold" the oxidation electron, was known to render hemoglobin incapable of
binding normal triplet O
2
as it occurs in the air. It was thus assumed that iron remained as Fe(II) when oxygen gas
was bound in the lungs. The iron chemistry in this previous classical model was elegant, but the required presence of
the required diamagnetic high-energy singlet oxygen was never explained. It was classically argued that the binding
of an oxygen molecule placed high-spin iron(II) in an octahedral field of strong-field ligands; this change in field
would increase the crystal field splitting energy, causing iron's electrons to pair into the low-spin configuration,
which would be diamagnetic in Fe(II). This forced low-spin pairing is indeed thought to happen in iron when oxygen
binds, but is not enough to explain iron's change in size. Extraction of an additional electron from iron by oxygen is
required to explain both iron's smaller size and observed increased oxidation state, and oxygen's weaker bond.
It should be noted that the assignment of a whole-number oxidation state is a formalism, as the covalent bonds are
not required to have perfect bond orders involving whole electron transfer. Thus, all three models for paramagnetic
Hb-O
2
may contribute to some small degree (by resonance) to the actual electronic configuration of Hb-O
2
.
However, the model of iron in Hb-O
2
being Fe(III) is more correct than the classical idea that it remains Fe(II).
Binding for ligands other than oxygen
Besides the oxygen ligand, which binds to hemoglobin in a cooperative manner, hemoglobin ligands also include
competitive inhibitors such as carbon monoxide (CO) and allosteric ligands such as carbon dioxide (CO
2
) and nitric
oxide (NO). The carbon dioxide is bound to amino groups of the globin proteins as carbaminohemoglobin, and is
thought to account for about 10% of carbon dioxide transport in mammals. Nitric oxide is bound to specific thiol
groups in the globin protein to form an S-nitrosothiol which dissociates into free nitric oxide and thiol again, as the
hemoglobin releases oxygen from its heme site. This nitric oxide transport to peripheral tissues is hypothesized to
assist oxygen transport in tissues, by releasing vasodilatory nitric oxide to tissues in which oxygen levels are low.
[]
Hemoglobin
118
Cooperative
A schematic visual model of oxygen-binding
process, showing all four monomers and hemes,
and protein chains only as diagramatic coils, to
facilitate visualization into the molecule. Oxygen
is not shown in this model, but, for each of the
iron atoms, it binds to the iron (red sphere) in the
flat heme. For example, in the upper left of the
four hemes shown, oxygen binds at the left of the
iron atom shown in the upper left of diagram.
This causes the iron atom to move backward into
the heme which holds it (the iron moves upward
as it binds oxygen, in this illustration), tugging
the histidine residue (modeled as a red pentagon
on the right of the iron) closer, as it does. This, in
turn, pulls on the protein chain holding the
histidine.
When oxygen binds to the iron complex, it causes the iron atom to
move back toward the center of the plane of the porphyrin ring (see
moving diagram). At the same time, the imidazole side-chain of the
histidine residue interacting at the other pole of the iron is pulled
toward the porphyrin ring. This interaction forces the plane of the ring
sideways toward the outside of the tetramer, and also induces a strain
in the protein helix containing the histidine as it moves nearer to the
iron atom. This strain is transmitted to the remaining three monomers
in the tetramer, where it induces a similar conformational change in the
other heme sites such that binding of oxygen to these sites becomes
easier.
In the tetrameric form of normal adult hemoglobin, the binding of
oxygen is, thus, a cooperative process. The binding affinity of
hemoglobin for oxygen is increased by the oxygen saturation of the
molecule, with the first oxygens bound influencing the shape of the
binding sites for the next oxygens, in a way favorable for binding. This
positive cooperative binding is achieved through steric conformational
changes of the hemoglobin protein complex as discussed above; i.e.,
when one subunit protein in hemoglobin becomes oxygenated, a
conformational or structural change in the whole complex is initiated,
causing the other subunits to gain an increased affinity for oxygen. As
a consequence, the oxygen binding curve of hemoglobin is sigmoidal,
or S-shaped, as opposed to the normal hyperbolic curve associated with
noncooperative binding.
The dynamic mechanism of the cooperativity in hemoglobin and its relation with the low-frequency resonance has
been discussed.
[]
Competitive
The binding of oxygen is affected by molecules such as carbon monoxide (CO) (for example, from tobacco smoking,
car exhaust, and incomplete combustion in furnaces). CO competes with oxygen at the heme binding site.
Hemoglobin binding affinity for CO is 250 times greater than its affinity for oxygen,
[30]
meaning that small amounts
of CO dramatically reduce hemoglobin's ability to transport oxygen. Since carbon monoxide is a colorless, odorless
and tasteless gas, and poses a potentially fatal threat, detectors have become commercially available to warn of
dangerous levels in residences. When hemoglobin combines with CO, it forms a very bright red compound called
carboxyhemoglobin, which may cause the skin of CO poisoning victims to appear pink in death, instead of white or
blue. When inspired air contains CO levels as low as 0.02%, headache and nausea occur; if the CO concentration is
increased to 0.1%, unconsciousness will follow. In heavy smokers, up to 20% of the oxygen-active sites can be
blocked by CO.
In similar fashion, hemoglobin also has competitive binding affinity for cyanide (CN

), sulfur monoxide (SO), nitric

oxide (NO), and sulfide (S
2
), including hydrogen sulfide (H
2
S). All of these bind to iron in heme without changing
its oxidation state, but they nevertheless inhibit oxygen-binding, causing grave toxicity.
The iron atom in the heme group must initially be in the ferrous (Fe
2+
) oxidation state to support oxygen and other
gases' binding and transport (it temporarily switches to ferric during the time oxygen is bound, as explained above).
Initial oxidation to the ferric (Fe
3+
) state without oxygen converts hemoglobin into "hemiglobin" or methemoglobin
Hemoglobin
119
(pronounced "MET-hemoglobin"), which cannot bind oxygen. Hemoglobin in normal red blood cells is protected by
a reduction system to keep this from happening. Nitric oxide is capable of converting a small fraction of hemoglobin
to methemoglobin in red blood cells. The latter reaction is a remnant activity of the more ancient nitric oxide
dioxygenase function of globins.
Allosteric
Carbon dioxide occupies a different binding site on the hemoglobin. Carbon dioxide is more readily dissolved in
deoxygenated blood, facilitating its removal from the body after the oxygen has been released to tissues undergoing
metabolism. This increased affinity for carbon dioxide by the venous blood is known as the Haldane effect. Through
the enzyme carbonic anhydrase, carbon dioxide reacts with water to give carbonic acid, which decomposes into
bicarbonate and protons:
CO
2
+ H
2
O H
2
CO
3
HCO
3
-
+ H
+
The sigmoidal shape of hemoglobin's oxygen-dissociation curve results
from cooperative binding of oxygen to hemoglobin.
Hence blood with high carbon dioxide levels is
also lower in pH (more acidic). Hemoglobin can
bind protons and carbon dioxide, which causes a
conformational change in the protein and facilitates
the release of oxygen. Protons bind at various
places on the protein, while carbon dioxide binds
at the -amino group.
[31]
Carbon dioxide binds to
hemoglobin and forms carbaminohemoglobin.
[32]
This decrease in hemoglobin's affinity for oxygen
by the binding of carbon dioxide and acid is known
as the Bohr effect (shifts the O
2
-saturation curve to
the right). Conversely, when the carbon dioxide
levels in the blood decrease (i.e., in the lung
capillaries), carbon dioxide and protons are
released from hemoglobin, increasing the oxygen
affinity of the protein. A reduction in the total
binding capacity of hemoglobin to oxygen (i.e.
shifting the curve down, not just to the right) due to reduced pH is called the root effect. This is seen in bony fish.
It is necessary for hemoglobin to release the oxygen that it binds; if not, there is no point in binding it. The sigmoidal
curve of hemoglobin makes it efficient in binding (taking up O
2
in lungs), and efficient in unloading (unloading O
2
in tissues).
[33]
In people acclimated to high altitudes, the concentration of 2,3-Bisphosphoglycerate (2,3-BPG) in the blood is
increased, which allows these individuals to deliver a larger amount of oxygen to tissues under conditions of lower
oxygen tension. This phenomenon, where molecule Y affects the binding of molecule X to a transport molecule Z, is
called a heterotropic allosteric effect.
Animals other than humans use different molecules to bind to hemoglobin and change its O
2
affinity under
unfavorable conditions. Fish use both ATP and GTP. These bind to a phosphate "pocket" on the fish hemoglobin
molecule, which stabilizes the tense state and therefore decreases oxygen affinity.
[]
GTP reduces hemoglobin oxygen
affinity much more than ATP, which is thought to be due to an extra hydrogen bond formed that further stabilizes the
tense state.
[]
Under hypoxic conditions, the concentration of both ATP and GTP is reduced in fish red blood cells to
increase oxygen affinity.
[]
A variant hemoglobin, called fetal hemoglobin (HbF,
2

2
), is found in the developing fetus, and binds oxygen with
greater affinity than adult hemoglobin. This means that the oxygen binding curve for fetal hemoglobin is left-shifted
Hemoglobin
120
(i.e., a higher percentage of hemoglobin has oxygen bound to it at lower oxygen tension), in comparison to that of
adult hemoglobin. As a result, fetal blood in the placenta is able to take oxygen from maternal blood.
Hemoglobin also carries nitric oxide in the globin part of the molecule. This improves oxygen delivery in the
periphery and contributes to the control of respiration. NO binds reversibly to a specific cysteine residue in globin;
the binding depends on the state (R or T) of the hemoglobin. The resulting S-nitrosylated hemoglobin influences
various NO-related activities such as the control of vascular resistance, blood pressure and respiration. NO is not
released in the cytoplasm of erythrocytes but transported by an anion exchanger called AE1 out of them.
[34]
A study was performed to examine the influence of the form of hemoglobin (Hb) on the partitioning of inhaled
volatile organic compounds (VOCs) into [human and animal] blood. Benzene was the prototypic VOC used in the
investigations for this research due to the similar properties it shares with many other VOCs. To be specific, this
study analyses the influence of the water solubility of Hb on the partitioning coefficient (PC) of a VOC as compared
to the influence of the "species" or form of Hb. The different forms of blood used include: human hemoglobin
(HbA), rat Hb, and sickle-cell hemoglobin (HbS). Rat Hb contains little water and is in a quasi-crystalline form,
found inside the red blood cells (RBC), meaning they are more hydrophobic than human Hb, which are
water-soluble. Sickle-cell hemoglobin (HbS) is water-soluble, however it can become water-insoluble, forming
hydrophobic polymers, when deoxygenated. The findings state that the benzene PC for rat Hb was much higher than
human that for Hb; however, the tests that measured the PCs of the oxygenated and deoxygenated forms of HbA and
HbS did not differ, indicating that the affinity of benzene was not affected by the water solubility of Hb.
[35]
Types in humans
Hemoglobin variants are a part of the normal embryonic and fetal development, but may also be pathologic mutant
forms of hemoglobin in a population, caused by variations in genetics. Some well-known hemoglobin variants such
as sickle-cell anemia are responsible for diseases, and are considered hemoglobinopathies. Other variants cause no
detectable pathology, and are thus considered non-pathological variants.
[][]
In the embryo:
Gower 1 (
2

2
)
Gower 2 (
2

2
) (PDB 1A9W
[36]
)
Hemoglobin Portland (
2

2
).
In the fetus:
Hemoglobin F (
2

2
) (PDB 1FDH
[37]
).
In adults:
Hemoglobin A (
2

2
) (PDB 1BZ0
[38]
) - The most common with a normal amount over 95%
Hemoglobin A
2
(
2

2
) - chain synthesis begins late in the third trimester and in adults, it has a normal range of
1.5-3.5%
Hemoglobin F (
2

2
) - In adults Hemoglobin F is restricted to a limited population of red cells called F-cells.
However, the level of Hb F can be elevated in persons with sickle-cell disease and beta-thalassemia.
Hemoglobin
121
Gene expression of hemoglobin before and after birth. Also identifies the types of
cells and organs in which the gene expression (data on Wood W.G., (1976). Br.
Med. Bull. 32, 282.)
Variant forms that cause disease:
Hemoglobin H (
4
) - A variant form of
hemoglobin, formed by a tetramer of
chains, which may be present in variants
of thalassemia.
Hemoglobin Barts (
4
) - A variant form
of hemoglobin, formed by a tetramer of
chains, which may be present in variants
of thalassemia.
Hemoglobin S (
2

S
2
) - A variant form
of hemoglobin found in people with
sickle cell disease. There is a variation in
the -chain gene, causing a change in the
properties of hemoglobin, which results
in sickling of red blood cells.
Hemoglobin C (
2

C
2
) - Another variant
due to a variation in the -chain gene.
This variant causes a mild chronic hemolytic anemia.
Hemoglobin E (
2

E
2
) - Another variant due to a variation in the -chain gene. This variant causes a mild chronic
hemolytic anemia.
Hemoglobin AS - A heterozygous form causing Sickle cell trait with one adult gene and one sickle cell disease
gene
Hemoglobin SC disease - A compound heterozygous form with one sickle gene and another encoding
Hemoglobin C.
Degradation in vertebrate animals
When red cells reach the end of their life due to aging or defects, they are broken down in spleen, the hemoglobin
molecule is broken up and the iron gets recycled. This process also produces one molecule of carbon monoxide for
every molecule of heme degraded.
[39]
This is one of the few natural sources of carbon monoxide production in the
human body, and is responsible for the normal blood levels of carbon monoxide even in people breathing pure air.
The other major final product of heme degradation is bilirubin. Increased levels of this chemical are detected in the
blood if red cells are being destroyed more rapidly than usual. Improperly degraded hemoglobin protein or
hemoglobin that has been released from the blood cells too rapidly can clog small blood vessels, especially the
delicate blood filtering vessels of the kidneys, causing kidney damage. Iron is removed from heme and salvaged for
later use, it is stored as hemosiderin or ferritin in tissues and transported in plasma by beta globulins as transferins.
When the porphyrin ring is broken up, the fragments are normally secreted as a yellow pigment called bilirubin,
which is secreted into the intestines as bile. Intestines metabolise bilirubin into urobilinogen. Urobilinogen leaves the
body in faeces, in a pigment called stercobilin. Globulin is metabolised into amino acids which are then released into
circulation.
Hemoglobin
122
Role in disease
In sickle cell hemoglobin (HbS) glutamic acid in
position 6 (in beta chain) is mutated to valine.
This change allows the deoxygenated form of the
hemoglobin to stick to itself.
Hemoglobin deficiency can be caused either by decreased amount of
hemoglobin molecules, as in anemia, or by decreased ability of each
molecule to bind oxygen at the same partial pressure of oxygen.
Hemoglobinopathies (genetic defects resulting in abnormal structure of
the hemoglobin molecule)
[40]
may cause both. In any case, hemoglobin
deficiency decreases blood oxygen-carrying capacity. Hemoglobin
deficiency is, in general, strictly distinguished from hypoxemia,
defined as decreased partial pressure of oxygen in blood,
[41][42][43][44]
although both are causes of hypoxia (insufficient oxygen supply to
tissues).
Other common causes of low hemoglobin include loss of blood,
nutritional deficiency, bone marrow problems, chemotherapy, kidney
failure, or abnormal hemoglobin (such as that of sickle-cell disease).
High hemoglobin levels may be caused by exposure to high altitudes,
smoking, dehydration, or tumors.
[24]
The ability of each hemoglobin molecule to carry oxygen is normally
modified by altered blood pH or CO
2
, causing an altered
oxygenhemoglobin dissociation curve. However, it can also be
pathologically altered in, e.g., carbon monoxide poisoning.
Decrease of hemoglobin, with or without an absolute decrease of red
blood cells, leads to symptoms of anemia. Anemia has many different
causes, although iron deficiency and its resultant iron deficiency
anemia are the most common causes in the Western world. As absence of iron decreases heme synthesis, red blood
cells in iron deficiency anemia are hypochromic (lacking the red hemoglobin pigment) and microcytic (smaller than
normal). Other anemias are rarer. In hemolysis (accelerated breakdown of red blood cells), associated jaundice is
caused by the hemoglobin metabolite bilirubin, and the circulating hemoglobin can cause renal failure.
Some mutations in the globin chain are associated with the hemoglobinopathies, such as sickle-cell disease and
thalassemia. Other mutations, as discussed at the beginning of the article, are benign and are referred to merely as
hemoglobin variants.
There is a group of genetic disorders, known as the porphyrias that are characterized by errors in metabolic pathways
of heme synthesis. King George III of the United Kingdom was probably the most famous porphyria sufferer.
To a small extent, hemoglobin A slowly combines with glucose at the terminal valine (an alpha aminoacid) of each
chain. The resulting molecule is often referred to as Hb A
1c
. As the concentration of glucose in the blood increases,
the percentage of Hb A that turns into Hb A
1c
increases. In diabetics whose glucose usually runs high, the percent Hb
A
1c
also runs high. Because of the slow rate of Hb A combination with glucose, the Hb A
1c
percentage is
representative of glucose level in the blood averaged over a longer time (the half-life of red blood cells, which is
typically 5055 days).
Glycosylated hemoglobin is the form of hemoglobin to which glucose is bound. The binding of glucose to amino
acids in the hemoglobin takes place spontaneously (without the help of an enzyme) in many proteins, and is not
known to serve a useful purpose. However, the binding to hemoglobin does serve as a record for average blood
glucose levels over the lifetime of red cells, which is approximately 120 days. The levels of glycosylated hemoglobin
are therefore measured in order to monitor the long-term control of the chronic disease of type 2 diabetes mellitus
(T2DM). Poor control of T2DM results in high levels of glycosylated hemoglobin in the red blood cells. The normal
Hemoglobin
123
reference range is approximately 45.9 %. Though difficult to obtain, values less than 7% are recommended for
people with T2DM. Levels greater than 9% are associated with poor control of the glycosylated hemoglobin, and
levels greater than 12% are associated with very poor control. Diabetics who keep their glycosylated hemoglobin
levels close to 7% have a much better chance of avoiding the complications that may accompany diabetes (than
those whose levels are 8% or higher).
[45]
In addition, increased glycosylation of hemoglobin increases its affinity for
oxygen, therefore preventing its release at the tissue and inducing a level of hypoxia in extreme cases.
[46]
Elevated levels of hemoglobin are associated with increased numbers or sizes of red blood cells, called
polycythemia. This elevation may be caused by congenital heart disease, cor pulmonale, pulmonary fibrosis, too
much erythropoietin, or polycythemia vera.
[47]
A recent study done in Pondicherry, India, shows its importance in coronary artery disease.
[48]
Diagnostic uses
Hemoglobin concentration measurement is among the most commonly performed blood tests, usually as part of a
complete blood count. For example it is typically tested before or after blood donation. Results are reported in g/L,
g/dL or mol/L. 1 g/dL equals about 0.6206 mmol/L, although the latter units are not used as often due to uncertainty
regarding the polymeric state of the molecule.
[49]
This conversion factor, using the single globin unit molecular
weight of 16,000 Da, is more common for hemoglobin concentration in blood. For MCHC the conversion factor
0.155, which uses the tetramer weight of 64,500 Da, is more common.
[50]
Normal levels are:
Men: 13.8 to 18.0 g/dL (138 to 180 g/L, or 8.56 to 11.17mmol/L)
Women: 12.1 to 15.1 g/dL (121 to 151 g/L, or 7.51 to 9.37mmol/L)
Children: 11 to 16 g/dL (111 to 160 g/L, or 6.83 to 9.93mmol/L)
Pregnant women: 11 to 14 g/dL (110 to 140 g/L, or 6.83 to 8.69mmol/L)
[51][52]
Normal values of hemoglobin in the 1st and 3rd trimesters of pregnant women must be at least 11 g/dL and at least
10.5 g/dL during the 2nd trimester.
[53]
Dehydration or hyperhydration can greatly influence measured hemoglobin levels. Albumin can indicate hydration
status.
If the concentration is below normal, this is called anemia. Anemias are classified by the size of red blood cells, the
cells that contain hemoglobin in vertebrates. The anemia is called "microcytic" if red cells are small, "macrocytic" if
they are large, and "normocytic" otherwise.
Hematocrit, the proportion of blood volume occupied by red blood cells, is typically about three times the
hemoglobin concentration measured in g/dL. For example, if the hemoglobin is measured at 17 g/dL, that compares
with a hematocrit of 51%.
[54]
Laboratory hemoglobin test methods require a blood sample (arterial, venous, or capillary) and analysis on
hematology analyzer and CO-oximeter. Additionally, a new noninvasive hemoglobin (SpHb) test method called
Pulse CO-Oximetry is also available with comparable accuracy to invasive methods.
[55]
Concentrations of oxy- and deoxyhemoglobin can be measured continuously, regionally and noninvasively using
NIRS.
[56][57][58][59][60]
NIRS can be used both on the head as on muscles. This technique is often used for research
in e.g. elite sports training, ergonomics, rehabilition, patient monitoring, neonatal research, functional brain
monitoring, brain computer interface, urology (bladder contraction), neurology (Neurovascular coupling) and more.
Long-term control of blood sugar concentration can be measured by the concentration of Hb A
1c
. Measuring it
directly would require many samples because blood sugar levels vary widely through the day. Hb A
1c
is the product
of the irreversible reaction of hemoglobin A with glucose. A higher glucose concentration results in more Hb A
1c
.
Because the reaction is slow, the Hb A
1c
proportion represents glucose level in blood averaged over the half-life of
red blood cells, is typically 5055 days. An Hb A
1c
proportion of 6.0% or less show good long-term glucose control,
while values above 7.0% are elevated. This test is especially useful for diabetics.
[61]
Hemoglobin
124
The functional magnetic resonance imaging (fMRI) machine uses the signal from deoxyhemoglobin, which is
sensitive to magnetic fields since it is paramagnetic. Combined measurement with NIRS shows good correlation
with both the oxy- and deoxyhemoglobin signal compared to the BOLD signal.
[62]
Analogues in non-vertebrate organisms
A variety of oxygen-transport and -binding proteins exist in organisms throughout the animal and plant kingdoms.
Organisms including bacteria, protozoans, and fungi all have hemoglobin-like proteins whose known and predicted
roles include the reversible binding of gaseous ligands. Since many of these proteins contain globins and the heme
moiety (iron in a flat porphyrin support), they are often called hemoglobins, even if their overall tertiary structure is
very different from that of vertebrate hemoglobin. In particular, the distinction of "myoglobin" and hemoglobin in
lower animals is often impossible, because some of these organisms do not contain muscles. Or, they may have a
recognizable separate circulatory system but not one that deals with oxygen transport (for example, many insects and
other arthropods). In all these groups, heme/globin-containing molecules (even monomeric globin ones) that deal
with gas-binding are referred to as oxyhemoglobins. In addition to dealing with transport and sensing of oxygen,
they may also deal with NO, CO
2
, sulfide compounds, and even O
2
scavenging in environments that must be
anaerobic. They may even deal with detoxification of chlorinated materials in a way analogous to heme-containing
P450 enzymes and peroxidases.
The giant tube worm Riftia pachyptila showing
red hemoglobin-containing plumes
The structure of hemoglobins varies across species. Hemoglobin
occurs in all kingdoms of organisms, but not in all organisms.
Primitive species such as bacteria, protozoa, algae, and plants often
have single-globin hemoglobins. Many nematode worms, molluscs,
and crustaceans contain very large multisubunit molecules, much
larger than those in vertebrates. In particular, chimeric hemoglobins
found in fungi and giant annelids may contain both globin and other
types of proteins.
[63]
One of the most striking occurrences and uses of hemoglobin in
organisms is in the giant tube worm (Riftia pachyptila, also called
Vestimentifera), which can reach 2.4 meters length and populates ocean volcanic vents. Instead of a digestive tract,
these worms contain a population of bacteria constituting half the organism's weight. The bacteria react with H
2
S
from the vent and O
2
from the water to produce energy to make food from H
2
O and CO
2
. The worms end with a
deep red fan-like structure ("plume"), which extends into the water and absorbs H
2
S and O
2
for the bacteria, and CO
2
for use as synthetic raw material similar to photosynthetic plants. The structures are bright-red due to their
containing several extraordinarily complex hemoglobins that have up to 144 globin chains, each including associated
heme structures. These hemoglobins are remarkable for being able to carry oxygen in the presence of sulfide, and
even to carry sulfide, without being completely "poisoned" or inhibited by it as hemoglobins in most other species
are.
[64][65]
Hemoglobin
125
Other oxygen-binding proteins
Myoglobin
Found in the muscle tissue of many vertebrates, including humans, it gives muscle tissue a distinct red or dark
gray color. It is very similar to hemoglobin in structure and sequence, but is not a tetramer; instead, it is a
monomer that lacks cooperative binding. It is used to store oxygen rather than transport it.
Hemocyanin
The second most common oxygen-transporting protein found in nature, it is found in the blood of many
arthropods and molluscs. Uses copper prosthetic groups instead of iron heme groups and is blue in color when
oxygenated.
Hemerythrin
Some marine invertebrates and a few species of annelid use this iron-containing non-heme protein to carry
oxygen in their blood. Appears pink/violet when oxygenated, clear when not.
Chlorocruorin
Found in many annelids, it is very similar to erythrocruorin, but the heme group is significantly different in
structure. Appears green when deoxygenated and red when oxygenated.
Vanabins
Also known as vanadium chromagens, they are found in the blood of sea squirts. There were once
hypothesized to use the rare metal vanadium as an oxygen binding prosthetic group. However, although they
do contain vanadium by preference, they apparently bind little oxygen, and thus have some other function,
which has not been elucidated (sea squirts also contain some hemoglobin). They may act as toxins.
Erythrocruorin
Found in many annelids, including earthworms, it is a giant free-floating blood protein containing many
dozenspossibly hundredsof iron- and heme-bearing protein subunits bound together into a single protein
complex with a molecular mass greater than 3.5 million daltons.
Pinnaglobin
Only seen in the mollusc Pinna squamosa. Brown manganese-based porphyrin protein.
Leghemoglobin
In leguminous plants, such as alfalfa or soybeans, the nitrogen fixing bacteria in the roots are protected from
oxygen by this iron heme containing oxygen-binding protein. The specific enzyme protected is nitrogenase,
which is unable to reduce nitrogen gas in the presence of free oxygen.
Coboglobin
A synthetic cobalt-based porphyrin. Coboprotein would appear colorless when oxygenated, but yellow when
in veins.
Presence in nonerythroid cells
Some nonerythroid cells (i.e., cells other than the red blood cell line) contain hemoglobin. In the brain, these include
the A9 dopaminergic neurons in the substantia nigra, astrocytes in the cerebral cortex and hippocampus, and in all
mature oligodendrocytes.
[]
It has been suggested that brain hemoglobin in these cells may enable the "storage of
oxygen to provide a homeostatic mechanism in anoxic conditions, which is especially important for A9 DA neurons
that have an elevated metabolism with a high requirement for energy production".
[]
It has been noted further that "A9
dopaminergic neurons may be at particular risk since in addition to their high mitochondrial activity they are under
intense oxidative stress caused by the production of hydrogen peroxide via autoxidation and/or monoamine oxidase
(MAO)-mediated deamination of dopamine and the subsequent reaction of accessible ferrous iron to generate highly
Hemoglobin
126
toxic hydroxyl radicals".
[]
This may explain the risk of these cells for degeneration in Parkinson's disease.
[]
The
hemoglobin-derived iron in these cells is not the cause of the post-mortem darkness of these cells (origin of the Latin
name, substantia nigra), but rather is due to neuromelanin.
Outside the brain, hemoglobin has non-oxygen-carrying functions as an antioxidant and a regulator of iron
metabolism in macrophages,
[66]
alveolar cells,
[67]
and mesangial cells in the kidney.
[68]
In history, art and music
The planet Mars
Historically, the color of blood was associated with rust, as ancient Romans
associated the planet Mars with the god of war since Mars is orange-red. The
color of Mars is due to the iron oxide in the Martian soil, but the red in blood
is not due to the iron in hemoglobin and its oxides, which is a common
misconception. The red is due to the porphyrin moiety of hemoglobin to
which the iron is bound, not the iron itself,
[69]
although the ligation and redox
state of the iron can influence the pi to pi* or n to pi* electronic transitions of
the porphyrin and hence its optical characteristics.
Heart of Steel (Hemoglobin) (2005) by Julian Voss-Andreae. The images show the 5'
(1.60m) tall sculpture right after installation, after 10 days, and after several months of
exposure to the elements.
Artist Julian Voss-Andreae created a
sculpture called "Heart of Steel
(Hemoglobin)" in 2005, based on the
protein's backbone. The sculpture was
made from glass and weathering steel.
The intentional rusting of the initially
shiny work of art mirrors hemoglobin's
fundamental chemical reaction of
oxygen binding to iron.
[70][71]
Rock band Placebo recorded a song
called "Haemoglobin" with the lyrics
"Haemoglobin is the key to a healthy
heartbeat". French rap artist MC Solaar
also had a successful single titled "La Concubine de L'Hemoglobin" in 1994.
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[61] This Hb A
1c
level is only useful in individuals who have red blood cells (RBCs) with normal survivals (i.e., normal half-life). In individuals
with abnormal RBCs, whether due to abnormal hemoglobin molecules (such as Hemoglobin S in Sickle Cell Anemia) or RBC membrane
defects - or other problems, the RBC half-life is frequently shortened. In these individuals, an alternative test called "fructosamine level" can
be used. It measures the degree of glycation (glucose binding) to albumin, the most common blood protein, and reflects average blood glucose
levels over the previous 18-21 days, which is the half-life of albumin molecules in the circulation.
Hemoglobin
128
Further reading
Campbell, MK (1999). Biochemistry (Third Edition).
Harcourt. ISBN0-03-024426-9
Eshaghian, S; Horwich, TB; Fonarow, GC (January 2006).
"An unexpected inverse relationship between HbA1c levels
and mortality in patients with diabetes and advanced systolic
heart failure". Am Heart J 151 (1): 91. doi:
10.1016/j.ahj.2005.10.008 (http:/ / dx. doi.org/ 10.1016/ j.
ahj.2005.10. 008). PMID 16368297 (http:/ / www.ncbi.
nlm.nih.gov/ pubmed/ 16368297).
Ganong, WF (2003). Review of Medical Physiology
(Twenty-First Edition). Lange. ISBN0-07-140236-5.
Hager, T (1995). Force of Nature: The Life of Linus Pauling.
Simon and Schuster. ISBN0-684-80909-5.
Hardison, RC (June 11, 1996). "A brief history of hemoglobins: plant,
animal, protist, and bacteria" (http:/ / www. pubmedcentral. gov/
articlerender. fcgi?tool=pubmed& pubmedid=8650150). Proc Natl Acad Sci
USA 93 (12): 56759. doi: 10.1073/pnas.93.12.5675 (http:/ / dx. doi. org/ 10.
1073/ pnas. 93. 12. 5675). PMC 39118 (http:/ / www. ncbi. nlm. nih. gov/
pmc/ articles/ PMC39118). PMID 8650150 (http:/ / www. ncbi. nlm. nih.
gov/ pubmed/ 8650150).
Kneipp, J; Balakrishnan, G; Chen, R, Shen TJ, Sahu SC, Ho NT,
Giovannelli JL, Simplaceanu V, Ho C, Spiro TG; Shen, TJ; Sahu, SC; Ho,
NT; Giovannelli, JL; Simplaceanu, V et al. (November 22, 2005).
"Dynamics of allostery in hemoglobin: roles of the penultimate tyrosine H
bonds". J Mol Biol 356 (2): 33553. doi: 10.1016/j.jmb.2005.11.006 (http:/ /
dx. doi. org/ 10. 1016/ j. jmb. 2005. 11. 006). PMID 16368110 (http:/ /
www. ncbi. nlm. nih. gov/ pubmed/ 16368110) .
Steinberg, MH (2001). Disorders of Hemoglobin: Genetics,
Pathophysiology, and Clinical Management (http:/ / books. google. com/
books?vid=ISBN0521632668). Cambridge University Press.
ISBN0-521-63266-8.
External links
Hemoglobin - Test, Levels and Information (http:/ / www. medicinenet. com/ hemoglobin/ article. htm) on
MedicineNet
Interactive hemoglobin saturation curves (http:/ / www. altitude. org/ hemoglobin_saturation. php)
Interactive models of hemoglobin (http:/ / www. ufp. pt/ ~pedros/ anim/ 2frame-hben. htm) (Requires MDL
Chime (http:/ / www. mdl. com/ products/ framework/ chime/ ))
National Anemia Action Council (http:/ / www. anemia. org/ ) - anemia.org
New hemoglobin type causes mock diagnosis with pulse oxymeters (http:/ / www. life-of-science. net/ medicine/
news/ new-hemoglobin-type-discovered-causing-mock-diagnosis-of-cardiac-insufficiency. html)
129
Enzyme mechanisms
Enzyme catalysis
Enzyme catalysis is the catalysis of chemical reactions by specialized proteins known as enzymes. Catalysis of
biochemical reactions in the cell is vital due to the very low reaction rates of the uncatalysed reactions.
[citation needed]
The mechanism of enzyme catalysis is similar in principle to other types of chemical catalysis. By providing an
alternative reaction route the enzyme reduces the energy required to reach the highest energy transition state of the
reaction. The reduction of activation energy (Ea) increases the number of reactant molecules with enough energy to
reach the activation energy and form the product.
Induced fit
Diagrams to show the induced fit hypothesis of enzyme action
The favored model for the
enzyme-substrate interaction is the
induced fit model.
[1]
This model
proposes that the initial interaction
between enzyme and substrate is
relatively weak, but that these weak
interactions rapidly induce
conformational changes in the enzyme
that strengthen binding.
The advantages of the induced fit
mechanism arise due to the stabilizing effect of strong enzyme binding. There are two different mechanisms of
substrate binding: uniform binding, which has strong substrate binding, and differential binding, which has strong
transition state binding. The stabilizing effect of uniform binding increases both substrate and transition state binding
affinity, while differential binding increases only transition state binding affinity. Both are used by enzymes and
have been evolutionarily chosen to minimize the Ea of the reaction. Enzymes which are saturated, that is, have a
high affinity substrate binding, require differential binding to reduce the Ea, whereas small substrate unbound
enzymes may use either differential or uniform binding.
Enzyme catalysis
130
The different mechanisms of substrate binding
These effects have led to most proteins using the differential
binding mechanism to reduce the Ea, so most proteins have high
affinity of the enzyme to the transition state. Differential binding is
carried out by the induced fit mechanism - the substrate first binds
weakly, then the enzyme changes conformation increasing the
affinity to the transition state and stabilizing it, so reducing the
activation energy to reach it.
It is important to clarify, however, that the induced fit concept cannot be used to rationalize catalysis. That is, the
chemical catalysis is defined as the reduction of Ea

(when the system is already in the ES

) relative to Ea

in the
uncatalyzed reaction in water (without the enzyme). The induced fit only suggests that the barrier is lower in the
closed form of the enzyme but does not tell us what the reason for the barrier reduction is.
Induced fit may be beneficial to the fidelity of molecular recognition in the presence of competition and noise via the
conformational proofreading mechanism .
[2]
Mechanisms of an alternative reaction route
These conformational changes also bring catalytic residues in the active site close to the chemical bonds in the
substrate that will be altered in the reaction. After binding takes place, one or more mechanisms of catalysis lowers
the energy of the reaction's transition state, by providing an alternative chemical pathway for the reaction. There are
six possible mechanisms of "over the barrier" catalysis as well as a "through the barrier" mechanism:
Bond strain
This is the principal effect of induced fit binding, where the affinity of the enzyme to the transition state is greater
than to the substrate itself. This induces structural rearrangements which strain substrate bonds into a position closer
to the conformation of the transition state, so lowering the energy difference between the substrate and transition
state and helping catalyze the reaction.
However, the strain effect is, in fact, a ground state destabilization effect, rather than transition state stabilization
effect.
[3][4]
Furthermore, enzymes are very flexible and they cannot apply large strain effect.
[5]
In addition to bond strain in the substrate, bond strain may also be induced within the enzyme itself to activate
residues in the active site.
Enzyme catalysis
131
For example:
Substrate, bound substrate, and transition state conformations of lysozyme.
The substrate, on binding, is distorted from the half chair conformation of the hexose ring (because of the steric hindrance with amino acids of the
protein forcing the equatorial c6 to be in the axial position) into the chair conformation
[6]
Proximity and orientation
This increases the rate of the reaction as enzyme-substrate interactions align reactive chemical groups and hold them
close together. This reduces the entropy of the reactants and thus makes reactions such as ligations or addition
reactions more favorable, there is a reduction in the overall loss of entropy when two reactants become a single
product.
This effect is analogous to an effective increase in concentration of the reagents. The binding of the reagents to the
enzyme gives the reaction intramolecular character, which gives a massive rate increase.
For example:
Similar reactions will occur far faster if the reaction is intramolecular.
The effective concentration of acetate in the intramolecular reaction can be estimated as k
2
/k
1
= 2 x 10
5
Molar.
However, the situation might be more complex, since modern computational studies have established that traditional
examples of proximity effects cannot be related directly to enzyme entropic effects.
[7][8][9]
Also, the original entropic
proposal
[10]
has been found to largely overestimate the contribution of orientation entropy to catalysis.
[11]
Enzyme catalysis
132
Proton donors or acceptors
Proton donors and acceptors, i.e. acids and base may donate and accept protons in order to stabilize developing
charges in the transition state.This typically has the effect of activating nucleophile and electrophile groups, or
stabilizing leaving groups. Histidine is often the residue involved in these acid/base reactions, since it has a pKa
close to neutral pH and can therefore both accept and donate protons.
Many reaction mechanisms involving acid/base catalysis assume a substantially altered pKa. This alteration of pKa
is possible through the local environment of the residue.
Conditions Acids Bases
Hydrophobic environment Increase pKa Decrease pKa
Adjacent residues of like charge Increase pKa Decrease pKa
Salt bridge (and hydrogen
bond) formation
Decrease pKa Increase pKa
pKa can also be influenced significantly by the surrounding environment, to the extent that residues which are basic
in solution may act as proton donors, and vice versa.
For example:
Serine protease catalytic mechanism
The initial step of the serine protease catalytic mechanism involves the histidine of the active site accepting a proton from the serine residue. This
prepares the serine as a nucleophile to attack the amide bond of the substrate. This mechanism includes donation of a proton from serine (a base,
pKa 14) to histidine (an acid, pKa 6), made possible due to the local environment of the bases.
It is important to clarify that the modification of the pKas is a pure part of the electrostatic mechanism.
[4]
Furthermore, the catalytic effect of the above example is mainly associated with the reduction of the pKa of the
oxyanion and the increase in the pKa of the histidine, while the proton transfer from the serine to the histidine is not
catalyzed significantly, since it is not the rate determining barrier.
[12]
Enzyme catalysis
133
Electrostatic catalysis
Stabilization of charged transition states can also be by residues in the active site forming ionic bonds (or partial
ionic charge interactions) with the intermediate. These bonds can either come from acidic or basic side chains found
on amino acids such as lysine, arginine, aspartic acid or glutamic acid or come from metal cofactors such as zinc.
Metal ions are particularly effective and can reduce the pKa of water enough to make it an effective nucleophile.
Systematic computer simulation studies established that electrostatic effects give, by far, the largest contribution to
catalysis.
[4]
In particular, it has been found that enzyme provides an environment which is more polar than water,
and that the ionic transition states are stabilized by fixed dipoles. This is very different from transition state
stabilization in water, where the water molecules must pay with "reorganization energy".
[13]
In order to stabilize
ionic and charged states. Thus, the catalysis is associated with the fact that the enzyme polar groups are preorganized
[14]
Binding of substrate usually excludes water from the active site, thereby lowering the local dielectric constant to that
of an organic solvent. This strengthens the electrostatic interactions between the charged/polar substrates and the
active sites. In addition, studies have shown that the charge distributions about the active sites are arranged so as to
stabilize the transition states of the catalyzed reactions. In several enzymes, these charge distributions apparently
serve to guide polar substrates toward their binding sites so that the rates of these enzymatic reactions are greater
than their apparent diffusion-controlled limits.
For example:
Carboxypeptidase catalytic mechanism
The tetrahedral intermediate is stabilised by a partial ionic bond between the Zn
2+
ion and the negative charge on the oxygen.
Covalent catalysis
Covalent catalysis involves the substrate forming a transient covalent bond with residues in the active site or with a
cofactor. This adds an additional covalent intermediate to the reaction, and helps to reduce the energy of later
transition states of the reaction. The covalent bond must, at a later stage in the reaction, be broken to regenerate the
enzyme. This mechanism is found in enzymes such as proteases like chymotrypsin and trypsin, where an
acyl-enzyme intermediate is formed. Schiff base formation using the free amine from a lysine residue is another
mechanism, as seen in the enzyme aldolase during glycolysis.
Some enzymes utilize non-amino acid cofactors such as pyridoxal phosphate (PLP) or thiamine pyrophosphate
(TPP) to form covalent intermediates with reactant molecules.
[15][16]
Such covalent intermediates function to reduce
the energy of later transition states, similar to how covalent intermediates formed with active site amino acid
residues allow stabilization, but the capabilities of cofactors allow enzymes to carryout reactions that amino acid side
Enzyme catalysis
134
residues alone could not. Enzymes utilizing such cofactors include the PLP-dependent enzyme aspartate
transaminase and the TPP-dependent enzyme pyruvate dehydrogenase.
[][]
It is important to clarify that covalent catalysis does correspond in most cases to simply the use of a specific
mechanism rather than to true catalysis.
[4]
For example, the energetics of the covalent bond to the serine molecule in
chymotrypsin should be compared to the well-understood covalent bond to the nucleophile in the uncatalyzed
solution reaction. A true proposal of a covalent catalysis (where the barrier is lower than the corresponding barrier in
solution) would require, for example, a partial covalent bond to the transition state by an enzyme group (e.g., a very
strong hydrogen bond), and such effects do not contribute significantly to catalysis.
Quantum tunneling
These traditional "over the barrier" mechanisms have been challenged in some cases by models and observations of
"through the barrier" mechanisms (quantum tunneling). Some enzymes operate with kinetics which are faster than
what would be predicted by the classical G

. In "through the barrier" models, a proton or an electron can tunnel

through activation barriers.
[17][]
Quantum tunneling for protons has been observed in tryptamine oxidation by
aromatic amine dehydrogenase.
[9]
Interestingly, quantum tunneling does not appear to provide a major catalytic advantage, since the tunneling
contributions are similar in the catalyzed and the uncatalyzed reactions in solution.
[][18][19][20]
However, the
tunneling contribution (typically enhancing rate constants by a factor of ~1000
[9]
compared to the rate of reaction for
the classical 'over the barrier' route) is likely crucial to the viability of biological organisms. This emphasizes the
general importance of tunneling reactions in biology.
In 1971-1972 the first quantum-mechanical model of enzyme catalysis was
formulated.
[21][22]
Wikipedia:Independent sources
Active enzyme
The binding energy of the enzyme-substrate complex cannot be considered as an external energy which is necessary
for the substrate activation. The enzyme of high energy content may firstly transfer some specific energetic group X
1
from catalytic site of the enzyme to the final place of the first bound reactant, then another group X
2
from the second
bound reactant (or from the second group of the single reactant) must be transferred to active site to finish substrate
conversion to product and enzyme regeneration.
[23]
We can present the whole enzymatic reaction as a two coupling reactions:
S
1
+ EX
1
=> S
1
EX
1
=> P
1
+ EP
2
(1)
S
2
+ EP
2
=> S
2
EP
2
=> P
2
+ EX
2
(2)
It may be seen from reaction (1) that the group X
1
of the active enzyme appears in the product due to possibility of
the exchange reaction inside enzyme to avoid both electrostatic inhibition and repulsion of atoms. So we represent
the active enzyme as a powerful reactant of the enzymatic reaction. The reaction (2) shows incomplete conversion of
the substrate because its group X
2
remains inside enzyme. This approach as idea had formerly proposed relying on
the hypothetical extremely high enzymatic conversions (catalytically perfect enzyme).
[24]
The crucial point for the verication of the present approach is that the catalyst must be a complex of the enzyme
with the transfer group of the reaction. This chemical aspect is supported by the well-studied mechanisms of the
several enzymatic reactions. Let us consider the reaction of peptide bond hydrolysis catalyzed by a pure protein
-chymotrypsin (an enzyme acting without a cofactor), which is a well-studied member of the serine proteases
family, see.
[25]
We present the experimental results for this reaction as two chemical steps:
Enzyme catalysis
135
S
1
+ EH => P
1
+ EP
2
(3)
EP
2
+ HOH => EH + P
2
(4)
where S
1
is a polypeptide, P
1
and P
2
are products. The rst chemical step (3) includes the formation of a covalent
acyl-enzyme intermediate. The second step (4) is the deacylation step. It is important to note that the group H+ ,
initially found on the enzyme, but not in water, appears in the product before the step of hydrolysis, therefore it may
be considered as an additional group of the enzymatic reaction.
Thus, the reaction (3) shows that the enzyme acts as a powerful reactant of the reaction. According to the proposed
concept, the H transport from the enzyme promotes the rst reactant conversion, breakdown of the rst initial
chemical bond (between groups P
1
and P
2
). The step of hydrolysis leads to a breakdown of the second chemical bond
and regeneration of the enzyme.
The proposed chemical mechanism does not depend on the concentration of the substrates or products in the
medium. However, a shift in their concentration mainly causes free energy changes in the rst and nal steps of the
reactions (1) and (2) due to the changes in the free energy content of every molecule, whether S or P, in water
solution. This approach is in accordance with the following mechanism of muscle contraction. The nal step of ATP
hydrolysis in skeletal muscle is the product release caused by the association of myosin heads with actin.
[26]
The
closing of the actin-binding cleft during the association reaction is structurally coupled with the opening of the
nucleotide-binding pocket on the myosin active site.
[27]
Notably, the nal steps of ATP hydrolysis include the fast release of phosphate and the slow release of ADP.
[28][29]
The release of a phosphate anion from bound ADP anion into water solution may be considered as an exergonic
reaction because the phosphate anion has low molecular mass.
Thus, we arrive at the conclusion that the primary release of the inorganic phosphate H
2
PO
4
-
leads to transformation
of a signicant part of the free energy of ATP hydrolysis into the kinetic energy of the solvated phosphate, producing
active streaming. This assumption of a local mechano-chemical transduction is in accord with Tiroshs mechanism of
muscle contraction, where the muscle force derives from an integrated action of active streaming created by ATP
hydrolysis.
[30][31]
Examples of catalytic mechanisms
In reality, most enzyme mechanisms involve a combination of several different types of catalysis.
Triose phosphate isomerase
Triose phosphate isomerase (EC 5.3.1.1
[32]
) catalyses the reversible interconvertion of the two triose phosphates
isomers dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate.
Trypsin
Trypsin (EC 3.4.21.4
[33]
) is a serine protease that cleaves protein substrates at lysine and arginine amino acid
residues.
Aldolase
Aldolase (EC 4.1.2.13
[34]
) catalyses the breakdown of fructose 1,6-bisphosphate (F-1,6-BP) into glyceraldehyde
3-phosphate and dihydroxyacetone phosphate (DHAP).
Enzyme catalysis
136
References
[3] [3] Jencks W.P. "Catalysis in Chemistry and Enzymology" 1987, Dover, New York
[4] [4] Warshel A., Sharma P.K., Kato M., Xiang Y., Liu H., and Olsson M.H.M. "Electrostatic Basis of Enzyme Catalysis", Chem. Rev. 2006, 106:
3210-3235.
[6] <fundamentals of biochemistry Voet, voet and Pratt 4th edition>, which is similar in shape to the transition state.
[13] [13] Marcus R. A. "On the Theory of Electron-Transfer Reactions. VI. Unified Treatment for Homogeneous and Electrode Reactions" J. Chem.
Phys. 1965 43:679-701
[14] [14] Warshel A. "Energetics of Enzyme Catalysis", Proc. Natl. Acad. Sci. USA, 1978 75: 5250
[15] [15] Toney, M. D. "Reaction specificity in pyridoxal enzymes." Archives of biochemistry and biophysics (2005) 433: 279-287
[16] Micronutrient Information Center, Oregon State University (http:/ / lpi. oregonstate. edu/ infocenter/ vitamins/ thiamin/ )
[21] Volkenshtein M.V., Dogonadze R.R., Madumarov A.K., Urushadze Z.D., Kharkats Yu.I. Theory of Enzyme Catalysis.- Molekuliarnaya
Biologia, Moscow, 6, 1972, 431-439
[22] Volkenshtein M.V., Dogonadze R.R., Madumarov A.K., Urushadze Z.D., Kharkats Yu.I. Electronic and Conformational Interactions in
Enzyme Catalysis. In: E.L. Andronikashvili (Ed.), Konformatsionnie Izmenenia Biopolimerov v Rastvorakh, Publishing House "Nauka",
Moscow, 1973, 153-157
[23] Foigel, A.G. (2011). "Is the enzyme a powerful reactant of the biochemical reaction?". Mol. Cell. Biochem 352: 87-89
[24] Fogel, A.G. (1982). "Cooperativity of enzymatic reactions and molecular aspects of energy transduction". Mol. Cell. Biochem 47: 5964
[25] Hengge, AC, Stein, RL. (2004). "Role of protein conformational mobility in enzyme catalysis: acylation of alpha-chymotrypsin by specic
peptide substrates". Biochemistry 43 : 742-747
[26] Lymn, RW, Taylor, EW. (1971). "Mechanism of adenosine triphosphate hydrolysis by actomyosin. Biochemistry 10: 46174624
[27] Holmes, KC, Angert, I, Kull, FG, Jahn, W, Schroder, RR. (2003). "Electron cryo-microscopy shows how strong binding of myosin to actin
releases nucleotide". Nature 425: 423427
[28] Siemankowski, RF, Wiseman, MO, White, HD. (1985). "ADP dissociation from actomyosin subfragment 1 is sufciently slow to limit the
unloaded shortening velocity in vertebrate muscle". Proc. Natl. Acad. Sci. USA 82: 658662
[29] White, HD, Belknap, B, Webb, MR. (1997). "Kinetics of nucleoside triphosphate cleavage and phosphate release steps by associated rabbit
skeletal actomyosin, measured using a novel uorescent probe for phosphate". Biochemistry 36: 1182811836
[30] Tirosh, R, Low, WZ, Oplatka, A. (1990). "Translational motion of actin laments in the presence of heavy meromyosin and MgATP as
measured by Doppler broadening of laser light scattering". Biochim. Biophys. Acta 1037: 274280
[31] Tirosh, R. (2006). "Ballistic protons and microwave-induced water solutions (solitons) in bioenergetic transformations". Int. J. Mol. Sci. 7:
320345
[32] http:/ / enzyme. expasy. org/ EC/ 5.3.1.1
[33] http:/ / enzyme. expasy. org/ EC/ 3.4.21.4
[34] http:/ / enzyme. expasy. org/ EC/ 4.1.2.13
Further reading
Alan Fersht, Structure and Mechanism in Protein Science : A Guide to Enzyme Catalysis and Protein Folding. W.
H. Freeman, 1998. ISBN 0-7167-3268-8
Dedicated issue of Philosophical Transactions B on Quantum catalysis in enzymes freely available. (http:/ /
publishing. royalsociety. org/ quantum-catalysis)
137
Enzyme kinetics
Enzyme kinetics
Dihydrofolate reductase from E. coli with its two substrates dihydrofolate
(right) and NADPH (left), bound in the active site. The protein is shown as a
ribbon diagram, with alpha helices in red, beta sheets in yellow and loops in
blue. Generated from 7DFR
[1]
.
Enzyme kinetics is the study of the chemical
reactions that are catalysed by enzymes. In
enzyme kinetics, the reaction rate is measured and
the effects of varying the conditions of the
reaction is investigated. Studying an enzyme's
kinetics in this way can reveal the catalytic
mechanism of this enzyme, its role in
metabolism, how its activity is controlled, and
how a drug or an agonist might inhibit the
enzyme.
Enzymes are usually protein molecules that
manipulate other molecules the enzymes'
substrates. These target molecules bind to an
enzyme's active site and are transformed into
products through a series of steps known as the
enzymatic mechanism. These mechanisms can be
divided into single-substrate and
multiple-substrate mechanisms. Kinetic studies
on enzymes that only bind one substrate, such as
triosephosphate isomerase, aim to measure the
affinity with which the enzyme binds this
substrate and the turnover rate. Some other
examples of enzymes are phosphofructokinase
and hexokinase, both of which are important for
cellular respiration (glycolysis).
When enzymes bind multiple substrates, such as
dihydrofolate reductase (shown right), enzyme kinetics can also show the sequence in which these substrates bind
and the sequence in which products are released. An example of enzymes that bind a single substrate and release
multiple products are proteases, which cleave one protein substrate into two polypeptide products. Others join two
substrates together, such as DNA polymerase linking a nucleotide to DNA. Although these mechanisms are often a
complex series of steps, there is typically one rate-determining step that determines the overall kinetics. This
rate-determining step may be a chemical reaction or a conformational change of the enzyme or substrates, such as
those involved in the release of product(s) from the enzyme.
Knowledge of the enzyme's structure is helpful in interpreting kinetic data. For example, the structure can suggest
how substrates and products bind during catalysis; what changes occur during the reaction; and even the role of
particular amino acid residues in the mechanism. Some enzymes change shape significantly during the mechanism;
in such cases, it is helpful to determine the enzyme structure with and without bound substrate analogues that do not
undergo the enzymatic reaction.
Enzyme kinetics
138
Not all biological catalysts are protein enzymes; RNA-based catalysts such as ribozymes and ribosomes are essential
to many cellular functions, such as RNA splicing and translation. The main difference between ribozymes and
enzymes is that RNA catalysts are composed of nucleotides, whereas enzymes are composed of amino acids.
Ribozymes also perform a more limited set of reactions, although their reaction mechanisms and kinetics can be
analysed and classified by the same methods.
General principles
As larger amounts of substrate are added to a reaction, the available enzyme
binding sites become filled to the limit of . Beyond this limit the enzyme is
saturated with substrate and the reaction rate ceases to increase.
The reaction catalysed by an enzyme uses
exactly the same reactants and produces
exactly the same products as the uncatalysed
reaction. Like other catalysts, enzymes do
not alter the position of equilibrium between
substrates and products.
[2]
However, unlike
uncatalysed chemical reactions,
enzyme-catalysed reactions display
saturation kinetics. For a given enzyme
concentration and for relatively low
substrate concentrations, the reaction rate
increases linearly with substrate
concentration; the enzyme molecules are
largely free to catalyse the reaction, and
increasing substrate concentration means an increasing rate at which the enzyme and substrate molecules encounter
one another. However, at relatively high substrate concentrations, the reaction rate asymptotically approaches the
theoretical maximum; the enzyme active sites are almost all occupied and the reaction rate is determined by the
intrinsic turnover rate of the enzyme. The substrate concentration midway between these two limiting cases is
denoted by K
M
.
The two most important kinetic properties of an enzyme are how quickly the enzyme becomes saturated with a
particular substrate, and the maximum rate it can achieve. Knowing these properties suggests what an enzyme might
do in the cell and can show how the enzyme will respond to changes in these conditions.
Enzyme kinetics
139
Enzyme assays
Progress curve for an enzyme reaction. The slope in the initial rate
period is the initial rate of reaction v. The MichaelisMenten
equation describes how this slope varies with the concentration of
substrate.
Enzyme assays are laboratory procedures that measure
the rate of enzyme reactions. Because enzymes are not
consumed by the reactions they catalyse, enzyme
assays usually follow changes in the concentration of
either substrates or products to measure the rate of
reaction. There are many methods of measurement.
Spectrophotometric assays observe change in the
absorbance of light between products and reactants;
radiometric assays involve the incorporation or release
of radioactivity to measure the amount of product made
over time. Spectrophotometric assays are most
convenient since they allow the rate of the reaction to
be measured continuously. Although radiometric assays
require the removal and counting of samples (i.e., they
are discontinuous assays) they are usually extremely
sensitive and can measure very low levels of enzyme
activity.
[3]
An analogous approach is to use mass
spectrometry to monitor the incorporation or release of stable isotopes as substrate is converted into product.
The most sensitive enzyme assays use lasers focused through a microscope to observe changes in single enzyme
molecules as they catalyse their reactions. These measurements either use changes in the fluorescence of cofactors
during an enzyme's reaction mechanism, or of fluorescent dyes added onto specific sites of the protein to report
movements that occur during catalysis.
[4]
These studies are providing a new view of the kinetics and dynamics of
single enzymes, as opposed to traditional enzyme kinetics, which observes the average behaviour of populations of
millions of enzyme molecules.
[5][6]
An example progress curve for an enzyme assay is shown above. The enzyme produces product at an initial rate that
is approximately linear for a short period after the start of the reaction. As the reaction proceeds and substrate is
consumed, the rate continuously slows (so long as substrate is not still at saturating levels). To measure the initial
(and maximal) rate, enzyme assays are typically carried out while the reaction has progressed only a few percent
towards total completion. The length of the initial rate period depends on the assay conditions and can range from
milliseconds to hours. However, equipment for rapidly mixing liquids allows fast kinetic measurements on initial
rates of less than one second.
[7]
These very rapid assays are essential for measuring pre-steady-state kinetics, which
are discussed below.
Most enzyme kinetics studies concentrate on this initial, approximately linear part of enzyme reactions. However, it
is also possible to measure the complete reaction curve and fit this data to a non-linear rate equation. This way of
measuring enzyme reactions is called progress-curve analysis.
[8]
This approach is useful as an alternative to rapid
kinetics when the initial rate is too fast to measure accurately.
Single-substrate reactions
Enzymes with single-substrate mechanisms include isomerases such as triosephosphateisomerase or
bisphosphoglycerate mutase, intramolecular lyases such as adenylate cyclase and the hammerhead ribozyme, an
RNA lyase.
[9]
However, some enzymes that only have a single substrate do not fall into this category of mechanisms.
Catalase is an example of this, as the enzyme reacts with a first molecule of hydrogen peroxide substrate, becomes
oxidised and is then reduced by a second molecule of substrate. Although a single substrate is involved, the existence
of a modified enzyme intermediate means that the mechanism of catalase is actually a pingpong mechanism, a type
Enzyme kinetics
140
of mechanism that is discussed in the Multi-substrate reactions section below.
MichaelisMenten kinetics
Saturation curve for an enzyme showing the relation between the concentration of
substrate and rate.
Single-substrate mechanism for an enzyme reaction. k
1
, k
1
and k
2
are the rate
constants for the individual steps.
As enzyme-catalysed reactions are
saturable, their rate of catalysis does not
show a linear response to increasing
substrate. If the initial rate of the reaction is
measured over a range of substrate
concentrations (denoted as [S]), the reaction
rate (v) increases as [S] increases, as shown
on the right. However, as [S] gets higher,
the enzyme becomes saturated with
substrate and the rate reaches V
max
, the
enzyme's maximum rate.
The MichaelisMenten kinetic model of a
single-substrate reaction is shown on the
right. There is an initial bimolecular reaction
between the enzyme E and substrate S to
form the enzymesubstrate complex ES.
Although the enzymatic mechanism for the
unimolecular reaction can
be quite complex, there is typically one
rate-determining enzymatic step that allows
this reaction to be modelled as a single
catalytic step with an apparent unimolecular
rate constant k
cat
. If the reaction path
proceeds over one or several intermediates,
k
cat
will be a function of several elementary
rate constants, whereas in the simplest case
of a single elementary reaction (e.g. no intermediates) it will be identical to the elementary unimolecular rate
constant k
2
. The apparent unimolecular rate constant k
cat
is also called turnover number and denotes the maximum
number of enzymatic reactions catalysed per second.
The MichaelisMenten equation
[10]
describes how the (initial) reaction rate v
0
depends on the position of the
substrate-binding equilibrium and the rate constant k
2
.
(MichaelisMenten equation)
with the constants
This MichaelisMenten equation is the basis for most single-substrate enzyme kinetics. Two crucial assumptions
underlie this equation (apart from the general assumption about the mechanism only involving no intermediate or
product inhibition, and there is no allostericity or cooperativity). The first assumption is the so-called
quasi-steady-state assumption (or pseudo-steady-state hypothesis), namely that the concentration of the
substrate-bound enzyme (and hence also the unbound enzyme) changes much more slowly than those of the product
Enzyme kinetics
141
and substrate and thus the change over time of the complex can be set to zero . The second assumption is that the
total enzyme concentration does not change over time, thus . A complete derivation can be found here.
The Michaelis constant K
M
is experimentally defined as the concentration at which the rate of the enzyme reaction is
half V
max
, which can be verified by substituting [S] = K
m
into the MichaelisMenten equation and can also be seen
graphically. If the rate-determining enzymatic step is slow compared to substrate dissociation ( ), the
Michaelis constant K
M
is roughly the dissociation constant K
D
of the ES complex.
If is small compared to then the term and also very little ES complex is
formed, thus . Therefore, the rate of product formation is
Thus the product formation rate depends on the enzyme concentration as well as on the substrate concentration, the
equation resembles a bimolecular reaction with a corresponding pseudo-second order rate constant . This
constant is a measure of catalytic efficiency. The most efficient enzymes reach a in the range of 10
8

10
10
M
1
s
1
. These enzymes are so efficient they effectively catalyse a reaction each time they encounter a
substrate molecule and have thus reached an upper theoretical limit for efficiency (diffusion limit); these enzymes
have often been termed perfect enzymes.
[11]
Direct use of the MichaelisMenten equation for time course kinetic analysis
The observed velocities predicted by the MichaelisMenten equation can be used to directly model the time course
disappearance of substrate and the production of product through incorporation of the MichaelisMenten equation
into the equation for first order chemical kinetics. This can only be achieved however if one recognises the problem
associated with the use of Euler's number in the description of first order chemical kinetics. i.e. e
-k
is a split constant
that introduces a systematic error into calculations and can be rewritten as a single constant which represents the
remaining substrate after each time period.
[12]
In 1983 Stuart Beal (and also independently Santiago Schnell and Claudio Mendoza in 1997) derived a closed form
solution for the time course kinetics analysis of the Michaelis-Menten mechanism.
[13][14]
The solution, known as the
Schnell-Mendoza equation, has the form:
where W[] is the Lambert-W function.
[15][16]
Enzyme kinetics
142
Linear plots of the MichaelisMenten equation
LineweaverBurk or double-reciprocal plot of kinetic data, showing the significance of
the axis intercepts and gradient.
The plot of v versus [S] above is not
linear; although initially linear at low
[S], it bends over to saturate at high
[S]. Before the modern era of nonlinear
curve-fitting on computers, this
nonlinearity could make it difficult to
estimate K
M
and V
max
accurately.
Therefore, several researchers
developed linearisations of the
MichaelisMenten equation, such as
the LineweaverBurk plot, the
EadieHofstee diagram and the
HanesWoolf plot. All of these linear
representations can be useful for
visualising data, but none should be
used to determine kinetic parameters,
as computer software is readily available that allows for more accurate determination by nonlinear regression
methods.
[17]
The LineweaverBurk plot or double reciprocal plot is a common way of illustrating kinetic data. This is produced
by taking the reciprocal of both sides of the MichaelisMenten equation. As shown on the right, this is a linear form
of the MichaelisMenten equation and produces a straight line with the equation y = mx + c with a y-intercept
equivalent to 1/V
max
and an x-intercept of the graph representing 1/K
M
.
Naturally, no experimental values can be taken at negative 1/[S]; the lower limiting value 1/[S] = 0 (the y-intercept)
corresponds to an infinite substrate concentration, where 1/v=1/V
max
as shown at the right; thus, the x-intercept is an
extrapolation of the experimental data taken at positive concentrations. More generally, the LineweaverBurk plot
skews the importance of measurements taken at low substrate concentrations and, thus, can yield inaccurate
estimates of V
max
and K
M
.
[]
A more accurate linear plotting method is the Eadie-Hofstee plot. In this case, v is
plotted against v/[S]. In the third common linear representation, the Hanes-Woolf plot, [S]/v is plotted against [S]. In
general, data normalisation can help diminish the amount of experimental work and can increase the reliability of the
output, and is suitable for both graphical and numerical analysis.
[18]
Practical significance of kinetic constants
The study of enzyme kinetics is important for two basic reasons. Firstly, it helps explain how enzymes work, and
secondly, it helps predict how enzymes behave in living organisms. The kinetic constants defined above, K
M
and
V
max
, are critical to attempts to understand how enzymes work together to control metabolism.
Making these predictions is not trivial, even for simple systems. For example, oxaloacetate is formed by malate
dehydrogenase within the mitochondrion. Oxaloacetate can then be consumed by citrate synthase,
phosphoenolpyruvate carboxykinase or aspartate aminotransferase, feeding into the citric acid cycle,
gluconeogenesis or aspartic acid biosynthesis, respectively. Being able to predict how much oxaloacetate goes into
which pathway requires knowledge of the concentration of oxaloacetate as well as the concentration and kinetics of
each of these enzymes. This aim of predicting the behaviour of metabolic pathways reaches its most complex
expression in the synthesis of huge amounts of kinetic and gene expression data into mathematical models of entire
Enzyme kinetics
143
organisms. Alternatively, one useful simplification of the metabolic modelling problem is to ignore the underlying
enzyme kinetics and only rely on information about the reaction network's stoichiometry, a technique called flux
balance analysis.
[19][20]
MichaelisMenten kinetics with intermediate
One could also consider the less simple case
where a complex with the enzyme and an intermediate exists and the intermediate is converted into product in a
second step. In this case we have a very similar equation
[21]
but the constants are different
We see that for the limiting case , thus when the last step from EI to E + P is much faster than the
previous step, we get again the original equation. Mathematically we have then and .
Multi-substrate reactions
Multi-substrate reactions follow complex rate equations that describe how the substrates bind and in what sequence.
The analysis of these reactions is much simpler if the concentration of substrate A is kept constant and substrate B
varied. Under these conditions, the enzyme behaves just like a single-substrate enzyme and a plot of v by [S] gives
apparent K
M
and V
max
constants for substrate B. If a set of these measurements is performed at different fixed
concentrations of A, these data can be used to work out what the mechanism of the reaction is. For an enzyme that
takes two substrates A and B and turns them into two products P and Q, there are two types of mechanism: ternary
complex and pingpong.
Ternary-complex mechanisms
Random-order ternary-complex mechanism for an enzyme reaction. The reaction path
is shown as a line and enzyme intermediates containing substrates A and B or
products P and Q are written below the line.
In these enzymes, both substrates bind to
the enzyme at the same time to produce an
EAB ternary complex. The order of
binding can either be random (in a
random mechanism) or substrates have to
bind in a particular sequence (in an
ordered mechanism). When a set of v by
[S] curves (fixed A, varying B) from an
enzyme with a ternary-complex
mechanism are plotted in a
LineweaverBurk plot, the set of lines
produced will intersect.
Enzyme kinetics
144
Enzymes with ternary-complex mechanisms include glutathione S-transferase,
[22]
dihydrofolate reductase
[23]
and
DNA polymerase.
[24]
The following links show short animations of the ternary-complex mechanisms of the enzymes
dihydrofolate reductase
[]
and DNA polymerase
[]
.
Pingpong mechanisms
Pingpong mechanism for an enzyme reaction. Intermediates contain substrates A
and B or products P and Q.
As shown on the right, enzymes with a
ping-pong mechanism can exist in two
states, E and a chemically modified form
of the enzyme E*; this modified enzyme
is known as an intermediate. In such
mechanisms, substrate A binds, changes
the enzyme to E* by, for example, transferring a chemical group to the active site, and is then released. Only after
the first substrate is released can substrate B bind and react with the modified enzyme, regenerating the unmodified
E form. When a set of v by [S] curves (fixed A, varying B) from an enzyme with a pingpong mechanism are plotted
in a LineweaverBurk plot, a set of parallel lines will be produced. This is called a secondary plot.
Enzymes with pingpong mechanisms include some oxidoreductases such as thioredoxin peroxidase,
[25]
transferases
such as acylneuraminate cytidylyltransferase
[26]
and serine proteases such as trypsin and chymotrypsin.
[27]
Serine
proteases are a very common and diverse family of enzymes, including digestive enzymes (trypsin, chymotrypsin,
and elastase), several enzymes of the blood clotting cascade and many others. In these serine proteases, the E*
intermediate is an acyl-enzyme species formed by the attack of an active site serine residue on a peptide bond in a
protein substrate. A short animation showing the mechanism of chymotrypsin is linked here.
[]
Non-MichaelisMenten kinetics
Saturation curve for an enzyme reaction showing sigmoid kinetics.
Some enzymes produce a sigmoid v by [S]
plot, which often indicates cooperative
binding of substrate to the active site. This
means that the binding of one substrate
molecule affects the binding of subsequent
substrate molecules. This behavior is most
common in multimeric enzymes with
several interacting active sites.
[28]
Here, the
mechanism of cooperation is similar to that
of hemoglobin, with binding of substrate to
one active site altering the affinity of the
other active sites for substrate molecules.
Positive cooperativity occurs when binding
of the first substrate molecule increases the
affinity of the other active sites for substrate.
Negative cooperativity occurs when binding
of the first substrate decreases the affinity of
the enzyme for other substrate molecules.
Allosteric enzymes include mammalian tyrosyl tRNA-synthetase, which shows negative cooperativity,
[29]
and
bacterial aspartate transcarbamoylase
[30]
and phosphofructokinase,
[31]
which show positive cooperativity.
Cooperativity is surprisingly common and can help regulate the responses of enzymes to changes in the
concentrations of their substrates. Positive cooperativity makes enzymes much more sensitive to [S] and their
Enzyme kinetics
145
activities can show large changes over a narrow range of substrate concentration. Conversely, negative cooperativity
makes enzymes insensitive to small changes in [S].
The Hill equation (biochemistry)
[32]
is often used to describe the degree of cooperativity quantitatively in
non-MichaelisMenten kinetics. The derived Hill coefficient n measures how much the binding of substrate to one
active site affects the binding of substrate to the other active sites. A Hill coefficient of <1 indicates negative
cooperativity and a coefficient of >1 indicates positive cooperativity.
Pre-steady-state kinetics
Pre-steady state progress curve, showing the burst phase of an enzyme reaction.
In the first moment after an enzyme is
mixed with substrate, no product has been
formed and no intermediates exist. The
study of the next few milliseconds of the
reaction is called Pre-steady-state kinetics
also referred to as Burst kinetics.
Pre-steady-state kinetics is therefore
concerned with the formation and
consumption of enzymesubstrate
intermediates (such as ES or E*) until their
steady-state concentrations are reached.
This approach was first applied to the
hydrolysis reaction catalysed by
chymotrypsin.
[33]
Often, the detection of an
intermediate is a vital piece of evidence in
investigations of what mechanism an enzyme follows. For example, in the pingpong mechanisms that are shown
above, rapid kinetic measurements can follow the release of product P and measure the formation of the modified
enzyme intermediate E*.
[]
In the case of chymotrypsin, this intermediate is formed by an attack on the substrate by
the nucleophilic serine in the active site and the formation of the acyl-enzyme intermediate.
In the figure to the right, the enzyme produces E* rapidly in the first few seconds of the reaction. The rate then slows
as steady state is reached. This rapid burst phase of the reaction measures a single turnover of the enzyme.
Consequently, the amount of product released in this burst, shown as the intercept on the y-axis of the graph, also
gives the amount of functional enzyme which is present in the assay.
[34]
Chemical mechanism
An important goal of measuring enzyme kinetics is to determine the chemical mechanism of an enzyme reaction, i.e.,
the sequence of chemical steps that transform substrate into product. The kinetic approaches discussed above will
show at what rates intermediates are formed and inter-converted, but they cannot identify exactly what these
intermediates are.
Kinetic measurements taken under various solution conditions or on slightly modified enzymes or substrates often
shed light on this chemical mechanism, as they reveal the rate-determining step or intermediates in the reaction. For
example, the breaking of a covalent bond to a hydrogen atom is a common rate-determining step. Which of the
possible hydrogen transfers is rate determining can be shown by measuring the kinetic effects of substituting each
hydrogen by deuterium, its stable isotope. The rate will change when the critical hydrogen is replaced, due to a
primary kinetic isotope effect, which occurs because bonds to deuterium are harder to break than bonds to
hydrogen.
[35]
It is also possible to measure similar effects with other isotope substitutions, such as
13
C/
12
C and
18
O/
16
O, but these effects are more subtle.
[36]
Enzyme kinetics
146
Isotopes can also be used to reveal the fate of various parts of the substrate molecules in the final products. For
example, it is sometimes difficult to discern the origin of an oxygen atom in the final product; since it may have
come from water or from part of the substrate. This may be determined by systematically substituting oxygen's stable
isotope
18
O into the various molecules that participate in the reaction and checking for the isotope in the product.
[37]
The chemical mechanism can also be elucidated by examining the kinetics and isotope effects under different pH
conditions,
[38]
by altering the metal ions or other bound cofactors,
[39]
by site-directed mutagenesis of conserved
amino acid residues, or by studying the behaviour of the enzyme in the presence of analogues of the substrate(s).
[40]
Enzyme inhibition and activation
Kinetic scheme for reversible enzyme inhibitors.
Enzyme inhibitors are molecules that reduce
or abolish enzyme activity, while enzyme
activators are molecules that increase the
catalytic rate of enzymes. These interactions
can be either reversible (i.e., removal of the
inhibitor restores enzyme activity) or
irreversible (i.e., the inhibitor permanently
inactivates the enzyme).
Reversible inhibitors
Traditionally reversible enzyme inhibitors
have been classified as competitive, uncompetitive, or non-competitive, according to their effects on K
m
and V
max
.
These different effects result from the inhibitor binding to the enzyme E, to the enzymesubstrate complex ES, or to
both, respectively. The division of these classes arises from a problem in their derivation and results in the need to
use two different binding constants for one binding event. The binding of an inhibitor and its effect on the enzymatic
activity are two distinctly different things, another problem the traditional equations fail to acknowledge. In
noncompetitive inhibition the binding of the inhibitor results in 100% inhibition of the enzyme only, and fails to
consider the possibility of anything in between.
[41]
The common form of the inhibitory term also obscures the
relationship between the inhibitor binding to the enzyme and its relationship to any other binding term be it the
MichaelisMenten equation or a dose response curve associated with ligand receptor binding. To demonstrate the
relationship the following rearrangement can be made:
Adding zero to the bottom ([I]-[I])
Dividing by [I]+K
i
Enzyme kinetics
147
This notation demonstrates that similar to the MichaelisMenten equation,where the rate of reaction depends on the
percent of the enzyme population interacting with substrate
fraction of the enzyme population bound by substrate
fraction of the enzyme population bound by inhibitor
the effect of the inhibitor is a result of the percent of the enzyme population interacting with inhibitor. The only
problem with this equation in its present form is that it assumes absolute inhibition of the enzyme with inhibitor
binding, when in fact there can be a wide range of effects anywhere from 100% inhibition of substrate turn over to
just >0%. To account for this the equation can be easily modified to allow for different degrees of inhibition by
including a delta V
max
term.
or
This term can then define the residual enzymatic activity present when the inhibitor is interacting with individual
enzymes in the population. However the inclusion of this term has the added value of allowing for the possibility of
activation if the secondary V
max
term turns out to be higher than the initial term. To account for the possibly of
activation as well the notation can then be rewritten replacing the inhibitor "I" with a modifier term denoted here as
"X".
While this terminology results in a simplified way of dealing with kinetic effects relating to the maximum velocity of
the MichaelisMenten equation, it highlights potential problems with the term used to describe effects relating to the
K
m
. The K
m
relating to the affinity of the enzyme for the substrate should in most cases relate to potential changes in
the binding site of the enzyme which would directly result from enzyme inhibitor interactions. As such a term similar
to the one proposed above to modulate V
max
should be appropriate in most situations:
[42][43]
Enzyme kinetics
148
Irreversible inhibitors
Enzyme inhibitors can also irreversibly inactivate enzymes, usually by covalently modifying active site residues.
These reactions, which may be called suicide substrates, follow exponential decay functions and are usually
saturable. Below saturation, they follow first order kinetics with respect to inhibitor.
Mechanisms of catalysis
The energy variation as a function of reaction coordinate shows the stabilisation of
the transition state by an enzyme.
The favoured model for the
enzymesubstrate interaction is the induced
fit model.
[44]
This model proposes that the
initial interaction between enzyme and
substrate is relatively weak, but that these
weak interactions rapidly induce
conformational changes in the enzyme that
strengthen binding. These conformational
changes also bring catalytic residues in the
active site close to the chemical bonds in the
substrate that will be altered in the
reaction.
[45]
Conformational changes can be
measured using circular dichroism or dual
polarisation interferometry. After binding
takes place, one or more mechanisms of
catalysis lower the energy of the reaction's
transition state by providing an alternative
chemical pathway for the reaction.
Mechanisms of catalysis include catalysis by bond strain; by proximity and orientation; by active-site proton donors
or acceptors; covalent catalysis and quantum tunnelling.
[][46]
Enzyme kinetics cannot prove which modes of catalysis are used by an enzyme. However, some kinetic data can
suggest possibilities to be examined by other techniques. For example, a pingpong mechanism with burst-phase
pre-steady-state kinetics would suggest covalent catalysis might be important in this enzyme's mechanism.
Alternatively, the observation of a strong pH effect on V
max
but not K
m
might indicate that a residue in the active site
needs to be in a particular ionisation state for catalysis to occur.
Software
ENZO
ENZO (Enzyme Kinetics) is a graphical interface tool for building kinetic models of enzyme catalyzed reactions.
ENZO automatically generates the corresponding differential equations from a stipulated enzyme reaction scheme.
These differential equations are processed by a numerical solver and a regression algorithm which fits the
coefficients of differential equations to experimentally observed time course curves. ENZO allows rapid evaluation
of rival reaction schemes and can be used for routine tests in enzyme kinetics.
[47]
Enzyme kinetics
149
Footnotes
.
^
Link: Interactive MichaelisMenten kinetics tutorial (Java required)
[48]
.
^
Link: dihydrofolate reductase mechanism (Gif)
[49]
.
^
Link: DNA polymerase mechanism (Gif)
[50]
.
^
Link: Chymotrypsin mechanism (Flash required)
[51]
References
[1] http:/ / www. rcsb.org/ pdb/ explore. do?structureId=7DFR
[10] Michaelis L. and Menten M.L. Kinetik der Invertinwirkung Biochem. Z. 1913; 49:333369 English translation (http:/ / web. lemoyne. edu/
~giunta/ menten.html) Accessed 6 April 2007
[12] Walsh R, Martin E, Darvesh S. A method to describe enzyme-catalyzed reactions by combining steady state and time course enzyme kinetic
parameters. Biochim Biophys Acta. 2010 Jan;1800:15
[21] for a complete derivation, see here
[32] Hill, A. V. The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves. J. Physiol. (Lond.), 1910 40,
ivvii.
[47] ENZO server (http:/ / enzo. cmm. ki. si/ )
[48] http:/ / cti. itc. virginia. edu/ ~cmg/ Demo/ scriptFrame.html
[49] http:/ / chem-faculty. ucsd. edu/ kraut/ dhfr. html
[50] http:/ / chem-faculty. ucsd. edu/ kraut/ dNTP.html
[51] http:/ / web.archive. org/ web/ 20070319235224/ http:/ / courses. cm. utexas. edu/ jrobertus/ ch339k/ overheads-2/ 06_21_chymotrypsin.
html
Further reading
Introductory
Cornish-Bowden, Athel (2004). Fundamentals of enzyme kinetics (3rd ed.). London: Portland Press.
ISBN1-85578-158-1.
Stevens, Lewis; Price, Nicholas C. (1999). Fundamentals of enzymology: the cell and molecular biology of
catalytic proteins. Oxford [Oxfordshire]: Oxford University Press. ISBN0-19-850229-X.
Bugg, Tim (2004). Introduction to Enzyme and Coenzyme Chemistry. Cambridge, MA: Blackwell Publishers.
ISBN1-4051-1452-5.
Advanced
Segel, Irwin H. (1993). Enzyme kinetics: behavior and analysis of rapid equilibrium and steady state enzyme
systems (New ed.). New York: Wiley. ISBN0-471-30309-7.
Fersht, Alan (1999). Structure and mechanism in protein science: a guide to enzyme catalysis and protein folding.
San Francisco: W.H. Freeman. ISBN0-7167-3268-8.
Santiago Schnell, Philip K. Maini (2004). "A century of enzyme kinetics: Reliability of the K
M
and v
max
estimates" (http:/ / web. archive. org/ web/ 20060221045110/ http:/ / www. informatics. indiana. edu/ schnell/
papers/ ctb8_169. pdf). Comments on Theoretical Biology 8 (23): 16987. doi: 10.1080/08948550302453 (http:/
/ dx. doi. org/ 10. 1080/ 08948550302453).
Walsh, Christopher (1979). Enzymatic reaction mechanisms. San Francisco: W. H. Freeman.
ISBN0-7167-0070-0.
Cleland, William Wallace; Cook, Paul (2007). Enzyme kinetics and mechanism. New York: Garland Science.
ISBN0-8153-4140-7.
Enzyme kinetics
150
External links
Animation of an enzyme assay (http:/ / www. kscience. co. uk/ animations/ model. swf) Shows effects of
manipulating assay conditions
MACiE (http:/ / www. ebi. ac. uk/ thornton-srv/ databases/ MACiE/ ) A database of enzyme reaction
mechanisms
ENZYME (http:/ / us. expasy. org/ enzyme/ ) Expasy enzyme nomenclature database
ENZO (http:/ / enzo. cmm. ki. si) Web application for easy construction and quick testing of kinetic models of
enzyme catalyzed reactions.
ExCatDB (http:/ / mbs. cbrc. jp/ EzCatDB/ ) A database of enzyme catalytic mechanisms
BRENDA (http:/ / www. brenda-enzymes. info/ ) Comprehensive enzyme database, giving substrates,
inhibitors and reaction diagrams
SABIO-RK (http:/ / sabio. h-its. org) A database of reaction kinetics
Joseph Kraut's Research Group, University of California San Diego (http:/ / chem-faculty. ucsd. edu/ kraut/ dhfr.
html) Animations of several enzyme reaction mechanisms
Symbolism and Terminology in Enzyme Kinetics (http:/ / www. chem. qmul. ac. uk/ iubmb/ kinetics/ ) A
comprehensive explanation of concepts and terminology in enzyme kinetics
An introduction to enzyme kinetics (http:/ / web. archive. org/ web/ 20040612065857/ http:/ / orion1. paisley. ac.
uk/ kinetics/ contents. html) An accessible set of on-line tutorials on enzyme kinetics
Enzyme kinetics animated tutorial (http:/ / www. wiley. com/ college/ pratt/ 0471393878/ student/ animations/
enzyme_kinetics/ index. html) An animated tutorial with audio
151
Lipids and membranes
Lipid
Structures of some common lipids. At the top are cholesterol
[]
and oleic acid.
[1]
The
middle structure is a triglyceride composed of oleoyl, stearoyl, and palmitoyl chains
attached to a glycerol backbone. At the bottom is the common phospholipid,
phosphatidylcholine.
[2]
Lipids constitute a group of naturally
occurring molecules that include fats,
waxes, sterols, fat-soluble vitamins
(such as vitamins A, D, E, and K),
monoglycerides, diglycerides,
triglycerides, phospholipids, and
others. The main biological functions
of lipids include energy storage,
signaling, and acting as structural
components of cell membranes.
[][]
Lipids have found applications in
cosmetic and food industries as well as
in nanotechnology.
[3]
Lipids may be broadly defined as
hydrophobic or amphiphilic small
molecules; the amphiphilic nature of
some lipids allows them to form
structures such as vesicles, liposomes,
or membranes in an aqueous
environment. Biological lipids
originate entirely or in part from two
distinct types of biochemical subunits
or "building-blocks": ketoacyl and
isoprene groups.
[]
Using this approach,
lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids,
saccharolipids, and polyketides (derived from condensation of ketoacyl subunits); and sterol lipids and prenol lipids
(derived from condensation of isoprene subunits).
[]
Although the term lipid is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides.
Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and
phospholipids), as well as other sterol-containing metabolites such as cholesterol.
[4]
Although humans and other
mammals use various biosynthetic pathways to both break down and synthesize lipids, some essential lipids cannot
be made this way and must be obtained from the diet.
Lipid
152
Categories of lipids
Fatty acids
Fatty acids, or fatty acid residues when they form part of a lipid, are a diverse group of molecules synthesized by
chain-elongation of an acetyl-CoA primer with malonyl-CoA or methylmalonyl-CoA groups in a process called fatty
acid synthesis.
[][]
They are made of a hydrocarbon chain that terminates with a carboxylic acid group; this
arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in
water. The fatty acid structure is one of the most fundamental categories of biological lipids, and is commonly used
as a building-block of more structurally complex lipids.
[5]
The carbon chain, typically between four and 24 carbons
long,
[]
may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens,
nitrogen, and sulfur. Where a double bond exists, there is the possibility of either a cis or trans geometric isomerism,
which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain to bend, an
effect that is compounded with more double bonds in the chain. This in turn plays an important role in the structure
and function of cell membranes.
[6]
Most naturally occurring fatty acids are of the cis configuration, although the
trans form does exist in some natural and partially hydrogenated fats and oils.
[]
Examples of biologically important fatty acids are the eicosanoids, derived primarily from arachidonic acid and
eicosapentaenoic acid, that include prostaglandins, leukotrienes, and thromboxanes. Docosahexaenoic acid is also
important in biological systems, particularly with respect to sight.
[][7]
Other major lipid classes in the fatty acid
category are the fatty esters and fatty amides. Fatty esters include important biochemical intermediates such as wax
esters, fatty acid thioester coenzyme A derivatives, fatty acid thioester ACP derivatives and fatty acid carnitines. The
fatty amides include N-acyl ethanolamines, such as the cannabinoid neurotransmitter anandamide.
[]
Glycerolipids
Glycerolipids are composed mainly of mono-, di-, and tri-substituted glycerols,
[8]
the most well-known being the
fatty acid triesters of glycerol, called triglycerides. The word "triacylglycerol" is sometimes used synonymously with
"triglyceride", though the latter lipid contain no hydroxyl group. In these compounds, the three hydroxyl groups of
glycerol are each esterified, typically by different fatty acids. Because they function as an energy store, these lipids
comprise the bulk of storage fat in animal tissues. The hydrolysis of the ester bonds of triglycerides and the release
of glycerol and fatty acids from adipose tissue are the initial steps in metabolising fat.
[9]
Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence
of one or more sugar residues attached to glycerol via a glycosidic linkage. Examples of structures in this category
are the digalactosyldiacylglycerols found in plant membranes
[]
and seminolipid from mammalian sperm cells.
[]
Glycerophospholipids
Glycerophospholipids, usually referred to as phospholipids, are ubiquitous in nature and are key components of the
lipid bilayer of cells,
[]
as well as being involved in metabolism and cell signaling.
[]
Neural tissue (including the
brain) contains relatively high amounts of glycerophospholipids, and alterations in their composition has been
implicated in various neurological disorders.
[]
Glycerophospholipids may be subdivided into distinct classes, based
on the nature of the polar headgroup at the sn-3 position of the glycerol backbone in eukaryotes and eubacteria, or
the sn-1 position in the case of archaebacteria.
[]
Lipid
153
Phosphatidylethanolamine
Examples of glycerophospholipids found in biological membranes are
phosphatidylcholine (also known as PC, GPCho or lecithin),
phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS
or GPSer). In addition to serving as a primary component of cellular
membranes and binding sites for intra- and intercellular proteins, some
glycerophospholipids in eukaryotic cells, such as phosphatidylinositols
and phosphatidic acids are either precursors of or, themselves,
membrane-derived second messengers.
[10]
Typically, one or both of these hydroxyl groups are acylated with
long-chain fatty acids, but there are also alkyl-linked and 1Z-alkenyl-linked (plasmalogen) glycerophospholipids, as
well as dialkylether variants in archaebacteria.
[]
Sphingolipids
Sphingolipids are a complicated family of compounds
[11]
that share a common structural feature, a sphingoid base
backbone that is synthesized de novo from the amino acid serine and a long-chain fatty acyl CoA, then converted
into ceramides, phosphosphingolipids, glycosphingolipids and other compounds. The major sphingoid base of
mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of
sphingoid base derivatives with an amide-linked fatty acid. The fatty acids are typically saturated or
mono-unsaturated with chain lengths from 16 to 26 carbon atoms.
[12]
Sphingomyelin
The major phosphosphingolipids of mammals are sphingomyelins
(ceramide phosphocholines),
[13]
whereas insects contain mainly
ceramide phosphoethanolamines
[]
and fungi have phytoceramide
phosphoinositols and mannose-containing headgroups.
[]
The
glycosphingolipids are a diverse family of molecules composed of one
or more sugar residues linked via a glycosidic bond to the sphingoid
base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.
Sterol lipids
Sterol lipids, such as cholesterol and its derivatives, are an important component of membrane lipids,
[14]
along with
the glycerophospholipids and sphingomyelins. The steroids, all derived from the same fused four-ring core structure,
have different biological roles as hormones and signaling molecules. The eighteen-carbon (C18) steroids include the
estrogen family whereas the C19 steroids comprise the androgens such as testosterone and androsterone. The C21
subclass includes the progestogens as well as the glucocorticoids and mineralocorticoids.
[15]
The secosteroids,
comprising various forms of vitamin D, are characterized by cleavage of the B ring of the core structure.
[]
Other
examples of sterols are the bile acids and their conjugates,
[16]
which in mammals are oxidized derivatives of
cholesterol and are synthesized in the liver. The plant equivalents are the phytosterols, such as -sitosterol,
stigmasterol, and brassicasterol; the latter compound is also used as a biomarker for algal growth.
[17]
The
predominant sterol in fungal cell membranes is ergosterol.
[]
Prenol lipids
Prenol lipids are synthesized from the five-carbon-unit precursors isopentenyl diphosphate and dimethylallyl
diphosphate that are produced mainly via the mevalonic acid (MVA) pathway.
[18]
The simple isoprenoids (linear
alcohols, diphosphates, etc.) are formed by the successive addition of C5 units, and are classified according to
number of these terpene units. Structures containing greater than 40 carbons are known as polyterpenes. Carotenoids
are important simple isoprenoids that function as antioxidants and as precursors of vitamin A.
[]
Another biologically
important class of molecules is exemplified by the quinones and hydroquinones, which contain an isoprenoid tail
attached to a quinonoid core of non-isoprenoid origin.
[]
Vitamin E and vitamin K, as well as the ubiquinones, are
Lipid
154
examples of this class. Prokaryotes synthesize polyprenols (called bactoprenols) in which the terminal isoprenoid
unit attached to oxygen remains unsaturated, whereas in animal polyprenols (dolichols) the terminal isoprenoid is
reduced.
[]
Saccharolipids
Structure of the saccharolipid Kdo
2
-Lipid A.
[]
Glucosamine residues in blue, Kdo
residues in red, acyl chains in black and phosphate groups in green.
Saccharolipids describe compounds in
which fatty acids are linked directly to a
sugar backbone, forming structures that are
compatible with membrane bilayers. In the
saccharolipids, a monosaccharide substitutes
for the glycerol backbone present in
glycerolipids and glycerophospholipids. The
most familiar saccharolipids are the acylated
glucosamine precursors of the LipidA
component of the lipopolysaccharides in
Gram-negative bacteria. Typical lipidA
molecules are disaccharides of glucosamine,
which are derivatized with as many as seven
fatty-acyl chains. The minimal
lipopolysaccharide required for growth in E.
coli is Kdo
2
-Lipid A, a hexa-acylated
disaccharide of glucosamine that is
glycosylated with two
3-deoxy-D-manno-octulosonic acid (Kdo)
residues.
[]
Polyketides
Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as
iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a
large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources,
and have great structural diversity.
[19][]
Many polyketides are cyclic molecules whose backbones are often further
modified by glycosylation, methylation, hydroxylation, oxidation, and/or other processes. Many commonly used
anti-microbial, anti-parasitic, and anti-cancer agents are polyketides or polyketide derivatives, such as
erythromycins, tetracyclines, avermectins, and antitumor epothilones.
[]
Biological functions
Membranes
Eukaryotic cells are compartmentalized into membrane-bound organelles that carry out different biological
functions. The glycerophospholipids are the main structural component of biological membranes, such as the cellular
plasma membrane and the intracellular membranes of organelles; in animal cells the plasma membrane physically
separates the intracellular components from the extracellular environment. The glycerophospholipids are
amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to
two fatty acid-derived "tails" by ester linkages and to one "head" group by a phosphate ester linkage. While
glycerophospholipids are the major component of biological membranes, other non-glyceride lipid components such
Lipid
155
as sphingomyelin and sterols (mainly cholesterol in animal cell membranes) are also found in biological
membranes.
[20]
In plants and algae, the galactosyldiacylglycerols,
[21]
and sulfoquinovosyldiacylglycerol,
[]
which
lack a phosphate group, are important components of membranes of chloroplasts and related organelles and are the
most abundant lipids in photosynthetic tissues, including those of higher plants, algae and certain bacteria.
Bilayers have been found to exhibit high levels of birefringence, which can be used to probe the degree of order (or
disruption) within the bilayer using techniques such as dual polarization interferometry and Circular dichroism.
Self-organization of phospholipids: a spherical liposome, a micelle,
and a lipid bilayer.
A biological membrane is a form of lipid bilayer. The
formation of lipid bilayers is an energetically preferred
process when the glycerophospholipids described
above are in an aqueous environment.
[22]
This is known
as the hydrophobic effect. In an aqueous system, the
polar heads of lipids align towards the polar, aqueous
environment, while the hydrophobic tails minimize
their contact with water and tend to cluster together,
forming a vesicle; depending on the concentration of
the lipid, this biophysical interaction may result in the
formation of micelles, liposomes, or lipid bilayers.
Other aggregations are also observed and form part of
the polymorphism of amphiphile (lipid) behavior.
Phase behavior is an area of study within biophysics
and is the subject of current academic research.
[][]
Micelles and bilayers form in the polar medium by a
process known as the hydrophobic effect.
[23]
When
dissolving a lipophilic or amphiphilic substance in a
polar environment, the polar molecules (i.e., water in
an aqueous solution) become more ordered around the
dissolved lipophilic substance, since the polar
molecules cannot form hydrogen bonds to the
lipophilic areas of the amphiphile. So in an aqueous environment, the water molecules form an ordered "clathrate"
cage around the dissolved lipophilic molecule.
[24]
Energy storage
Triglycerides, stored in adipose tissue, are a major form of energy storage both in animals and plants. The adipocyte,
or fat cell, is designed for continuous synthesis and breakdown of triglycerides in animals, with breakdown
controlled mainly by the activation of hormone-sensitive enzyme lipase.
[25]
The complete oxidation of fatty acids
provides high caloric content, about 9kcal/g, compared with 4kcal/g for the breakdown of carbohydrates and
proteins. Migratory birds that must fly long distances without eating use stored energy of triglycerides to fuel their
flights.
[26]
Signaling
In recent years, evidence has emerged showing that lipid signaling is a vital part of the cell signaling.
[27][28]
Lipid
signaling may occur via activation of G protein-coupled or nuclear receptors, and members of several different lipid
categories have been identified as signaling molecules and cellular messengers.
[29]
These include
sphingosine-1-phosphate, a sphingolipid derived from ceramide that is a potent messenger molecule involved in
regulating calcium mobilization,
[]
cell growth, and apoptosis;
[]
diacylglycerol (DAG) and the phosphatidylinositol
Lipid
156
phosphates (PIPs), involved in calcium-mediated activation of protein kinase C;
[]
the prostaglandins, which are one
type of fatty-acid derived eicosanoid involved in inflammation and immunity;
[]
the steroid hormones such as
estrogen, testosterone and cortisol, which modulate a host of functions such as reproduction, metabolism and blood
pressure; and the oxysterols such as 25-hydroxy-cholesterol that are liver X receptor agonists.
[]
Phosphatidylserine
lipids are known to be involved in signaling for the phagocytosis of apoptotic cells and/or pieces of cells. They
accomplish this by being exposed to the extracellular face of the cell membrane after the inactivation of flippases
which place them exclusively on the cytosolic side and the activation of scramblases, which scramble the orientation
of the phospholipids. After this occurs, other cells recognize the phosphatidylserines and phagocytosize the cells or
cell fragments exposing them.
[citation needed]
Other functions
The "fat-soluble" vitamins (A, D, E and K) which are isoprene-based lipids are essential nutrients stored in the
liver and fatty tissues, with a diverse range of functions. Acyl-carnitines are involved in the transport and metabolism
of fatty acids in and out of mitochondria, where they undergo beta oxidation.
[30]
Polyprenols and their
phosphorylated derivatives also play important transport roles, in this case the transport of oligosaccharides across
membranes. Polyprenol phosphate sugars and polyprenol diphosphate sugars function in extra-cytoplasmic
glycosylation reactions, in extracellular polysaccharide biosynthesis (for instance, peptidoglycan polymerization in
bacteria), and in eukaryotic protein N-glycosylation.
[31][32]
Cardiolipins are a subclass of glycerophospholipids
containing four acyl chains and three glycerol groups that are particularly abundant in the inner mitochondrial
membrane.
[33][34][]
They are believed to activate enzymes involved with oxidative phosphorylation.
[35]
Lipids also
form the basis of steroid hormones.
[36]
Metabolism
The major dietary lipids for humans and other animals are animal and plant triglycerides, sterols, and membrane
phospholipids. The process of lipid metabolism synthesizes and degrades the lipid stores and produces the structural
and functional lipids characteristic of individual tissues.
Biosynthesis
In animals, when there is an oversupply of dietary carbohydrate, the excess carbohydrate is converted to
triglycerides. This involves the synthesis of fatty acids from acetyl-CoA and the esterification of fatty acids in the
production of triglycerides, a process called lipogenesis.
[37]
Fatty acids are made by fatty acid synthases that
polymerize and then reduce acetyl-CoA units. The acyl chains in the fatty acids are extended by a cycle of reactions
that add the acetyl group, reduce it to an alcohol, dehydrate it to an alkene group and then reduce it again to an
alkane group. The enzymes of fatty acid biosynthesis are divided into two groups, in animals and fungi all these fatty
acid synthase reactions are carried out by a single multifunctional protein,
[38]
while in plant plastids and bacteria
separate enzymes perform each step in the pathway.
[39][40]
The fatty acids may be subsequently converted to
triglycerides that are packaged in lipoproteins and secreted from the liver.
The synthesis of unsaturated fatty acids involves a desaturation reaction, whereby a double bond is introduced into
the fatty acyl chain. For example, in humans, the desaturation of stearic acid by stearoyl-CoA desaturase-1 produces
oleic acid. The doubly unsaturated fatty acid linoleic acid as well as the triply unsaturated -linolenic acid cannot be
synthesized in mammalian tissues, and are therefore essential fatty acids and must be obtained from the diet.
[41]
Triglyceride synthesis takes place in the endoplasmic reticulum by metabolic pathways in which acyl groups in fatty
acyl-CoAs are transferred to the hydroxyl groups of glycerol-3-phosphate and diacylglycerol.
[42]
Terpenes and isoprenoids, including the carotenoids, are made by the assembly and modification of isoprene units
donated from the reactive precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate.
[]
These precursors
can be made in different ways. In animals and archaea, the mevalonate pathway produces these compounds from
Lipid
157
acetyl-CoA,
[43]
while in plants and bacteria the non-mevalonate pathway uses pyruvate and glyceraldehyde
3-phosphate as substrates.
[][44]
One important reaction that uses these activated isoprene donors is steroid
biosynthesis. Here, the isoprene units are joined together to make squalene and then folded up and formed into a set
of rings to make lanosterol.
[]
Lanosterol can then be converted into other steroids such as cholesterol and
ergosterol.
[][45]
Degradation
Beta oxidation is the metabolic process by which fatty acids are broken down in the mitochondria and/or in
peroxisomes to generate acetyl-CoA. For the most part, fatty acids are oxidized by a mechanism that is similar to,
but not identical with, a reversal of the process of fatty acid synthesis. That is, two-carbon fragments are removed
sequentially from the carboxyl end of the acid after steps of dehydrogenation, hydration, and oxidation to form a
beta-keto acid, which is split by thiolysis. The acetyl-CoA is then ultimately converted into ATP, CO
2
, and H
2
O
using the citric acid cycle and the electron transport chain.
Hence the Krebs Cycle can start at acetyl-CoA when fat is being broken down for energy if there is little or no
glucose available.
The energy yield of the complete oxidation of the fatty acid palmitate is 106 ATP.
[46]
Unsaturated and odd-chain
fatty acids require additional enzymatic steps for degradation.
Nutrition and health
Most of the fat found in food is in the form of triglycerides, cholesterol, and phospholipids. Some dietary fat is
necessary to facilitate absorption of fat-soluble vitamins (A, D, E, and K) and carotenoids.
[47]
Humans and other
mammals have a dietary requirement for certain essential fatty acids, such as linoleic acid (an omega-6 fatty acid)
and alpha-linolenic acid (an omega-3 fatty acid) because they cannot be synthesized from simple precursors in the
diet.
[41]
Both of these fatty acids are 18-carbon polyunsaturated fatty acids differing in the number and position of
the double bonds. Most vegetable oils are rich in linoleic acid (safflower, sunflower, and corn oils). Alpha-linolenic
acid is found in the green leaves of plants, and in selected seeds, nuts, and legumes (in particular flax, rapeseed,
walnut, and soy).
[]
Fish oils are particularly rich in the longer-chain omega-3 fatty acids eicosapentaenoic acid (EPA)
and docosahexaenoic acid (DHA).
[48]
A large number of studies have shown positive health benefits associated with
consumption of omega-3 fatty acids on infant development, cancer, cardiovascular diseases, and various mental
illnesses, such as depression, attention-deficit hyperactivity disorder, and dementia.
[][]
In contrast, it is now
well-established that consumption of trans fats, such as those present in partially hydrogenated vegetable oils, are a
risk factor for cardiovascular disease.
[][][]
A few studies have suggested that total dietary fat intake is linked to an increased risk of obesity
[][49]
and
diabetes.
[50][]
However, a number of very large studies, including the Women's Health Initiative Dietary
Modification Trial, an eight-year study of 49,000 women, the Nurses' Health Study and the Health Professionals
Follow-up Study, revealed no such links.
[][][]
None of these studies suggested any connection between percentage of
calories from fat and risk of cancer, heart disease, or weight gain. The Nutrition Source, a website maintained by the
Department of Nutrition at the Harvard School of Public Health, summarizes the current evidence on the impact of
dietary fat: "Detailed researchmuch of it done at Harvardshows that the total amount of fat in the diet isn't really
linked with weight or disease."
[]
Lipid
158
References
[1] Stryer et al., p. 328.
[2] Stryer et al., p. 330.
[6] Devlin, pp. 19395.
[9] van Holde and Mathews, p. 63031.
[10] [10] van Holde and Mathews, p. 844.
[11] Merrill AH, Sandhoff K. (2002). "Sphingolipids: metabolism and cell signaling",in New Comprehensive Biochemistry: Biochemistry of
Lipids, Lipoproteins,and Membranes, Vance, D.E. and Vance, J.E., eds. Elsevier Science, NY. Ch. 14.
[12] Devlin, pp. 42122.
[15] Stryer et al., p. 749.
[20] Stryer et al., pp. 329331
[21] Heinz E.(1996). Plant glycolipids: structure, isolation and analysis. in Advances in Lipid Methodology - 3, pp. 211332 (ed. W.W. Christie,
Oily Press, Dundee)
[22] Stryer et al., pp. 33334.
[26] Stryer et al., p. 619.
[36] http:/ / www.elmhurst. edu/ ~chm/ vchembook/ 556steroids. html
[37] Stryer et al., p. 634.
[41] Stryer et al., p. 643.
[42] Stryer et al., pp. 73339.
[46] Stryer et al., pp. 62526.
[47] [47] Bhagavan, p. 903.
[48] [48] Bhagavan, p. 388.
Bibliography
Bhagavan NV (2002). Medical Biochemistry (http:/ / books. google. com/ ?id=vT9YttFTPi0C&
printsec=frontcover). San Diego: Harcourt/Academic Press. ISBN0-12-095440-0.
Devlin TM (1997). Textbook of Biochemistry: With Clinical Correlations (4th ed.). Chichester: John Wiley &
Sons. ISBN0-471-17053-4.
Stryer L, Berg JM, Tymoczko JL (2007). Biochemistry (6th ed.). San Francisco: W.H. Freeman.
ISBN0-7167-8724-5.
Van Holde KE, Mathews CK (1996). Biochemistry (2nd ed.). Menlo Park, Calif: Benjamin/Cummings Pub. Co.
ISBN0-8053-3931-0.
External links
Introductory
List of lipid-related web sites (http:/ / www. cyberlipid. org/ cyberlip/ link0041. htm)
Nature Lipidomics Gateway (http:/ / www. lipidmaps. org/ ) - Round-up and summaries of recent lipid research
Lipid Library (http:/ / www. lipidlibrary. co. uk/ ) - General reference on lipid chemistry and biochemistry
Cyberlipid.org (http:/ / www. cyberlipid. org/ ) - Resources and history for lipids.
Molecular Computer Simulations (http:/ / www. fos. su. se/ ~sasha/ Lipid_membranes. html) - Modeling of Lipid
Membranes
Lipids, Membranes and Vesicle Trafficking (http:/ / www. biochemweb. org/ lipids_membranes. shtml) - The
Virtual Library of Biochemistry and Cell Biology
Nomenclature
IUPAC nomenclature of lipids (http:/ / www. chem. qmul. ac. uk/ iupac/ lipid/ )
IUPAC glossary entry for the lipid class of molecules (http:/ / www. chem. qmul. ac. uk/ iupac/ class/ lipid. html)
Databases
LIPID MAPS (http:/ / www. lipidmaps. org/ data/ databases. html) - Comprehensive lipid and lipid-associated
gene/protein databases.
Lipid
159
LipidBank (http:/ / lipidbank. jp/ ) - Japanese database of lipids and related properties, spectral data and
references.
LIPIDAT (http:/ / www. lipidat. tcd. ie/ ) - Database composed mainly of phospholipids and associated
thermodynamic data.
General
ApolloLipids (http:/ / www. apollolipids. org/ ) - Provides dyslipidemia and cardiovascular disease prevention and
treatment information as well as continuing medical education programs
National Lipid Association (http:/ / www. lipid. org/ ) - Professional medical education organization for health
care professionals who seek to prevent morbidity and mortality stemming from dyslipidemias and other
cholesterol-related disorders.
Biological membrane
Cross section view of the structures that can be formed
by phospholipids in aqueous solutions
A biological membrane or biomembrane is an enclosing or
separating membrane that acts as a selective barrier, within or
around a cell. It consists of a lipid bilayer with embedded proteins
that may constitute close to 50% of membrane content.
[1]
The
cellular membranes should not be confused with isolating tissues
formed by layers of cells, such as mucous and basement
membranes.
Function
Membranes in cells typically define enclosed spaces or
compartments in which cells may maintain a chemical or
biochemical environment that differs from the outside. For
example, the membrane around peroxisomes shields the rest of the
cell from peroxides, and the cell membrane separates a cell from
its surrounding medium. Most organelles are defined by such
membranes, and are called "membrane-bound" organelles.
Probably the most important feature of a biomembrane is that it is
a selectively permeable structure. This means that the size, charge, and other chemical properties of the atoms and
molecules attempting to cross it will determine whether they succeed in doing so. Selective permeability is essential
for effective separation of a cell or organelle from its surroundings. Biological membranes also have certain
mechanical or elastic properties.
Particles that are required for cellular function but are unable to diffuse freely across a membrane enter through a
membrane transport protein or are taken in by means of endocytosis.
Diversity of biological membranes
Many types of specialized plasma membranes can separate cell from external environment: apical, basolateral,
presynaptic and postsynaptic ones, membranes of flagella, cilia, microvillus, filopodia and lamellipodia, the
sarcolemma of muscle cells, as well as specialized myelin and dendritic spine membranes of neurons. Plasma
membranes can also form different types of "supramembrane" structures such as caveola, postsynaptic density,
podosome, invadopodium, desmosome, hemidesmosome, focal adhesion, and cell junctions. These types of
membranes differ in lipid and protein composition.
Biological membrane
160
Distinct types of membranes also create intracellular organelles: endosome; smooth and rough endoplasmic
reticulum; sarcoplasmic reticulum; Golgi apparatus; lysosome; mitochondrion (inner and outer membranes); nucleus
(inner and outer membranes); peroxisome; vacuole; cytoplasmic granules; cell vesicles (phagosome, autophagosome,
clathrin-coated vesicles, COPI-coated and COPII-coated vesicles) and secretory vesicles (including synaptosome,
acrosomes, melanosomes, and chromaffin granules).
Different types of biological membranes have diverse lipid and protein compositions. The content of membranes
defines their physical and biological properties. Some components of membranes play a key role in medicine, such
as the efflux pumps that pump drugs out of a cell.
References
von Heijne G, Rees D (August 2008). "Membranes: reading between the lines" (http:/ / linkinghub. elsevier. com/
retrieve/ pii/ S0959-440X(08)00091-2). Curr. Opin. Struct. Biol. 18 (4): 4035. doi: 10.1016/j.sbi.2008.06.003
(http:/ / dx. doi. org/ 10. 1016/ j.sbi. 2008. 06. 003). PMID 18634876 (http:/ / www. ncbi. nlm. nih. gov/
pubmed/ 18634876).
External links
Membranes (http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2011/ MB_cgi?mode=& term=Membranes) at the US
National Library of Medicine Medical Subject Headings (MeSH)
Membrane protein
161
Membrane protein
Crystal structure of Potassium channel KvAP. Calculated hydrocarbon
boundaries of the lipid bilayer are indicated by red and blue dots.
Membrane proteins constitute one of the three
main protein classes, with the other classes being
the fibrous and globular proteins. Membrane
proteins are attached to, or associated with the
membrane of a cell or an organelle. These
proteins are specifically targeted to different
types of biological membranes
[1]
They are also
the target of over 50% of all modern medicinal
drugs.
[]
It is estimated that 20-30% of all genes in
most genomes encode membrane proteins.
[2]
Function
Membrane proteins perform a variety of functions
vital to the survival of organisms:
[3]
Membrane receptor proteins relay signals
between the cell's internal and external
environments.
Transport proteins move molecules and ions
across the membrane. They can be categorised
according to the Transporter Classification
database.
Membrane enzymes for example
Oxidoreductases, Transferases and
Hydrolases.
Cell adhesion molecules allow cells to identify each other and interact. For example proteins involved in immune
response.
Topology
The topology of an integral membrane protein describes the number of transmembrane segments, as well as the
orientation in the membrane.
[4]
Membrane proteins have several different topologies:
[]
A slightly different classification is to divide all membrane proteins to integral and amphitropic.
[]
The amphitropic
are proteins that can exist in two alternative states: a water-soluble and a lipid bilayer-bound. The amphitropic
protein category includes water-soluble channel-forming polypeptide toxins, which associate irreversibly with
membranes, but excludes peripheral proteins that interact with other membrane proteins rather than with lipid
bilayer.
Membrane protein
162
Integral membrane proteins
Schematic representation of transmembrane proteins: 1. a single transmembrane
-helix (bitopic membrane protein) 2. a polytopic transmembrane -helical protein
3. a polytopic transmembrane -sheet protein
The membrane is represented in light brown.
Integral membrane proteins are permanently
attached to the membrane. Such proteins can
be separated from the biological membranes
only using detergents, nonpolar solvents, or
sometimes denaturing agents. They can be
classified according to their relationship
with the bilayer:
Integral polytopic proteins, also known
as "transmembrane proteins," are integral
membrane proteins which span across the
membrane at least once. They have one
of two tertiary structures:
Helix bundle proteins which are
present in all types of biological
membranes;
Beta barrel proteins which are found
only in outer membranes of Gram-negative bacteria, lipid-rich cell walls of a few Gram-positive bacteria, and
outer membranes of mitochondria and chloroplasts.
Integral monotopic proteins are integral membrane proteins which are attached to only one side of the membrane
and do not span the whole way across.
Peripheral membrane proteins
Schematic representation of the different types of interaction between monotopic
membrane proteins and the cell membrane: 1. interaction by an amphipathic -helix
parallel to the membrane plane (in-plane membrane helix) 2. interaction by a hydrophobic
loop 3. interaction by a covalently bound membrane lipid (lipidation) 4. electrostatic or
ionic interactions with membrane lipids (e.g. through a calcium ion)
Peripheral membrane proteins are
temporarily attached either to the lipid
bilayer or to integral proteins by a
combination of hydrophobic,
electrostatic, and other non-covalent
interactions. Peripheral proteins
dissociate following treatment with a
polar reagent, such as a solution with
an elevated pH or high salt
concentrations.
Integral and peripheral proteins may be
post-translationally modified, with
added fatty acid or prenyl chains, or
GPI (glycosylphosphatidylinositol), which may be anchored in the lipid bilayer.
Polypeptide toxins
Polypeptide toxins and many antibacterial peptides, such as colicins or hemolysins, and certain proteins involved in
apoptosis, are sometimes considered a separate category. These proteins are water-soluble but can aggregate and
associate irreversibly with the lipid bilayer and become reversibly or irreversibly membrane-associated.
Membrane protein
163
3D Structure
Increase in the number of 3D structures of
membrane proteins known
The most common tertiary structures are Helix bundle and Beta barrel.
The portion of the membrane proteins that are attached to the lipid
bilayer are consisting of hydrophobic amino acids only. This is done so
that the peptide bonds' carbonyl and amine will react with each other
instead of the hydrophobic surrounding. The portion of the protein that
is not touching the lipid bilayer and is protruding out of the cell
membrane are usually hydrophilic amino acids.
[5]
Membrane proteins have hydrophobic surfaces, are relatively flexible
and are expressed at relatively low levels. This creates difficulties in
obtaining enough protein and then growing crystals. Hence despite the
significant functional importance of membrane proteins, determining
atomic resolution structures for these proteins is more difficult than
globular proteins.
[6]
As of January 2013 less than 0.1% of protein structures determined were membrane proteins
despite being 20-30% of the total proteome.
[7]
Many of the successful membrane protein structures are characterized by X-ray crystallography and are very large
structures in which the interactions with the membrane mimetic environments can be anticipated to be small in
comparison to those within the protein structures. The small domains are particularly sensitive to the influence of
membrane mimetic environments, potentially leading to non-native structures. Fortunately, there are many sample
preparation conditions that can be chosen for crystallization and for solution NMR. All membrane protein structural
biology should be subjected to careful scrutiny; through a combination of structural methodologies it should be
possible to achieve an understanding of the native functional state for membrane protein structures.
[8]
Coevolution
information has been successfully exploited for prediction of multiple large (membrane) protein structures.
[9][10][11]
Due to this difficulty and the importance of this class of proteins methods of protein structure prediction based on
hydropathy plots and the positive inside rule have been developed.
[12][13]
References
White, Stephen. General Principle of Membrane Protein Folding and Stability. Stephen White Laboratory
Homepage. 10 Nov. 2009. web.
[1] Classification of membrane proteins with known 3D structure to different membrane types (http:/ / opm. phar. umich. edu/ atlas. php)
[5] White, Stephen. General Principle of Membrane Protein Folding and Stability. Stephen White Laboratory Homepage. 10 Nov. 2009. web.
[7] Membrane Proteins of known 3D Structure (http:/ / blanco. biomol. uci. edu/ mpstruc/ )
[8] [8] Cross, Timothy, Mukesh Sharma, Myunggi Yi, Huan-Xiang Zhou (2010). "Influence of Solubilizing Environments on Membrane Protein
Structures"
[13] State of the art in membrane protein prediction (http:/ / gepard. bioinformatik. uni-saarland. de/ old_html/ html/
MembraneBioinformaticsSS06/ SuggestedReadingLecture6/ Chen_Review. pdf)
Membrane protein
164
External links
Organizations
Membrane Protein Structural Dynamics Consortium (http:/ / memprotein. org)
Transmembrane Protein Center (http:/ / www. uwmembraneproteins. org)
Membrane protein databases
List of transmembrane proteins of known 3D structure (http:/ / blanco. biomol. uci. edu/
Membrane_Proteins_xtal. html)
TCDB (http:/ / www. tcdb. org/ ) - Transporter Classification database
Orientations of Proteins in Membranes (OPM) database (http:/ / opm. phar. umich. edu/ ) 3D structures of integral
and peripheral membrane proteins arranged in the lipid bilayer
Membrane PDB (http:/ / www. mpdb. tcd. ie/ ) Database of 3D structures of integral membrane proteins and
hydrophobic peptides with an emphasis on crystallization conditions
Protein Data Bank of Transmembrane Proteins (http:/ / pdbtm.enzim. hu/ ) 3D models of all transmembrane
proteins currently in PDB. Approximate positions of membrane boundary planes were calculated for each PDB
entry.
TransportDB (http:/ / www. membranetransport. org/ ) Genomics-oriented database of transporters from TIGR
Membrane targeting domains (MeTaDoR) (http:/ / proteomics. bioengr. uic. edu/ metador/ MeTaDoR. html)
Further reading
The Human Membrane Proteome (http:/ / www. biomedcentral. com/ 1741-7007/ 7/ 50) - A comprehensive
article covering the transmembrane protein component of the human proteome
Cell membrane
165
Cell membrane
Illustration of a Eukaryotic cell membrane
The cell membrane is a biological
membrane that separates the interior of
all cells from the outside environment.
[1]
The cell membrane is selectively
permeable to ions and organic molecules
and controls the movement of substances
in and out of cells.
[]
The basic function of
the cell membrane is to protect the cell
from its surroundings. It consists of the
lipid bilayer with embedded proteins.
Cell membranes are involved in a variety
of cellular processes such as cell
adhesion, ion conductivity and cell
signaling and serve as the attachment
surface for several extracellular
structures, including the cell wall,
glycocalyx, and intracellular
cytoskeleton. Cell membranes can be
artificially reassembled.
[][][]
Function
A detailed diagram of the cell membrane
The cell membrane or plasma
membrane surrounds the cytoplasm of
living cells, physically separating the
intracellular components from the
extracellular environment. Fungi,
bacteria and plants also have the cell
wall which provides a mechanical
support for the cell and precludes the
passage of larger molecules. The cell
membrane also plays a role in
anchoring the cytoskeleton to provide
shape to the cell, and in attaching to the extracellular matrix and other cells to help group cells together to form
tissues.
The membrane is selectively permeable and able to regulate what enters and exits the cell, thus facilitating the
transport of materials needed for survival. The movement of substances across the membrane can be either "passive",
occurring without the input of cellular energy, or active, requiring the cell to expend energy in transporting it. The
Cell membrane
166
membrane also maintains the cell potential. The cell membrane thus works as a selective filter that allows only
certain things to come inside or go outside the cell. The cell employs a number of transport mechanisms that involve
biological membranes:
1. Passive diffusion and osmosis: Some substances (small molecules, ions) such as carbon dioxide (CO
2
) and oxygen
(O
2
), can move across the plasma membrane by diffusion, which is a passive transport process. Because the
membrane acts as a barrier for certain molecules and ions, they can occur in different concentrations on the two sides
of the membrane. Such a concentration gradient across a semipermeable membrane sets up an osmotic flow for the
water.
2. Transmembrane protein channels and transporters: Nutrients, such as sugars or amino acids, must enter the cell,
and certain products of metabolism must leave the cell. Such molecules are pumped across the membrane by
transmembrane transporters or diffuse through protein channels such as Aquaporins (in the case of water (H
2
O)).
These proteins, also called permeases, are usually quite specific, recognizing and transporting only a limited food
group of chemical substances, often even only a single substance.
3. Endocytosis: Endocytosis is the process in which cells absorb molecules by engulfing them. The plasma
membrane creates a small deformation inward, called an invagination, in which the substance to be transported is
captured. The deformation then pinches off from the membrane on the inside of the cell, creating a vesicle
containing the captured substance. Endocytosis is a pathway for internalizing solid particles (cell eating or
phagocytosis), small molecules and ions (cell drinking or pinocytosis), and macromolecules. Endocytosis requires
energy and is thus a form of active transport.
4. Exocytosis: Just as material can be brought into the cell by invagination and formation of a vesicle, the membrane
of a vesicle can be fused with the plasma membrane, extruding its contents to the surrounding medium. This is the
process of exocytosis. Exocytosis occurs in various cells to remove undigested residues of substances brought in by
endocytosis, to secrete substances such as hormones and enzymes, and to transport a substance completely across a
cellular barrier. In the process of exocytosis, the undigested waste-containing food vacuole or the secretory vesicle
budded from Golgi apparatus, is first moved by cytoskeleton from the interior of the cell to the surface. The vesicle
membrane comes in contact with the plasma membrane. The lipid molecules of the two bilayers rearrange
themselves and the two membranes are, thus, fused. A passage is formed in the fused membrane and the vesicles
discharges its contents outside the cell.
Prokaryotes
Gram-negative bacteria have a plasma membrane and an outer membrane separated by a periplasmic space. Other
prokaryotes have only a plasma membrane. Prokaryotic cells are also surrounded by a cell wall composed of
peptidoglycan (amino acids and sugars). Some eukaryotic cells also have cells walls, but none that are made of
peptidoglycan.
Structure
Fluid mosaic model
According to the fluid mosaic model of S.J. Singer and G.L. Nicolson (1972), which replaced the earlier model of
Davson and Danielli, biological membranes can be considered as a two-dimensional liquid in which lipid and protein
molecules diffuse more or less easily.
[2]
Although the lipid bilayers that form the basis of the membranes do indeed
form two-dimensional liquids by themselves, the plasma membrane also contains a large quantity of proteins, which
provide more structure. Examples of such structures are protein-protein complexes, pickets and fences formed by the
actin-based cytoskeleton, and potentially lipid rafts.
Cell membrane
167
Lipid bilayer
Diagram of the arrangement of amphipathic lipid
molecules to form a lipid bilayer. The yellow polar
head groups separate the grey hydrophobic tails from
the aqueous cytosolic and extracellular environments.
Lipid bilayers form through the process of self-assembly. The cell
membrane consists primarily of a thin layer of amphipathic
phospholipids which spontaneously arrange so that the
hydrophobic "tail" regions are isolated from the surrounding polar
fluid, causing the more hydrophilic "head" regions to associate
with the intracellular (cytosolic) and extracellular faces of the
resulting bilayer. This forms a continuous, spherical lipid bilayer.
Forces such as van der Waals, electrostatic, hydrogen bonds, and
noncovalent interactions all contribute to the formation of the lipid
bilayer. Overall, hydrophobic interactions are the major driving
force in the formation of lipid bilayers.
Lipid bilayers are generally impermeable to ions and polar molecules. The arrangement of hydrophilic heads and
hydrophobic tails of the lipid bilayer prevent polar solutes (ex. amino acids, nucleic acids, carbohydrates, proteins,
and ions) from diffusing across the membrane, but generally allows for the passive diffusion of hydrophobic
molecules. This affords the cell the ability to control the movement of these substances via transmembrane protein
complexes such as pores, channels and gates.
Flippases and scramblases concentrate phosphatidyl serine, which carries a negative charge, on the inner membrane.
Along with NANA, this creates an extra barrier to charged moieties moving through the membrane.
Membranes serve diverse functions in eukaryotic and prokaryotic cells. One important role is to regulate the
movement of materials into and out of cells. The phospholipid bilayer structure (fluid mosaic model) with specific
membrane proteins accounts for the selective permeability of the membrane and passive and active transport
mechanisms. In addition, membranes in prokaryotes and in the mitochondria and chloroplasts of eukaryotes facilitate
the synthesis of ATP through chemiosmosis.
Membrane polarity
Alpha intercalated cell
The apical membrane of a polarized cell is
the surface of the plasma membrane that
faces inward to the lumen. This is
particularly evident in epithelial and
endothelial cells, but also describes other
polarized cells, such as neurons. The
basolateral membrane of a polarized cell is
the surface of the plasma membrane that
forms its basal and lateral surfaces. It faces
outwards, towards the interstitium, and
away from the lumen. Basolateral
membrane is a compound phrase referring to
the terms "basal (base) membrane" and
"lateral (side) membrane", which, especially
in epithelial cells, are identical in
composition and activity. Proteins (such as
ion channels and pumps) are free to move from the basal to the lateral surface of the cell or vice versa in accordance
with the fluid mosaic model. Tight junctions join epithelial cells near their apical surface to prevent the migration of
proteins from the basolateral membrane to the apical membrane. The basal and lateral surfaces thus remain roughly
Cell membrane
168
equivalentWikipedia:Please clarify to one another, yet distinct from the apical surface.
Membrane structures
Cell membrane can form different types of "supramembrane" structures such as caveola, postsynaptic density,
podosome, invadopodium, focal adhesion, and different types of cell junctions. These structures are usually
responsible for cell adhesion, communication, endocytosis and exocytosis. They can be visualized by electron
microscopy or fluorescence microscopy. They are composed of specific proteins, such as integrins and cadherins.
Cytoskeleton
The cytoskeleton is found underlying the cell membrane in the cytoplasm and provides a scaffolding for membrane
proteins to anchor to, as well as forming organelles that extend from the cell. Indeed, cytoskeletal elements interact
extensively and intimately with the cell membrane.
[3]
Anchoring proteins restricts them to a particular cell surface
for example, the apical surface of epithelial cells that line the vertebrate gut and limits how far they may diffuse
within the bilayer. The cytoskeleton is able to form appendage-like organelles, such as cilia, which are
microtubule-based extensions covered by the cell membrane, and filopodia, which are actin-based extensions. These
extensions are ensheathed in membrane and project from the surface of the cell in order to sense the external
environment and/or make contact with the substrate or other cells. The apical surfaces of epithelial cells are dense
with actin-based finger-like projections known as microvilli, which increase cell surface area and thereby increase
the absorption rate of nutrients. Localized decoupling of the cytoskeleton and cell membrane results in formation of
a bleb.
Composition
Cell membranes contain a variety of biological molecules, notably lipids and proteins. Material is incorporated into
the membrane, or deleted from it, by a variety of mechanisms:
Fusion of intracellular vesicles with the membrane (exocytosis) not only excretes the contents of the vesicle but
also incorporates the vesicle membrane's components into the cell membrane. The membrane may form blebs
around extracellular material that pinch off to become vesicles (endocytosis).
If a membrane is continuous with a tubular structure made of membrane material, then material from the tube can
be drawn into the membrane continuously.
Although the concentration of membrane components in the aqueous phase is low (stable membrane components
have low solubility in water), there is an exchange of molecules between the lipid and aqueous phases.
Cell membrane
169
Lipids
Examples of the major membrane phospholipids and glycolipids: phosphatidylcholine
(PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylinositol (PtdIns),
phosphatidylserine (PtdSer).
The cell membrane consists of three
classes of amphipathic lipids:
phospholipids, glycolipids, and
cholesterols. The amount of each depends
upon the type of cell, but in the majority
of cases phospholipids are the most
abundant.
[]
In RBC studies, 30% of the
plasma membrane is lipid.
The fatty chains in phospholipids and
glycolipids usually contain an even
number of carbon atoms, typically
between 16 and 20. The 16- and
18-carbon fatty acids are the most
common. Fatty acids may be saturated or
unsaturated, with the configuration of the
double bonds nearly always "cis". The
length and the degree of unsaturation of
fatty acid chains have a profound effect
on membrane fluidity
[]
as unsaturated
lipids create a kink, preventing the fatty
acids from packing together as tightly,
thus decreasing the melting temperature
(increasing the fluidity) of the membrane.
The ability of some organisms to regulate the fluidity of their cell membranes by altering lipid composition is called
homeoviscous adaptation.
The entire membrane is held together via non-covalent interaction of hydrophobic tails, however the structure is
quite fluid and not fixed rigidly in place. Under physiological conditions phospholipid molecules in the cell
membrane are in the liquid crystalline state. It means the lipid molecules are free to diffuse and exhibit rapid lateral
diffusion along the layer in which they are present. However, the exchange of phospholipid molecules between
intracellular and extracellular leaflets of the bilayer is a very slow process. Lipid rafts and caveolae are examples of
cholesterol-enriched microdomains in the cell membrane.
In animal cells cholesterol is normally found dispersed in varying degrees throughout cell membranes, in the
irregular spaces between the hydrophobic tails of the membrane lipids, where it confers a stiffening and
strengthening effect on the membrane.
[]
Phospholipids forming lipid vesicles
Lipid vesicles or liposomes are circular pockets that are enclosed by a lipid bilayer. These structures are used in
laboratories to study the effects of chemicals in cells by delivering these chemicals directly to the cell, as well as
getting more insight into cell membrane permeability. Lipid vesicles and liposomes are formed by first suspending a
lipid in an aqueous solution then agitating the mixture through sonication, resulting in a vesicle. By measuring the
rate of efflux from that of the inside of the vesicle to the ambient solution, allows researcher to better understand
membrane permeability. Vesicles can be formed with molecules and ions inside the vesicle by forming the vesicle
with the desired molecule or ion present in the solution. Proteins can also be embedded into the membrane through
solubilizing the desired proteins in the presence of detergents and attaching them to the phospholipids in which the
Cell membrane
170
liposome is formed. These provide researchers with a tool to examine various membrane protein functions.
Carbohydrates
Plasma membranes also contain carbohydrates, predominantly glycoproteins, but with some glycolipids
(cerebrosides and gangliosides). For the most part, no glycosylation occurs on membranes within the cell; rather
generally glycosylation occurs on the extracellular surface of the plasma membrane. The glycocalyx is an important
feature in all cells, especially epithelia with microvilli. Recent data suggest the glycocalyx participates in cell
adhesion, lymphocyte homing, and many others. The penultimate sugar is galactose and the terminal sugar is sialic
acid, as the sugar backbone is modified in the golgi apparatus. Sialic acid carries a negative charge, providing an
external barrier to charged particles.
Proteins
Type Description Examples
Integral proteins
or
transmembrane
proteins
Span the membrane and have a hydrophilic cytosolic domain, which interacts with internal
molecules, a hydrophobic membrane-spanning domain that anchors it within the cell
membrane, and a hydrophilic extracellular domain that interacts with external molecules. The
hydrophobic domain consists of one, multiple, or a combination of -helices and sheet
protein motifs.
Ion channels, proton
pumps, G
protein-coupled
receptor
Lipid anchored
proteins
Covalently bound to single or multiple lipid molecules; hydrophobically insert into the cell
membrane and anchor the protein. The protein itself is not in contact with the membrane.
G proteins
Peripheral
proteins
Attached to integral membrane proteins, or associated with peripheral regions of the lipid
bilayer. These proteins tend to have only temporary interactions with biological membranes,
and once reacted, the molecule dissociates to carry on its work in the cytoplasm.
Some enzymes, some
hormones
The cell membrane has large content of proteins, typically around 50% of membrane volume
[]
These proteins are
important for cell because they are responsible for various biological activities. Approximately a third of the genes in
yeast code specifically for them, and this number is even higher in multicellular organisms.
[]
The cell membrane, being exposed to the outside environment, is an important site of cellcell communication. As
such, a large variety of protein receptors and identification proteins, such as antigens, are present on the surface of
the membrane. Functions of membrane proteins can also include cellcell contact, surface recognition, cytoskeleton
contact, signaling, enzymatic activity, or transporting substances across the membrane.
Most membrane proteins must be inserted in some way into the membrane. For this to occur, an N-terminus "signal
sequence" of amino acids directs proteins to the endoplasmic reticulum, which inserts the proteins into a lipid
bilayer. Once inserted, the proteins are then transported to their final destination in vesicles, where the vesicle fuses
with the target membrane.
Variation
The cell membrane has different lipid and protein compositions in distinct types of cells and may have therefore
specific names for certain cell types:
Sarcolemma in myocytes
Oolemma in oocytes
Axolemma in neuronal processes - axons
Historically, the plasma membrane was also referred to as the plasmalemma
Cell membrane
171
Permeability
The permeability of a membrane is the rate of passive diffusion of molecules through the membrane. These
molecules are known as permeant molecules. Permeability depends mainly on the electric charge and polarity of the
molecule and to a lesser extent the molar mass of the molecule. Due to the cell membrane's hydrophobic nature,
small electrically neutral molecules pass through the membrane more easily than charged, large ones. The inability
of charged molecules to pass through the cell membrane results in pH partition of substances throughout the fluid
compartments of the body.
References
[1] Kimball's Biology Pages (http:/ / users. rcn.com/ jkimball. ma. ultranet/ BiologyPages/ C/ CellMembranes. html), Cell Membranes
External links
Lipids, Membranes and Vesicle Trafficking - The Virtual Library of Biochemistry and Cell Biology (http:/ /
www. biochemweb. org/ lipids_membranes. shtml)
Cell membrane protein extraction protocol (http:/ / www. westernblotting. org/ protocol membrane extraction.
htm)
Membrane homeostasis, tension regulation, mechanosensitive membrane exchange and membrane traffic (http:/ /
www. phys. unsw. edu. au/ ~jw/ tension. html)
3D structures of proteins associated with plasma membrane of eukaryotic cells (http:/ / opm. phar. umich. edu/
localization. php?localization=Eukaryotic plasma membrane)
Lipid composition and proteins of some eukariotic membranes (http:/ / opm. phar. umich. edu/ atlas.
php?membrane=Eukaryotic plasma membrane)
(http:/ / www. etap. org/ demo/ biology1/ instruction3tutor. html)
172
Carbohydrate structure
Carbohydrate
Lactose is a disaccharide found in milk. It consists of a molecule of D-galactose and a
molecule of D-glucose bonded by beta-1-4 glycosidic linkage. It has a formula of
C
12
H
22
O
11
.
A carbohydrate is an organic compound
that consists only of carbon, hydrogen,
and oxygen, usually with a
hydrogen:oxygen atom ratio of 2:1 (as in
water); in other words, with the empirical
formula C
m
(H
2
O)
n
(where m could be
different from n). Some exceptions exist;
for example, deoxyribose, a component of
DNA, has the empirical formula
C
5
H
10
O
4
. Carbohydrates are not
technically hydrates of carbon;
structurally it is more accurate to view
them as polyhydroxy aldehydes and ketones.
The term is most common in biochemistry, where it is a synonym of saccharide. The carbohydrates (saccharides)
are divided into four chemical groupings: monosaccharides, disaccharides, oligosaccharides, and polysaccharides. In
general, the monosaccharides and disaccharides, which are smaller (lower molecular weight) carbohydrates, are
commonly referred to as sugars.
[1]
The word saccharide comes from the Greek word (skkharon),
meaning "sugar." While the scientific nomenclature of carbohydrates is complex, the names of the monosaccharides
and disaccharides very often end in the suffix -ose. For example, blood sugar is the monosaccharide glucose, table
sugar is the disaccharide sucrose, and milk sugar is the disaccharide lactose (see illustration).
Carbohydrates perform numerous roles in living organisms. Polysaccharides serve for the storage of energy (e.g.,
starch and glycogen), and as structural components (e.g., cellulose in plants and chitin in arthropods). The 5-carbon
monosaccharide ribose is an important component of coenzymes (e.g., ATP, FAD, and NAD) and the backbone of
the genetic molecule known as RNA. The related deoxyribose is a component of DNA. Saccharides and their
derivatives include many other important biomolecules that play key roles in the immune system, fertilization,
preventing pathogenesis, blood clotting, and development.
[2]
In food science and in many informal contexts, the term carbohydrate often means any food that is particularly rich
in the complex carbohydrate starch (such as cereals, bread, and pasta) or simple carbohydrates, such as sugar (found
in candy, jams, and desserts).
Carbohydrate
173
Structure
Formerly the name "carbohydrate" was used in chemistry for any compound with the formula C
m
(H
2
O)
n
. Following
this definition, some chemists considered formaldehyde (CH
2
O) to be the simplest carbohydrate,
[3]
while others
claimed that title for glycolaldehyde.
[4]
Today the term is generally understood in the biochemistry sense, which
excludes compounds with only one or two carbons.
Natural saccharides are generally built of simple carbohydrates called monosaccharides with general formula
(CH
2
O)
n
where n is three or more. A typical monosaccharide has the structure H-(CHOH)
x
(C=O)-(CHOH)
y
-H, that
is, an aldehyde or ketone with many hydroxyl groups added, usually one on each carbon atom that is not part of the
aldehyde or ketone functional group. Examples of monosaccharides are glucose, fructose, and glyceraldehydes.
However, some biological substances commonly called "monosaccharides" do not conform to this formula (e.g.,
uronic acids and deoxy-sugars such as fucose), and there are many chemicals that do conform to this formula but are
not considered to be monosaccharides (e.g., formaldehyde CH
2
O and inositol (CH
2
O)
6
).
[5]
The open-chain form of a monosaccharide often coexists with a closed ring form where the aldehyde/ketone
carbonyl group carbon (C=O) and hydroxyl group (-OH) react forming a hemiacetal with a new C-O-C bridge.
Monosaccharides can be linked together into what are called polysaccharides (or oligosaccharides) in a large variety
of ways. Many carbohydrates contain one or more modified monosaccharide units that have had one or more groups
replaced or removed. For example, deoxyribose, a component of DNA, is a modified version of ribose; chitin is
composed of repeating units of N-acetyl glucosamine, a nitrogen-containing form of glucose.
Monosaccharides
D-glucose is an aldohexose with the
formula (CH
2
O)
6
. The red atoms
highlight the aldehyde group, and
the blue atoms highlight the
asymmetric center furthest from the
aldehyde; because this -OH is on
the right of the Fischer projection,
this is a D sugar.
Monosaccharides are the simplest carbohydrates in that they cannot be hydrolyzed
to smaller carbohydrates. They are aldehydes or ketones with two or more
hydroxyl groups. The general chemical formula of an unmodified monosaccharide
is (CH
2
O)
n
, literally a "carbon hydrate." Monosaccharides are important fuel
molecules as well as building blocks for nucleic acids. The smallest
monosaccharides, for which n=3, are dihydroxyacetone and D- and
L-glyceraldehydes.
Classification of monosaccharides

The and anomers of glucose. Note the position of the hydroxyl group (red or
green) on the anomeric carbon relative to the CH
2
OH group bound to carbon 5:
they are either on the opposite sides (), or the same side ().
Monosaccharides are classified according to three different characteristics: the
placement of its carbonyl group, the number of carbon atoms it contains, and its
chiral handedness. If the carbonyl group is an aldehyde, the monosaccharide is an
aldose; if the carbonyl group is a ketone, the monosaccharide is a ketose. Monosaccharides with three carbon atoms
are called trioses, those with four are called tetroses, five are called pentoses, six are hexoses, and so on.
[6]
These two
systems of classification are often combined. For example, glucose is an aldohexose (a six-carbon aldehyde), ribose
is an aldopentose (a five-carbon aldehyde), and fructose is a ketohexose (a six-carbon ketone).
Carbohydrate
174
Each carbon atom bearing a hydroxyl group (-OH), with the exception of the first and last carbons, are asymmetric,
making them stereo centers with two possible configurations each (R or S). Because of this asymmetry, a number of
isomers may exist for any given monosaccharide formula. The aldohexose D-glucose, for example, has the formula
(CH
2
O)
6
, of which all but two of its six carbons atoms are stereogenic, making D-glucose one of 2
4
=16 possible
stereoisomers. In the case of glyceraldehydes, an aldotriose, there is one pair of possible stereoisomers, which are
enantiomers and epimers. 1, 3-dihydroxyacetone, the ketose corresponding to the aldose glyceraldehydes, is a
symmetric molecule with no stereo centers). The assignment of D or L is made according to the orientation of the
asymmetric carbon furthest from the carbonyl group: in a standard Fischer projection if the hydroxyl group is on the
right the molecule is a D sugar, otherwise it is an L sugar. The "D-" and "L-" prefixes should not be confused with
"d-" or "l-", which indicate the direction that the sugar rotates plane polarized light. This usage of "d-" and "l-" is no
longer followed in carbohydrate chemistry.
[7]
Ring-straight chain isomerism
Glucose can exist in both a straight-chain and ring
form.
The aldehyde or ketone group of a straight-chain monosaccharide
will react reversibly with a hydroxyl group on a different carbon
atom to form a hemiacetal or hemiketal, forming a heterocyclic
ring with an oxygen bridge between two carbon atoms. Rings with
five and six atoms are called furanose and pyranose forms,
respectively, and exist in equilibrium with the straight-chain
form.
[]
During the conversion from straight-chain form to the cyclic form,
the carbon atom containing the carbonyl oxygen, called the
anomeric carbon, becomes a stereogenic center with two possible
configurations: The oxygen atom may take a position either above
or below the plane of the ring. The resulting possible pair of
stereoisomers is called anomers. In the anomer, the -OH
substituent on the anomeric carbon rests on the opposite side (trans) of the ring from the CH
2
OH side branch. The
alternative form, in which the CH
2
OH substituent and the anomeric hydroxyl are on the same side (cis) of the plane
of the ring, is called the anomer.
Carbohydrate
175
Use in living organisms
Monosaccharides are the major source of fuel for metabolism, being used both as an energy source (glucose being
the most important in nature) and in biosynthesis. When monosaccharides are not immediately needed by many cells
they are often converted to more space-efficient forms, often polysaccharides. In many animals, including humans,
this storage form is glycogen, especially in liver and muscle cells. In plants, starch, is used for the same purpose.
Disaccharides
Sucrose, also known as table sugar, is a common disaccharide. It is
composed of two monosaccharides: D-glucose (left) and D-fructose
(right).
Two joined monosaccharides are called a disaccharide
and these are the simplest polysaccharides. Examples
include sucrose and lactose. They are composed of two
monosaccharide units bound together by a covalent
bond known as a glycosidic linkage formed via a
dehydration reaction, resulting in the loss of a hydrogen
atom from one monosaccharide and a hydroxyl group
from the other. The formula of unmodified
disaccharides is C
12
H
22
O
11
. Although there are
numerous kinds of disaccharides, a handful of
disaccharides are particularly notable.
Sucrose, pictured to the right, is the most abundant disaccharide, and the main form in which carbohydrates are
transported in plants. It is composed of one D-glucose molecule and one D-fructose molecule. The systematic name
for sucrose, O--D-glucopyranosyl-(12)-D-fructofuranoside, indicates four things:
Its monosaccharides: glucose and fructose
Their ring types: glucose is a pyranose, and fructose is a furanose
How they are linked together: the oxygen on carbon number 1 (C1) of -D-glucose is linked to the C2 of
D-fructose.
The -oside suffix indicates that the anomeric carbon of both monosaccharides participates in the glycosidic bond.
Lactose, a disaccharide composed of one D-galactose molecule and one D-glucose molecule, occurs naturally in
mammalian milk. The systematic name for lactose is O--D-galactopyranosyl-(14)-D-glucopyranose. Other
notable disaccharides include maltose (two D-glucoses linked -1,4) and cellulobiose (two D-glucoses linked -1,4).
Disaccharides can be classified into two types.They are reducing and non-reducing disaccharides. If the functional
group is present in bonding with another sugar unit, it is called a reducing disaccharide or biose.
Carbohydrate
176
Nutrition
Grain products: rich sources of carbohydrates
Foods high in carbohydrate include fruits, sweets, soft drinks, breads,
pastas, beans, potatoes, bran, rice, and cereals. Carbohydrates are a
common source of energy in living organisms; however, no
carbohydrate is an essential nutrient in humans.
[]
Carbohydrates are not necessary building blocks of other molecules,
and the body can obtain all its energy from protein and fats.
[][8]
The
brain and neurons generally cannot burn fat for energy, but use glucose
or ketones. Humans can synthesize some glucose (in a set of processes
known as gluconeogenesis) from specific amino acids, from the
glycerol backbone in triglycerides and in some cases from fatty acids.
Carbohydrate and protein contain 4 calories per gram, while fats
contain 9 calories per gram. In the case of protein, this is somewhat
misleading as only some amino acids are usable for fuel.
Organisms typically cannot metabolize all types of carbohydrate to
yield energy. Glucose is a nearly universal and accessible source of
calories. Many organisms also have the ability to metabolize other
monosaccharides and Disaccharides, though glucose is preferred. In
Escherichia coli, for example, the lac operon will express enzymes for the digestion of lactose when it is present, but
if both lactose and glucose are present the lac operon is repressed, resulting in the glucose being used first (see:
Diauxie). Polysaccharides are also common sources of energy. Many organisms can easily break down starches into
glucose, however, most organisms cannot metabolize cellulose or other polysaccharides like chitin and
arabinoxylans. These carbohydrates types can be metabolized by some bacteria and protists. Ruminants and termites,
for example, use microorganisms to process cellulose. Even though these complex carbohydrates are not very
digestible, they represent an important dietary element for humans, called dietary fiber. Fiber enhances digestion,
among other benefits.
[9]
Based on the effects on risk of heart disease and obesity,
[10]
the Institute of Medicine recommends that American
and Canadian adults get between 4565% of dietary energy from carbohydrates.
[11]
The Food and Agriculture
Organization and World Health Organization jointly recommend that national dietary guidelines set a goal of
5575% of total energy from carbohydrates, but only 10% directly from sugars (their term for simple
carbohydrates).
[12]
Classification
Nutritionists often refer to carbohydrates as either simple or complex. However, the exact delineation of these
categories can be ambiguous. The term complex carbohydrate was first used in the U.S. Senate Select Committee on
Nutrition and Human Needs publication Dietary Goals for the United States (1977) where it was intended to
distinguish sugars from other carbohydrates (which were perceived to be nutritionally superior).
[13]
However, the
report put "fruit, vegetables and whole-grains" in the complex carbohydrate column, despite the fact that these may
contain sugars as well as polysaccharides. This confusion persists as today some nutritionists use the term complex
carbohydrate to refer to any sort of digestible saccharide present in a whole food, where fiber, vitamins and minerals
are also found (as opposed to processed carbohydrates, which provide calories but few other nutrients). The standard
usage, however, is to classify carbohydrates chemically: simple if they are sugars (monosaccharides and
disaccharides) and complex if they are polysaccharides (or oligosaccharides).
[]
In any case, the simple vs. complex chemical distinction has little value for determining the nutritional quality of
carbohydrates.
[]
Some simple carbohydrates (e.g. fructose) are digested very slowly, while some complex
Carbohydrate
177
carbohydrates (starches), especially if processed, raise blood sugar rapidly. The speed of digestion is determined by a
variety of factors including which other nutrients are consumed with the carbohydrate, how the food is prepared,
individual differences in metabolism, and the chemistry of the carbohydrate.
[]
The USDA's Dietary Guidelines for Americans 2010 call for moderate- to high-carbohydrate consumption from a
balanced diet that includes six one-ounce servings of grain foods each day, at least half from whole grain sources and
the rest from enriched.
[14]
The glycemic index (GI) and glycemic load concepts have been developed to characterize food behavior during
human digestion. They rank carbohydrate-rich foods based on the rapidity and magnitude of their effect on blood
glucose levels. Glycemic index is a measure of how quickly food glucose is absorbed, while glycemic load is a
measure of the total absorbable glucose in foods. The insulin index is a similar, more recent classification method
that ranks foods based on their effects on blood insulin levels, which are caused by glucose (or starch) and some
amino acids in food.
Metabolism
Catabolism
Catabolism is the metabolic reaction which cells undergo to extract energy. There are two major metabolic pathways
of monosaccharide catabolism: glycolysis and the citric acid cycle.
In glycolysis, oligo/polysaccharides are cleaved first to smaller monosaccharides by enzymes called glycoside
hydrolases. The monosaccharide units can then enter into monosaccharide catabolism. In some cases, as with
humans, not all carbohydrate types are usable as the digestive and metabolic enzymes necessary are not present.
Carbohydrate chemistry
Carbohydrate chemistry is a large and economically important branch of organic chemistry. Some of the main
organic reactions that involve carbohydrates are:
Carbohydrate acetalisation
Cyanohydrin reaction
Lobry-de Bruyn-van Ekenstein transformation
Amadori rearrangement
Nef reaction
Wohl degradation
KoenigsKnorr reaction
References
[3] John Merle Coulter, Charler Reid Barnes, Henry Chandler Cowles (1930), A Textbook of Botany for Colleges and Universities (http:/ /
books.google. com.br/ books?id=WyZnVpCiTHIC& pg=PA375& dq=simplest+ carbohydrate)"
[4] Carl A. Burtis, Edward R. Ashwood, Norbert W. Tietz (2000), Tietz fundamentals of clinical chemistry (http:/ / books. google. com/
books?id=l5hqAAAAMAAJ& q=simplest+ carbohydrate)
[5] Matthews, C. E.; K. E. Van Holde; K. G. Ahern (1999) Biochemistry. 3rd edition. Benjamin Cummings. ISBN 0-8053-3066-6
[11] Food and Nutrition Board (2002/2005). Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein,
and Amino Acids (http:/ / newton. nap.edu/ books/ 0309085373/ html). Washington, D.C.: The National Academies Press. Page 769 (http:/ /
newton.nap.edu/ books/ 0309085373/ html/ 769.html). ISBN 0-309-08537-3.
[12] Joint WHO/FAO expert consultation (2003). (http:/ / www. webcitation. org/ query?id=1304266103156369) (PDF). Geneva: World Health
Organization. pp. 5556. ISBN 92-4-120916-X.
[13] Joint WHO/FAO expert consultation (1998), Carbohydrates in human nutrition, chapter 1 (http:/ / www. fao. org/ docrep/ W8079E/
w8079e07. htm). ISBN 92-5-104114-8.
[14] DHHS and USDA, Dietary Guidelines for Americans 2010 (http:/ / www. cnpp. usda. gov/ DietaryGuidelines. htm).
Carbohydrate
178
External links
Carbohydrates, including interactive models and animations (http:/ / www2. ufp. pt/ ~pedros/ bq/ carb_en. htm)
(Requires MDL Chime (http:/ / www. mdl. com/ products/ framework/ chime/ ))
IUPAC-IUBMB Joint Commission on Biochemical Nomenclature (JCBN): Carbohydrate Nomenclature (http:/ /
www. chem. qmw. ac. uk/ iupac/ 2carb/ )
Carbohydrates detailed (http:/ / www. cem. msu. edu/ ~reusch/ VirtualText/ carbhyd. htm)
Complex And Simple Carbohydrates (http:/ / evilcyber. com/ nutrition/ complex-and-simple-carbohydrates/ )
Explanation of the differences
Carbohydrates and Glycosylation The Virtual Library of Biochemistry and Cell Biology (http:/ / www.
biochemweb. org/ carbohydrates. shtml)
Functional Glycomics Gateway (http:/ / www. functionalglycomics. org/ ), a collaboration between the
Consortium for Functional Glycomics and Nature Publishing Group
Wine Carbohydrates (http:/ / www. wineclubwizard. com/ wine-carbohydrates. html)
Polysaccharide
3D structure of cellulose, a beta-glucan polysaccharide.
Polysaccharides are long carbohydrate molecules of
monosaccharide units joined together by glycosidic
bonds. They range in structure from linear to highly
branched. Polysaccharides are often quite
heterogeneous, containing slight modifications of the
repeating unit. Depending on the structure, these
macromolecules can have distinct properties from their
monosaccharide building blocks. They may be
amorphous or even insoluble in water.
[][]
When all the monosaccharides in a polysaccharide are the same type, the polysaccharide is called a
homopolysaccharide or homoglycan, but when more than one type of monosaccharide is present they are called
heteropolysaccharides or heteroglycans.
[1][2]
Examples include storage polysaccharides such as starch and glycogen, and structural polysaccharides such as
cellulose and chitin.
Polysaccharides have a general formula of C
x
(H
2
O)
y
where x is usually a large number between 200 and 2500.
Considering that the repeating units in the polymer backbone are often six-carbon monosaccharides, the general
formula can also be represented as (C
6
H
10
O
5
)
n
where 40n3000.
Polysaccharide
179
Structure
Natural saccharides are generally built of simple carbohydrates called monosaccharides with general formula
(CH
2
O)
n
where n is three or more. A typical monosaccharide has the structure H-(CHOH)
x
(C=O)-(CHOH)
y
-H, that
is, an aldehyde or ketone with many hydroxyl groups added, usually one on each carbon atom that is not part of the
aldehyde or ketone functional group. Examples of monosaccharides are glucose, fructose, and glyceraldehyde
[3]
Amylose is a linear polymer of glucose mainly linked with (14) bonds. It can be made
of several thousands of glucose units. It is one of the two components of starch, the other
being amylopectin.
Polysaccharides are composed of long
chains of monosaccharide units bound
together by glycosidic bonds.
Polysaccharides contain more than ten
monosaccharide units. Definitions of
how large a carbohydrate must be to
fall into the categories polysaccharides
or oligosaccharides vary according to
personal opinion.
Polysaccharides is an important class
of biological polymers. Their function
in living organisms is usually either
structure- or storage-related. Starch (a
polymer of glucose) is used as a storage polysaccharide in plants, being found in the form of both amylose and the
branched amylopectin. In animals, the structurally similar glucose polymer is the more densely branched glycogen,
sometimes called 'animal starch'. Glycogen's properties allow it to be metabolized more quickly, which suits the
active lives of moving animals.
Cellulose and chitin are examples of structural polysaccharides. Cellulose is used in the cell walls of plants and other
organisms, and is said to be the most abundant organic molecule on earth.
[4]
It has many uses such as a significant
role in the paper and textile industries, and is used as a feedstock for the production of rayon (via the viscose
process), cellulose acetate, celluloid, and nitrocellulose. Chitin has a similar structure, but has nitrogen-containing
side branches, increasing its strength. It is found in arthropod exoskeletons and in the cell walls of some fungi. It also
has multiple uses, including surgical threads.
Polysaccharides also include callose or laminarin, chrysolaminarin, xylan, arabinoxylan, mannan, fucoidan and
galactomannan.
Function
Nutrition
Polysaccharides are common sources of energy. Many organisms can easily break down starches into glucose,
however, most organisms cannot metabolize cellulose or other polysaccharides like chitin and arabinoxylans. These
carbohydrates types can be metabolized by some bacteria and protists. Ruminants and termites, for example, use
microorganisms to process cellulose.
Even though these complex carbohydrates are not very digestible, they may comprise important dietary elements for
humans. Called dietary fiber, these carbohydrates enhance digestion among other benefits. The main action of
dietary fiber is to change the nature of the contents of the gastrointestinal tract, and to change how other nutrients
and chemicals are absorbed.
[][]
Soluble fiber binds to bile acids in the small intestine, making them less likely to
enter the body; this in turn lowers cholesterol levels in the blood.
[]
Soluble fiber also attenuates the absorption of
sugar, reduces sugar response after eating, normalizes blood lipid levels and, once fermented in the colon, produces
short-chain fatty acids as byproducts with wide-ranging physiological activities (discussion below). Although
Polysaccharide
180
insoluble fiber is associated with reduced diabetes risk, the mechanism by which this occurs is unknown.
[5]
Not yet formally proposed as an essential macronutrient, dietary fiber is nevertheless regarded as important for the
diet, with regulatory authorities in many developed countries recommending increases in fiber intake.
[][][6][7]
Storage polysaccharides
Starches
Starches are glucose polymers in which glucopyranose units are bonded by alpha-linkages. It is made up of a
mixture of amylose (1520%) and amylopectin (8085%). Amylose consists of a linear chain of several hundred
glucose molecules and Amylopectin is a branched molecule made of several thousand glucose units (every chain of
2430 glucose units is one unit of Amylopectin). Starches are insoluble in water. They can be digested by
hydrolysis, catalyzed by enzymes called amylases, which can break the alpha-linkages (glycosidic bonds). Humans
and other animals have amylases, so they can digest starches. Potato, rice, wheat, and maize are major sources of
starch in the human diet. The formations of starches are the ways that plants store glucose.
Glycogen
Schematic 2-D cross-sectional view of glycogen. A core protein of glycogenin is
surrounded by branches of glucose units. The entire globular granule may contain
approximately 30,000 glucose units.
[8]
Glycogen serves as the secondary long-term
energy storage in animal and fungal cells,
with the primary energy stores being held in
adipose tissue. Glycogen is made primarily
by the liver and the muscles, but can also be
made by glycogenesis within the brain and
stomach.
[9]
Glycogen is the analogue of starch, a
glucose polymer in plants, and is sometimes
referred to as animal starch, having a
similar structure to amylopectin but more
extensively branched and compact than
starch. Glycogen is a polymer of (14)
glycosidic bonds linked, with
(16)-linked branches. Glycogen is found
in the form of granules in the
cytosol/cytoplasm in many cell types, and
plays an important role in the glucose cycle.
Glycogen forms an energy reserve that can
be quickly mobilized to meet a sudden need
for glucose, but one that is less compact
than the less immediately available energy
reserves of triglycerides (lipids).
In the liver hepatocytes, glycogen can compose up to eight percent (100120g in an adult) of the fresh weight soon
after a meal.
[]
Only the glycogen stored in the liver
Polysaccharide
181
A view of the atomic structure of a single branched strand of glucose units in a
glycogen molecule.
can be made accessible to other organs. In
the muscles, glycogen is found in a low
concentration of one to two percent of the
muscle mass. The amount of glycogen
stored in the bodyespecially within the
muscles, liver, and red blood
cells
[10][11][12]
varies with physical
activity, basal metabolic rate, and eating
habits such as intermittent fasting. Small
amounts of glycogen are found in the
kidneys, and even smaller amounts in
certain glial cells in the brain and white
blood cells. The uterus also stores glycogen
during pregnancy, to nourish the embryo.
[]
Glycogen is composed of a branched chain
of glucose residues. It is stored in liver and
muscles.
It is an energy reserve for animals.
It is the chief form of carbohydrate stored
in animal body.
It is insoluble in water. It turns red when mixed with iodine.
It also yields glucose on hydrolysis.
Structural polysaccharides
Arabinoxylans
Arabinoxylans are found in both the primary and secondary cell walls of plants and are the copolymers of two
pentose sugars: arabinose and xylose.
Cellulose
The structural component of plants are formed primarily from cellulose. Wood is largely cellulose and lignin, while
paper and cotton are nearly pure cellulose. Cellulose is a polymer made with repeated glucose units bonded together
by beta-linkages. Humans and many other animals lack an enzyme to break the beta-linkages, so they do not digest
cellulose. Certain animals such as termites can digest cellulose, because bacteria possessing the enzyme are present
in their gut. Cellulose is insoluble in water. It does not change color when mixed with iodine. On hydrolysis, it yields
glucose. It is the most abundant carbohydrate in nature.
Chitin
Chitin is one of many naturally occurring polymers. It forms a structural component of many animals, such as
exoskeletons. Over time it is bio-degradable in the natural environment. Its breakdown may be catalyzed by enzymes
called chitinases, secreted by microorganisms such as bacteria and fungi, and produced by some plants. Some of
these microorganisms have receptors to simple sugars from the decomposition of chitin. If chitin is detected, they
then produce enzymes to digest it by cleaving the glycosidic bonds in order to convert it to simple sugars and
ammonia.
Polysaccharide
182
Chemically, chitin is closely related to chitosan (a more water-soluble derivative of chitin). It is also closely related
to cellulose in that it is a long unbranched chain of glucose derivatives. Both materials contribute structure and
strength, protecting the organism.
Pectins
Pectins are a family of complex polysaccharides that contain 1,4-linked -D-galactosyluronic acid residues. They are
present in most primary cell walls and in the non-woody parts of terrestrial plants.
Acidic polysaccharides
Acidic polysaccharides are polysaccharides that contain carboxyl groups, phosphate groups and/or sulfuric ester
groups.
Bacterial capsular polysaccharides
Pathogenic bacteria commonly produce a thick, mucous-like, layer of polysaccharide. This "capsule" cloaks
antigenic proteins on the bacterial surface that would otherwise provoke an immune response and thereby lead to the
destruction of the bacteria. Capsular polysaccharides are water soluble, commonly acidic, and have molecular
weights on the order of 100-2000 kDa. They are linear and consist of regularly repeating subunits of one to six
monosaccharides. There is enormous structural diversity; nearly two hundred different polysaccharides are produced
by E. coli alone. Mixtures of capsular polysaccharides, either conjugated or native are used as vaccines.
Bacteria and many other microbes, including fungi and algae, often secrete polysaccharides as an evolutionary
adaptation to help them adhere to surfaces and to prevent them from drying out. Humans have developed some of
these polysaccharides into useful products, including xanthan gum, dextran, welan gum, gellan gum, diutan gum and
pullulan.
Most of these polysaccharides exhibit useful visco-elastic properties when dissolved in water at very low levels.
[13]
This makes various liquids used in everyday life, such as some foods, lotions, cleaners, and paints, viscous when
stationary, but much more free-flowing when even slight shear is applied by stirring or shaking, pouring, wiping, or
brushing. This property is named pseudoplasticity or shear thinning; the study of such matters is called rheology.
Viscosity of Welan gum
[14]
Shear Rate (rpm) Viscosity (cP)
0.3 23330
0.5 16000
1 11000
2 5500
4 3250
5 2900
10 1700
20 900
50 520
100 310
Aqueous solutions of the polysaccharide alone have a curious behavior when stirred: after stirring ceases, the
solution initially continues to swirl due to momentum, then slows to a standstill due to viscosity and reverses
Polysaccharide
183
direction briefly before stopping. This recoil is due to the elastic effect of the polysaccharide chains, previously
stretched in solution, returning to their relaxed state.
Cell-surface polysaccharides play diverse roles in bacterial ecology and physiology. They serve as a barrier between
the cell wall and the environment, mediate host-pathogen interactions, and form structural components of biofilms.
These polysaccharides are synthesized from nucleotide-activated precursors (called nucleotide sugars) and, in most
cases, all the enzymes necessary for biosynthesis, assembly and transport of the completed polymer are encoded by
genes organized in dedicated clusters within the genome of the organism. Lipopolysaccharide is one of the most
important cell-surface polysaccharides, as it plays a key structural role in outer membrane integrity, as well as being
an important mediator of host-pathogen interactions.
The enzymes that make the A-band (homopolymeric) and B-band (heteropolymeric) O-antigens have been identified
and the metabolic pathways defined.
[15]
The exopolysaccharide alginate is a linear copolymer of -1,4-linked
D-mannuronic acid and L-guluronic acid residues, and is responsible for the mucoid phenotype of late-stage cystic
fibrosis disease. The pel and psl loci are two recently discovered gene clusters that also encode exopolysaccharides
found to be important for biofilm formation. Rhamnolipid is a biosurfactant whose production is tightly regulated at
the transcriptional level, but the precise role that it plays in disease is not well understood at present. Protein
glycosylation, particularly of pilin and flagellin, became a focus of research by several groups from about 2007, and
has been shown to be important for adhesion and invasion during bacterial infection.
[]
Chemical identification tests for polysaccharides
Periodic acid-Schiff stain (PAS)
Polysaccharides with unprotected vicinal diols or amino sugars (i.e. some OH groups replaced with amine) give a
positive Periodic acid-Schiff stain (PAS). The list of polysaccharides that stain with PAS is long. Although mucins
of epithelial origins stain with PAS, mucins of connective tissue origin have so many acidic substitutions that they do
not have enough glycol or amino-alcohol groups left to react with PAS.
References
[3] Matthews, C. E.; K. E. Van Holde; K. G. Ahern (1999) Biochemistry. 3rd edition. Benjamin Cummings. ISBN 0-8053-3066-6
[4] N.A.Campbell (1996) Biology (4th edition). Benjamin Cummings NY. p.23 ISBN 0-8053-1957-3
[8] Page 12 in: (http:/ / books. google.dk/ books?id=SRptlOx7yj4C& printsec=frontcover& hl=en) Exercise physiology: energy, nutrition, and
human performance By William D. McArdle, Frank I. Katch, Victor L. Katch Edition: 6, illustrated Published by Lippincott Williams &
Wilkins, 2006 ISBN 0-7817-4990-5, ISBN 978-0-7817-4990-9, 1068 pages
[9] [9] Anatomy and Physiology. Saladin, Kenneth S. McGraw-Hill, 2007.
[11] http:/ / jeb.biologists.org/ cgi/ reprint/ 129/ 1/ 141.pdf
[13] Viscosity of Welan Gum vs. Concentration in Water. http:/ / www. xydatasource. com/ xy-showdatasetpage. php?datasetcode=345115&
dsid=80
[14] http:/ / www.xydatasource. com/ xy-showdatasetpage. php?datasetcode=45615& dsid=76& searchtext=polysaccharide
External links
Polysaccharide Structure (http:/ / employees. csbsju. edu/ hjakubowski/ classes/ ch331/ cho/ complexoligosacch.
htm)
Applications and commercial sources of polysaccharides (http:/ / www. polysaccharidecenter. com)
European Polysaccharide Network of Excellence (http:/ / www. epnoe. eu)
184
Intermediary metabolism
185
Metabolism
Overview of metabolism
Structure of adenosine triphosphate (ATP), a
central intermediate in energy metabolism
Metabolism (from Greek: metabol, "change" or Greek:
metabolismos, "outthrow") is the set of life-sustaining
chemical transformations within the cells of living organisms. These
enzyme-catalyzed reactions allow organisms to grow and reproduce,
maintain their structures, and respond to their environments. The word
metabolism can also refer to all chemical reactions that occur in living
organisms, including digestion and the transport of substances into and
between different cells, in which case the set of reactions within the
cells is called intermediary metabolism or intermediate
metabolism.
Metabolism is usually divided into two categories. Catabolism breaks
down organic matter, for example to harvest energy in cellular respiration. Anabolism uses energy to construct
components of cells such as proteins and nucleic acids.
The chemical reactions of metabolism are organized into metabolic pathways, in which one chemical is transformed
through a series of steps into another chemical, by a sequence of enzymes. Enzymes are crucial to metabolism
because they allow organisms to drive desirable reactions that require energy and will not occur by themselves, by
coupling them to spontaneous reactions that release energy. As enzymes act as catalysts they allow these reactions to
proceed quickly and efficiently. Enzymes also allow the regulation of metabolic pathways in response to changes in
the cell's environment or signals from other cells.
The metabolism of an organism determines which substances it will find nutritious and which it will find poisonous.
For example, some prokaryotes use hydrogen sulfide as a nutrient, yet this gas is poisonous to animals.
[]
The speed
of metabolism, the metabolic rate, influences how much food an organism will require, and also affects how it is able
to obtain that food.
A striking feature of metabolism is the similarity of the basic metabolic pathways and components between even
vastly different species.
[1]
For example, the set of carboxylic acids that are best known as the intermediates in the
citric acid cycle are present in all known organisms, being found in species as diverse as the unicellular bacterium
Escherichia coli and huge multicellular organisms like elephants.
[]
These striking similarities in metabolic pathways
are likely due to their early appearance in evolutionary history, and being retained because of their efficacy.
[][]
Overview of metabolism
186
Key biochemicals
Structure of a triacylglycerol lipid
Most of the structures that make up animals, plants and
microbes are made from three basic classes of
molecule: amino acids, carbohydrates and lipids (often
called fats). As these molecules are vital for life,
metabolic reactions either focus on making these
molecules during the construction of cells and tissues,
or breaking them down and using them as a source of
energy, in the digestion and use of food. Many
important biochemicals can be joined together to make
polymers such as DNA and proteins. These
macromolecules are essential.
Type of molecule Name of monomer forms Name of polymer forms Examples of polymer forms
Amino acids Amino acids Proteins (also called polypeptides) Fibrous proteins and globular proteins
Carbohydrates Monosaccharides Polysaccharides Starch, glycogen and cellulose
Nucleic acids Nucleotides Polynucleotides DNA and RNA
Amino acids and proteins
Proteins are made of amino acids arranged in a linear chain and joined together by peptide bonds. Many proteins are
the enzymes that catalyze the chemical reactions in metabolism. Other proteins have structural or mechanical
functions, such as the proteins that form the cytoskeleton, a system of scaffolding that maintains the cell shape.
[2]
Proteins are also important in cell signaling, immune responses, cell adhesion, active transport across membranes,
and the cell cycle.
[]
Amino acids also contribute to cellular energy metabolism by providing a carbon source for
entry into the tricarboxylic acid cycle,
[3]
especially a when primary source of energy, such as glucose, is scarce, or
when cells undergo metabolic stress.
[4]
Lipids
Lipids are the most diverse group of biochemicals. Their main structural uses are as part of biological membranes
such as the cell membrane, or as a source of energy.
[]
Lipids are usually defined as hydrophobic or amphipathic
biological molecules that will dissolve in organic solvents such as benzene or chloroform.
[5]
The fats are a large
group of compounds that contain fatty acids and glycerol; a glycerol molecule attached to three fatty acid esters is a
triacylglyceride.
[6]
Several variations on this basic structure exist, including alternate backbones such as sphingosine
in the sphingolipids, and hydrophilic groups such as phosphate in phospholipids. Steroids such as cholesterol are
another major class of lipids that are made in cells.
[7]
Overview of metabolism
187
Carbohydrates
Glucose can exist in both a straight-chain and ring form.
Carbohydrates are aldehydes or ketones with many
hydroxyl groups that can exist as straight chains or
rings. Carbohydrates are the most abundant biological
molecules, and fill numerous roles, such as the storage
and transport of energy (starch, glycogen) and structural
components (cellulose in plants, chitin in animals).
[]
The basic carbohydrate units are called
monosaccharides and include galactose, fructose, and
most importantly glucose. Monosaccharides can be
linked together to form polysaccharides in almost
limitless ways.
[8]
Nucleotides
The two nucleic acids, DNA and RNA are polymers of
nucleotides, each nucleotide comprising a phosphate group, a ribose sugar group, and a nitrogenous base. Nucleic
acids are critical for the storage and use of genetic information, through the processes of transcription and protein
biosynthesis.
[]
This information is protected by DNA repair mechanisms and propagated through DNA replication.
Many viruses have an RNA genome, for example HIV, which uses reverse transcription to create a DNA template
from its viral RNA genome.
[9]
RNA in ribozymes such as spliceosomes and ribosomes is similar to enzymes as it
can catalyze chemical reactions. Individual nucleosides are made by attaching a nucleobase to a ribose sugar. These
bases are heterocyclic rings containing nitrogen, classified as purines or pyrimidines. Nucleotides also act as
coenzymes in metabolic group transfer reactions.
[]
Coenzymes
Structure of the coenzyme acetyl-CoA.The transferable acetyl group is bonded to
the sulfur atom at the extreme left.
Metabolism involves a vast array of
chemical reactions, but most fall under a
few basic types of reactions that involve the
transfer of functional groups.
[10]
This
common chemistry allows cells to use a
small set of metabolic intermediates to carry
chemical groups between different
reactions.
[]
These group-transfer
intermediates are called coenzymes. Each
class of group-transfer reactions is carried
out by a particular coenzyme, which is the substrate for a set of enzymes that produce it, and a set of enzymes that
consume it. These coenzymes are therefore continuously being made, consumed and then recycled.
[]
One central coenzyme is adenosine triphosphate (ATP), the universal energy currency of cells. This nucleotide is
used to transfer chemical energy between different chemical reactions. There is only a small amount of ATP in cells,
but as it is continuously regenerated, the human body can use about its own weight in ATP per day.
[]
ATP acts as a
bridge between catabolism and anabolism, with catabolic reactions generating ATP and anabolic reactions
consuming it. It also serves as a carrier of phosphate groups in phosphorylation reactions.
A vitamin is an organic compound needed in small quantities that cannot be made in the cells. In human nutrition,
most vitamins function as coenzymes after modification; for example, all water-soluble vitamins are phosphorylated
Overview of metabolism
188
or are coupled to nucleotides when they are used in cells.
[11]
Nicotinamide adenine dinucleotide (NADH), a
derivative of vitamin B
3
(niacin), is an important coenzyme that acts as a hydrogen acceptor. Hundreds of separate
types of dehydrogenases remove electrons from their substrates and reduce NAD
+
into NADH. This reduced form of
the coenzyme is then a substrate for any of the reductases in the cell that need to reduce their substrates.
[12]
Nicotinamide adenine dinucleotide exists in two related forms in the cell, NADH and NADPH. The NAD
+
/NADH
form is more important in catabolic reactions, while NADP
+
/NADPH is used in anabolic reactions.
Structure of hemoglobin. The protein subunits are in red and blue, and the
iron-containing heme groups in green. From PDB 1GZX
[1]
.
Minerals and cofactors
Inorganic elements play critical roles in
metabolism; some are abundant (e.g. sodium
and potassium) while others function at
minute concentrations. About 99% of a
mammal's mass is made up of the elements
carbon, nitrogen, calcium, sodium, chlorine,
potassium, hydrogen, phosphorus, oxygen
and sulfur.
[]
Organic compounds (proteins,
lipids and carbohydrates) contain the
majority of the carbon and nitrogen; most of
the oxygen and hydrogen is present as
water.
[]
The abundant inorganic elements act as
ionic electrolytes. The most important ions
are sodium, potassium, calcium,
magnesium, chloride, phosphate and the
organic ion bicarbonate. The maintenance of
precise gradients across cell membranes
maintains osmotic pressure and pH.
[13]
Ions
are also critical for nerve and muscle function, as action potentials in these tissues are produced by the exchange of
electrolytes between the extracellular fluid and the cytosol.
[14]
Electrolytes enter and leave cells through proteins in
the cell membrane called ion channels. For example, muscle contraction depends upon the movement of calcium,
sodium and potassium through ion channels in the cell membrane and T-tubules.
[15]
Transition metals are usually present as trace elements in organisms, with zinc and iron being most abundant.
[16][]
These metals are used in some proteins as cofactors and are essential for the activity of enzymes such as catalase and
oxygen-carrier proteins such as hemoglobin.
[17]
Metal cofactors are bound tightly to specific sites in proteins;
although enzyme cofactors can be modified during catalysis, they always return to their original state by the end of
the reaction catalyzed. Metal micronutrients are taken up into organisms by specific transporters and bind to storage
proteins such as ferritin or metallothionein when not being used.
[18][19]
Catabolism
Catabolism is the set of metabolic processes that break down large molecules. These include breaking down and
oxidizing food molecules. The purpose of the catabolic reactions is to provide the energy and components needed by
anabolic reactions. The exact nature of these catabolic reactions differ from organism to organism and organisms can
be classified based on their sources of energy and carbon (their primary nutritional groups), as shown in the table
below. Organic molecules are used as a source of energy by organotrophs, while lithotrophs use inorganic substrates
and phototrophs capture sunlight as chemical energy. However, all these different forms of metabolism depend on
Overview of metabolism
189
redox reactions that involve the transfer of electrons from reduced donor molecules such as organic molecules,
water, ammonia, hydrogen sulfide or ferrous ions to acceptor molecules such as oxygen, nitrate or sulfate.
[20]
In
animals these reactions involve complex organic molecules being broken down to simpler molecules, such as carbon
dioxide and water. In photosynthetic organisms such as plants and cyanobacteria, these electron-transfer reactions do
not release energy, but are used as a way of storing energy absorbed from sunlight.
[]
Classification of organisms based on their metabolism
Energy source sunlight photo- -troph
Preformed molecules chemo-
Electron donor organic compound organo-
inorganic compound litho-
Carbon source organic compound hetero-
inorganic compound auto-
The most common set of catabolic reactions in animals can be separated into three main stages. In the first, large
organic molecules such as proteins, polysaccharides or lipids are digested into their smaller components outside
cells. Next, these smaller molecules are taken up by cells and converted to yet smaller molecules, usually acetyl
coenzyme A (acetyl-CoA), which releases some energy. Finally, the acetyl group on the CoA is oxidised to water
and carbon dioxide in the citric acid cycle and electron transport chain, releasing the energy that is stored by
reducing the coenzyme nicotinamide adenine dinucleotide (NAD
+
) into NADH.
Digestion
Macromolecules such as starch, cellulose or proteins cannot be rapidly taken up by cells and must be broken into
their smaller units before they can be used in cell metabolism. Several common classes of enzymes digest these
polymers. These digestive enzymes include proteases that digest proteins into amino acids, as well as glycoside
hydrolases that digest polysaccharides into monosaccharides.
Microbes simply secrete digestive enzymes into their surroundings,
[21][22]
while animals only secrete these enzymes
from specialized cells in their guts.
[23]
The amino acids or sugars released by these extracellular enzymes are then
pumped into cells by specific active transport proteins.
[24][25]
Overview of metabolism
190
A simplified outline of the catabolism of proteins, carbohydrates and fats
Energy from organic compounds
Carbohydrate catabolism is the breakdown
of carbohydrates into smaller units.
Carbohydrates are usually taken into cells
once they have been digested into
monosaccharides.
[26]
Once inside, the major
route of breakdown is glycolysis, where
sugars such as glucose and fructose are
converted into pyruvate and some ATP is
generated.
[]
Pyruvate is an intermediate in
several metabolic pathways, but the majority
is converted to acetyl-CoA and fed into the
citric acid cycle. Although some more ATP
is generated in the citric acid cycle, the most
important product is NADH, which is made
from NAD
+
as the acetyl-CoA is oxidized.
This oxidation releases carbon dioxide as a
waste product. In anaerobic conditions,
glycolysis produces lactate, through the
enzyme lactate dehydrogenase re-oxidizing NADH to NAD+ for re-use in glycolysis. An alternative route for
glucose breakdown is the pentose phosphate pathway, which reduces the coenzyme NADPH and produces pentose
sugars such as ribose, the sugar component of nucleic acids.
Fats are catabolised by hydrolysis to free fatty acids and glycerol. The glycerol enters glycolysis and the fatty acids
are broken down by beta oxidation to release acetyl-CoA, which then is fed into the citric acid cycle. Fatty acids
release more energy upon oxidation than carbohydrates because carbohydrates contain more oxygen in their
structures.
Amino acids are either used to synthesize proteins and other biomolecules, or oxidized to urea and carbon dioxide as
a source of energy.
[27]
The oxidation pathway starts with the removal of the amino group by a transaminase. The
amino group is fed into the urea cycle, leaving a deaminated carbon skeleton in the form of a keto acid. Several of
these keto acids are intermediates in the citric acid cycle, for example the deamination of glutamate forms
-ketoglutarate.
[28]
The glucogenic amino acids can also be converted into glucose, through gluconeogenesis
(discussed below).
[29]
Energy transformations
Oxidative phosphorylation
In oxidative phosphorylation, the electrons removed from organic molecules in areas such as the protagon acid cycle
are transferred to oxygen and the energy released is used to make ATP. This is done in eukaryotes by a series of
proteins in the membranes of mitochondria called the electron transport chain. In prokaryotes, these proteins are
found in the cell's inner membrane.
[30]
These proteins use the energy released from passing electrons from reduced
molecules like NADH onto oxygen to pump protons across a membrane.
[31]
Overview of metabolism
191
Mechanism of ATP synthase. ATP is shown in red, ADP and
phosphate in pink and the rotating stalk subunit in black.
Pumping protons out of the mitochondria creates a proton
concentration difference across the membrane and generates
an electrochemical gradient.
[32]
This force drives protons back
into the mitochondrion through the base of an enzyme called
ATP synthase. The flow of protons makes the stalk subunit
rotate, causing the active site of the synthase domain to
change shape and phosphorylate adenosine diphosphate
turning it into ATP.
[]
Energy from inorganic compounds
Chemolithotrophy is a type of metabolism found in
prokaryotes where energy is obtained from the oxidation of
inorganic compounds. These organisms can use hydrogen,
[33]
reduced sulfur compounds (such as sulfide, hydrogen sulfide
and thiosulfate),
[]
ferrous iron (FeII)
[34]
or ammonia
[35]
as sources of reducing power and they gain energy from the
oxidation of these compounds with electron acceptors such as oxygen or nitrite.
[36]
These microbial processes are
important in global biogeochemical cycles such as acetogenesis, nitrification and denitrification and are critical for
soil fertility.
[37][38]
Energy from light
The energy in sunlight is captured by plants, cyanobacteria, purple bacteria, green sulfur bacteria and some protists.
This process is often coupled to the conversion of carbon dioxide into organic compounds, as part of photosynthesis,
which is discussed below. The energy capture and carbon fixation systems can however operate separately in
prokaryotes, as purple bacteria and green sulfur bacteria can use sunlight as a source of energy, while switching
between carbon fixation and the fermentation of organic compounds.
[39][40]
In many organisms the capture of solar energy is similar in principle to oxidative phosphorylation, as it involves
energy being stored as a proton concentration gradient and this proton motive force then driving ATP synthesis.
[]
The electrons needed to drive this electron transport chain come from light-gathering proteins called photosynthetic
reaction centres or rhodopsins. Reaction centers are classed into two types depending on the type of photosynthetic
pigment present, with most photosynthetic bacteria only having one type, while plants and cyanobacteria have
two.
[41]
In plants, algae, and cyanobacteria, photosystem II uses light energy to remove electrons from water, releasing
oxygen as a waste product. The electrons then flow to the cytochrome b6f complex, which uses their energy to pump
protons across the thylakoid membrane in the chloroplast.
[]
These protons move back through the membrane as they
drive the ATP synthase, as before. The electrons then flow through photosystem I and can then either be used to
reduce the coenzyme NADP
+
, for use in the Calvin cycle, which is discussed below, or recycled for further ATP
generation.
[42]
Anabolism
Anabolism is the set of constructive metabolic processes where the energy released by catabolism is used to
synthesize complex molecules. In general, the complex molecules that make up cellular structures are constructed
step-by-step from small and simple precursors. Anabolism involves three basic stages. Firstly, the production of
precursors such as amino acids, monosaccharides, isoprenoids and nucleotides, secondly, their activation into
reactive forms using energy from ATP, and thirdly, the assembly of these precursors into complex molecules such as
proteins, polysaccharides, lipids and nucleic acids.
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192
Organisms differ in how many of the molecules in their cells they can construct for themselves. Autotrophs such as
plants can construct the complex organic molecules in cells such as polysaccharides and proteins from simple
molecules like carbon dioxide and water. Heterotrophs, on the other hand, require a source of more complex
substances, such as monosaccharides and amino acids, to produce these complex molecules. Organisms can be
further classified by ultimate source of their energy: photoautotrophs and photoheterotrophs obtain energy from
light, whereas chemoautotrophs and chemoheterotrophs obtain energy from inorganic oxidation reactions.
Carbon fixation
Plant cells (bounded by purple walls) filled with
chloroplasts (green), which are the site of
photosynthesis
Photosynthesis is the synthesis of carbohydrates from sunlight and
carbon dioxide (CO
2
). In plants, cyanobacteria and algae, oxygenic
photosynthesis splits water, with oxygen produced as a waste product.
This process uses the ATP and NADPH produced by the
photosynthetic reaction centres, as described above, to convert CO
2
into glycerate 3-phosphate, which can then be converted into glucose.
This carbon-fixation reaction is carried out by the enzyme RuBisCO as
part of the Calvin Benson cycle.
[43]
Three types of photosynthesis
occur in plants, C3 carbon fixation, C4 carbon fixation and CAM
photosynthesis. These differ by the route that carbon dioxide takes to
the Calvin cycle, with C3 plants fixing CO
2
directly, while C4 and
CAM photosynthesis incorporate the CO
2
into other compounds first,
as adaptations to deal with intense sunlight and dry conditions.
[44]
In photosynthetic prokaryotes the mechanisms of carbon fixation are more diverse. Here, carbon dioxide can be
fixed by the Calvin Benson cycle, a reversed citric acid cycle,
[45]
or the carboxylation of acetyl-CoA.
[46][47]
Prokaryotic chemoautotrophs also fix CO
2
through the Calvin Benson cycle, but use energy from inorganic
compounds to drive the reaction.
[48]
Carbohydrates and glycans
In carbohydrate anabolism, simple organic acids can be converted into monosaccharides such as glucose and then
used to assemble polysaccharides such as starch. The generation of glucose from compounds like pyruvate, lactate,
glycerol, glycerate 3-phosphate and amino acids is called gluconeogenesis. Gluconeogenesis converts pyruvate to
glucose-6-phosphate through a series of intermediates, many of which are shared with glycolysis.
[]
However, this
pathway is not simply glycolysis run in reverse, as several steps are catalyzed by non-glycolytic enzymes. This is
important as it allows the formation and breakdown of glucose to be regulated separately, and prevents both
pathways from running simultaneously in a futile cycle.
[49][50]
Although fat is a common way of storing energy, in vertebrates such as humans the fatty acids in these stores cannot
be converted to glucose through gluconeogenesis as these organisms cannot convert acetyl-CoA into pyruvate; plants
do, but animals do not, have the necessary enzymatic machinery.
[]
As a result, after long-term starvation, vertebrates
need to produce ketone bodies from fatty acids to replace glucose in tissues such as the brain that cannot metabolize
fatty acids.
[51]
In other organisms such as plants and bacteria, this metabolic problem is solved using the glyoxylate
cycle, which bypasses the decarboxylation step in the citric acid cycle and allows the transformation of acetyl-CoA
to oxaloacetate, where it can be used for the production of glucose.
[][]
Polysaccharides and glycans are made by the sequential addition of monosaccharides by glycosyltransferase from a
reactive sugar-phosphate donor such as uridine diphosphate glucose (UDP-glucose) to an acceptor hydroxyl group
on the growing polysaccharide. As any of the hydroxyl groups on the ring of the substrate can be acceptors, the
polysaccharides produced can have straight or branched structures.
[52]
The polysaccharides produced can have
structural or metabolic functions themselves, or be transferred to lipids and proteins by enzymes called
Overview of metabolism
193
oligosaccharyltransferases.
[53][54]
Fatty acids, isoprenoids and steroids
Simplified version of the steroid synthesis pathway with the intermediates isopentenyl
pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate
(GPP) and squalene shown. Some intermediates are omitted for clarity.
Fatty acids are made by fatty acid
synthases that polymerize and then
reduce acetyl-CoA units. The acyl
chains in the fatty acids are extended
by a cycle of reactions that add the
acyl group, reduce it to an alcohol,
dehydrate it to an alkene group and
then reduce it again to an alkane group.
The enzymes of fatty acid biosynthesis
are divided into two groups, in animals
and fungi all these fatty acid synthase
reactions are carried out by a single
multifunctional type I protein,
[55]
while in plant plastids and bacteria
separate type II enzymes perform each
step in the pathway.
[56][57]
Terpenes and isoprenoids are a large
class of lipids that include the
carotenoids and form the largest class
of plant natural products.
[58]
These
compounds are made by the assembly
and modification of isoprene units
donated from the reactive precursors
isopentenyl pyrophosphate and
dimethylallyl pyrophosphate.
[]
These
precursors can be made in different ways. In animals and archaea, the mevalonate pathway produces these
compounds from acetyl-CoA,
[59]
while in plants and bacteria the non-mevalonate pathway uses pyruvate and
glyceraldehyde 3-phosphate as substrates.
[][60]
One important reaction that uses these activated isoprene donors is
steroid biosynthesis. Here, the isoprene units are joined together to make squalene and then folded up and formed
into a set of rings to make lanosterol.
[]
Lanosterol can then be converted into other steroids such as cholesterol and
ergosterol.
[][61]
Proteins
Organisms vary in their ability to synthesize the 20 common amino acids. Most bacteria and plants can synthesize all
twenty, but mammals can only synthesize eleven nonessential amino acids, so nine essential amino acids must be
obtained from food.
[]
Some simple parasites, such as the bacteria Mycoplasma pneumoniae, lack all amino acid
synthesis and take their amino acids directly from their hosts.
[62]
All amino acids are synthesized from intermediates
in glycolysis, the citric acid cycle, or the pentose phosphate pathway. Nitrogen is provided by glutamate and
glutamine. Amino acid synthesis depends on the formation of the appropriate alpha-keto acid, which is then
transaminated to form an amino acid.
[63]
Amino acids are made into proteins by being joined together in a chain by peptide bonds. Each different protein has
a unique sequence of amino acid residues: this is its primary structure. Just as the letters of the alphabet can be
Overview of metabolism
194
combined to form an almost endless variety of words, amino acids can be linked in varying sequences to form a huge
variety of proteins. Proteins are made from amino acids that have been activated by attachment to a transfer RNA
molecule through an ester bond. This aminoacyl-tRNA precursor is produced in an ATP-dependent reaction carried
out by an aminoacyl tRNA synthetase.
[64]
This aminoacyl-tRNA is then a substrate for the ribosome, which joins the
amino acid onto the elongating protein chain, using the sequence information in a messenger RNA.
[65]
Nucleotide synthesis and salvage
Nucleotides are made from amino acids, carbon dioxide and formic acid in pathways that require large amounts of
metabolic energy.
[66]
Consequently, most organisms have efficient systems to salvage preformed nucleotides.
[66][67]
Purines are synthesized as nucleosides (bases attached to ribose). Both adenine and guanine are made from the
precursor nucleoside inosine monophosphate, which is synthesized using atoms from the amino acids glycine,
glutamine, and aspartic acid, as well as formate transferred from the coenzyme tetrahydrofolate. Pyrimidines, on the
other hand, are synthesized from the base orotate, which is formed from glutamine and aspartate.
[68]
Xenobiotics and redox metabolism
All organisms are constantly exposed to compounds that they cannot use as foods and would be harmful if they
accumulated in cells, as they have no metabolic function. These potentially damaging compounds are called
xenobiotics.
[69]
Xenobiotics such as synthetic drugs, natural poisons and antibiotics are detoxified by a set of
xenobiotic-metabolizing enzymes. In humans, these include cytochrome P450 oxidases,
[70]
UDP-glucuronosyltransferases,
[71]
and glutathione S-transferases.
[72]
This system of enzymes acts in three stages to
firstly oxidize the xenobiotic (phase I) and then conjugate water-soluble groups onto the molecule (phase II). The
modified water-soluble xenobiotic can then be pumped out of cells and in multicellular organisms may be further
metabolized before being excreted (phase III). In ecology, these reactions are particularly important in microbial
biodegradation of pollutants and the bioremediation of contaminated land and oil spills.
[73]
Many of these microbial
reactions are shared with multicellular organisms, but due to the incredible diversity of types of microbes these
organisms are able to deal with a far wider range of xenobiotics than multicellular organisms, and can degrade even
persistent organic pollutants such as organochloride compounds.
[74]
A related problem for aerobic organisms is oxidative stress.
[]
Here, processes including oxidative phosphorylation
and the formation of disulfide bonds during protein folding produce reactive oxygen species such as hydrogen
peroxide.
[75]
These damaging oxidants are removed by antioxidant metabolites such as glutathione and enzymes
such as catalases and peroxidases.
[][]
Thermodynamics of living organisms
Living organisms must obey the laws of thermodynamics, which describe the transfer of heat and work. The second
law of thermodynamics states that in any closed system, the amount of entropy (disorder) will tend to increase.
Although living organisms' amazing complexity appears to contradict this law, life is possible as all organisms are
open systems that exchange matter and energy with their surroundings. Thus living systems are not in equilibrium,
but instead are dissipative systems that maintain their state of high complexity by causing a larger increase in the
entropy of their environments.
[76]
The metabolism of a cell achieves this by coupling the spontaneous processes of
catabolism to the non-spontaneous processes of anabolism. In thermodynamic terms, metabolism maintains order by
creating disorder.
[77]
Overview of metabolism
195
Regulation and control
As the environments of most organisms are constantly changing, the reactions of metabolism must be finely
regulated to maintain a constant set of conditions within cells, a condition called homeostasis.
[78][79]
Metabolic
regulation also allows organisms to respond to signals and interact actively with their environments.
[80]
Two closely
linked concepts are important for understanding how metabolic pathways are controlled. Firstly, the regulation of an
enzyme in a pathway is how its activity is increased and decreased in response to signals. Secondly, the control
exerted by this enzyme is the effect that these changes in its activity have on the overall rate of the pathway (the flux
through the pathway).
[]
For example, an enzyme may show large changes in activity (i.e. it is highly regulated) but if
these changes have little effect on the flux of a metabolic pathway, then this enzyme is not involved in the control of
the pathway.
[81]
Effect of insulin on glucose uptake and metabolism. Insulin binds to its receptor
(1), which in turn starts many protein activation cascades (2). These include:
translocation of Glut-4 transporter to the plasma membrane and influx of glucose
(3), glycogen synthesis (4), glycolysis (5) and fatty acid synthesis (6).
There are multiple levels of metabolic
regulation. In intrinsic regulation, the
metabolic pathway self-regulates to respond
to changes in the levels of substrates or
products; for example, a decrease in the
amount of product can increase the flux
through the pathway to compensate.
[]
This
type of regulation often involves allosteric
regulation of the activities of multiple
enzymes in the pathway.
[82]
Extrinsic
control involves a cell in a multicellular
organism changing its metabolism in
response to signals from other cells. These
signals are usually in the form of soluble
messengers such as hormones and growth
factors and are detected by specific receptors on the cell surface.
[83]
These signals are then transmitted inside the cell
by second messenger systems that often involved the phosphorylation of proteins.
[84]
A very well understood example of extrinsic control is the regulation of glucose metabolism by the hormone
insulin.
[85]
Insulin is produced in response to rises in blood glucose levels. Binding of the hormone to insulin
receptors on cells then activates a cascade of protein kinases that cause the cells to take up glucose and convert it into
storage molecules such as fatty acids and glycogen.
[86]
The metabolism of glycogen is controlled by activity of
phosphorylase, the enzyme that breaks down glycogen, and glycogen synthase, the enzyme that makes it. These
enzymes are regulated in a reciprocal fashion, with phosphorylation inhibiting glycogen synthase, but activating
phosphorylase. Insulin causes glycogen synthesis by activating protein phosphatases and producing a decrease in the
phosphorylation of these enzymes.
[87]
Overview of metabolism
196
Evolution
Evolutionary tree showing the common ancestry of organisms from all three domains of
life. Bacteria are colored blue, eukaryotes red, and archaea green. Relative positions of
some of the phyla included are shown around the tree.
The central pathways of metabolism
described above, such as glycolysis
and the citric acid cycle, are present in
all three domains of living things and
were present in the last universal
ancestor.
[][88]
This universal ancestral
cell was prokaryotic and probably a
methanogen that had extensive amino
acid, nucleotide, carbohydrate and
lipid metabolism.
[89][90]
The retention
of these ancient pathways during later
evolution may be the result of these
reactions being an optimal solution to
their particular metabolic problems,
with pathways such as glycolysis and
the citric acid cycle producing their
end products highly efficiently and in a
minimal number of steps.
[][]
Mutation
changes that affect non-coding DNA segments may merely affect the metabolic efficiency of the individual for
whom the mutation occurs.
[91]
The first pathways of enzyme-based metabolism may have been parts of purine
nucleotide metabolism, with previous metabolic pathways being part of the ancient RNA world.
[92]
Many models have been proposed to describe the mechanisms by which novel metabolic pathways evolve. These
include the sequential addition of novel enzymes to a short ancestral pathway, the duplication and then divergence of
entire pathways as well as the recruitment of pre-existing enzymes and their assembly into a novel reaction
pathway.
[93]
The relative importance of these mechanisms is unclear, but genomic studies have shown that enzymes
in a pathway are likely to have a shared ancestry, suggesting that many pathways have evolved in a step-by-step
fashion with novel functions being created from pre-existing steps in the pathway.
[94]
An alternative model comes
from studies that trace the evolution of proteins' structures in metabolic networks, this has suggested that enzymes
are pervasively recruited, borrowing enzymes to perform similar functions in different metabolic pathways (evident
in the MANET database)
[95]
These recruitment processes result in an evolutionary enzymatic mosaic.
[96]
A third
possibility is that some parts of metabolism might exist as "modules" that can be reused in different pathways and
perform similar functions on different molecules.
[97]
As well as the evolution of new metabolic pathways, evolution can also cause the loss of metabolic functions. For
example, in some parasites metabolic processes that are not essential for survival are lost and preformed amino acids,
nucleotides and carbohydrates may instead be scavenged from the host.
[98]
Similar reduced metabolic capabilities are
seen in endosymbiotic organisms.
[99]
Overview of metabolism
197
Investigation and manipulation
Metabolic network of the Arabidopsis thaliana citric acid cycle. Enzymes and
metabolites are shown as red squares and the interactions between them as black
lines.
Classically, metabolism is studied by a
reductionist approach that focuses on a
single metabolic pathway. Particularly
valuable is the use of radioactive tracers at
the whole-organism, tissue and cellular
levels, which define the paths from
precursors to final products by identifying
radioactively labelled intermediates and
products.
[100]
The enzymes that catalyze
these chemical reactions can then be
purified and their kinetics and responses to
inhibitors investigated. A parallel approach
is to identify the small molecules in a cell or
tissue; the complete set of these molecules is
called the metabolome. Overall, these
studies give a good view of the structure and
function of simple metabolic pathways, but
are inadequate when applied to more
complex systems such as the metabolism of
a complete cell.
[101]
An idea of the complexity of the metabolic
networks in cells that contain thousands of
different enzymes is given by the figure
showing the interactions between just 43 proteins and 40 metabolites to the right: the sequences of genomes provide
lists containing anything up to 45,000 genes.
[102]
However, it is now possible to use this genomic data to reconstruct
complete networks of biochemical reactions and produce more holistic mathematical models that may explain and
predict their behavior.
[103]
These models are especially powerful when used to integrate the pathway and metabolite
data obtained through classical methods with data on gene expression from proteomic and DNA microarray
studies.
[104]
Using these techniques, a model of human metabolism has now been produced, which will guide future
drug discovery and biochemical research.
[105]
These models are now being used in network analysis, to classify
human diseases into groups that share common proteins or metabolites.
[106][107]
Bacterial metabolic networks are a striking example of bow-tie
[][][]
organization, an architecture able to input a wide
range of nutrients and produce a large variety of products and complex macromolecules using a relatively few
intermediate common currencies.
A major technological application of this information is metabolic engineering. Here, organisms such as yeast, plants
or bacteria are genetically modified to make them more useful in biotechnology and aid the production of drugs such
as antibiotics or industrial chemicals such as 1,3-propanediol and shikimic acid.
[108]
These genetic modifications
usually aim to reduce the amount of energy used to produce the product, increase yields and reduce the production of
wastes.
[109]
Overview of metabolism
198
History
Santorio Santorio in his steelyard
balance, from Ars de statica medicina,
first published 1614
The term metabolism is derived from the Greek
"Metabolismos" for "change", or "overthrow".
[110]
The history of the
scientific study of metabolism spans several centuries and has moved from
examining whole animals in early studies, to examining individual metabolic
reactions in modern biochemistry. The first controlled experiments in human
metabolism were published by Santorio Santorio in 1614 in his book Ars de
statica medicina.
[111]
He described how he weighed himself before and after
eating, sleep, working, sex, fasting, drinking, and excreting. He found that
most of the food he took in was lost through what he called "insensible
perspiration".
In these early studies, the mechanisms of these metabolic processes had not
been identified and a vital force was thought to animate living tissue.
[112]
In
the 19th century, when studying the fermentation of sugar to alcohol by yeast,
Louis Pasteur concluded that fermentation was catalyzed by substances
within the yeast cells he called "ferments". He wrote that "alcoholic
fermentation is an act correlated with the life and organization of the yeast
cells, not with the death or putrefaction of the cells."
[113]
This discovery,
along with the publication by Friedrich Whler in 1828 of the chemical
synthesis of urea,
[114]
notable for being the first organic compound prepared from wholly inorganic precursors,
proved that the organic compounds and chemical reactions found in cells were no different in principle than any
other part of chemistry.
It was the discovery of enzymes at the beginning of the 20th century by Eduard Buchner that separated the study of
the chemical reactions of metabolism from the biological study of cells, and marked the beginnings of
biochemistry.
[115]
The mass of biochemical knowledge grew rapidly throughout the early 20th century. One of the
most prolific of these modern biochemists was Hans Krebs who made huge contributions to the study of
metabolism.
[116]
He discovered the urea cycle and later, working with Hans Kornberg, the citric acid cycle and the
glyoxylate cycle.
[117][]
Modern biochemical research has been greatly aided by the development of new techniques
such as chromatography, X-ray diffraction, NMR spectroscopy, radioisotopic labelling, electron microscopy and
molecular dynamics simulations. These techniques have allowed the discovery and detailed analysis of the many
molecules and metabolic pathways in cells.
References
[91] C.Michael Hogan. 2010. Mutation. ed. E.Monosson and C.J.Cleveland. Encyclopedia of Earth. National Council for Science and the
Environment. Washington DC (http:/ / www. eoearth.org/ article/ Mutation?topic=49496)
[112] Williams, H. S. (1904) A History of Science: in Five Volumes. Volume IV: Modern Development of the Chemical and Biological Sciences
(http:/ / etext.lib. virginia.edu/ toc/ modeng/ public/ Wil4Sci. html) Harper and Brothers (New York) Retrieved on 2007-03-26
[115] Eduard Buchner's 1907 Nobel lecture (http:/ / nobelprize. org/ nobel_prizes/ chemistry/ laureates/ 1907/ buchner-lecture. html) at http:/ /
nobelprize.org Accessed 2007-03-20
Further reading
Books about Metabolism:
Online books (http:/ / onlinebooks. library. upenn. edu/ webbin/ ftl?st=& su=Metabolism& library=OLBP),
Resources in your library (http:/ / onlinebooks. library. upenn. edu/ webbin/ ftl?st=& su=Metabolism),
Overview of metabolism
199
Resources in other libraries (http:/ / onlinebooks. library. upenn. edu/ webbin/ ftl?st=& su=Metabolism&
library=0CHOOSE0) Introductory
Rose, S. and Mileusnic, R., The Chemistry of Life. (Penguin Press Science, 1999), ISBN 0-14-027273-9
Schneider, E. D. and Sagan, D., Into the Cool: Energy Flow, Thermodynamics, and Life. (University Of Chicago
Press, 2005), ISBN 0-226-73936-8
Lane, N., Oxygen: The Molecule that Made the World. (Oxford University Press, USA, 2004), ISBN
0-19-860783-0
Advanced
Price, N. and Stevens, L., Fundamentals of Enzymology: Cell and Molecular Biology of Catalytic Proteins.
(Oxford University Press, 1999), ISBN 0-19-850229-X
Berg, J. Tymoczko, J. and Stryer, L., Biochemistry. (W. H. Freeman and Company, 2002), ISBN 0-7167-4955-6
Cox, M. and Nelson, D. L., Lehninger Principles of Biochemistry. (Palgrave Macmillan, 2004), ISBN
0-7167-4339-6
Brock, T. D. Madigan, M. T. Martinko, J. and Parker J., Brock's Biology of Microorganisms. (Benjamin
Cummings, 2002), ISBN 0-13-066271-2
Da Silva, J.J.R.F. and Williams, R. J. P., The Biological Chemistry of the Elements: The Inorganic Chemistry of
Life. (Clarendon Press, 1991), ISBN 0-19-855598-9
Nicholls, D. G. and Ferguson, S. J., Bioenergetics. (Academic Press Inc., 2002), ISBN 0-12-518121-3
External links
biochemical families: carbohydrates
alcohols
glycoproteins
glycosides
lipids
eicosanoids
fatty acids / intermediates
phospholipids
sphingolipids
steroids
nucleic acids
constituents / intermediates
proteins
amino acids / intermediates
tetrapyrroles / intermediates
200
Carbohydrate metabolism
Glycolysis
Glycolysis overview
Glycolysis (from glycose, an older
term
[1]
for glucose + -lysis
degradation) is the metabolic pathway
that converts glucose C
6
H
12
O
6
, into
pyruvate, CH
3
COCOO

+ H
+
. The
free energy released in this process is
used to form the high-energy
compounds ATP (adenosine
triphosphate) and NADH (reduced
nicotinamide adenine dinucleotide).
[2]
Glycolysis is a determined sequence of
ten reactions involving ten intermediate compounds (one of the steps involves two intermediates). The intermediates
provide entry points to glycolysis. For example, most monosaccharides, such as fructose, glucose, and galactose, can
be converted to one of these intermediates. The intermediates may also be directly useful. For example, the
intermediate dihydroxyacetone phosphate (DHAP) is a source of the glycerol that combines with fatty acids to form
fat.
It occurs, with variations, in nearly all organisms, both aerobic and anaerobic. The wide occurrence of glycolysis
indicates that it is one of the most ancient known metabolic pathways.
[3]
It occurs in the cytosol of the cell.
The most common type of glycolysis is the EmbdenMeyerhofParnas (EMP pathway), which was first discovered
by Gustav Embden, Otto Meyerhof, and Jakub Karol Parnas. Glycolysis also refers to other pathways, such as the
EntnerDoudoroff pathway and various heterofermentative and homofermentative pathways. However, the
discussion here will be limited to the EmbdenMeyerhofParnas pathway.
[4]
The entire glycolysis pathway can be separated into two phases:
[5]
1. The Preparatory Phase in which ATP is consumed and is hence also known as the investment phase
2. The Pay Off Phase in which ATP is produced.
Overview
The overall reaction of glycolysis is:
D-[Glucose] [Pyruvate]
+ 2 [NAD]
+
+ 2 [ADP] + 2 [P]
i
2
+ 2 [NADH] + 2 H
+
+ 2 [ATP] + 2 H
2
O
The use of symbols in this equation makes it appear unbalanced with respect to oxygen atoms, hydrogen atoms, and
charges. Atom balance is maintained by the two phosphate (P
i
) groups:
[]
each exists in the form of a hydrogen phosphate anion (HPO
4
2
), dissociating to contribute 2 H
+
overall
Glycolysis
201
each liberates an oxygen atom when it binds to an ADP (adenosine diphosphate) molecule, contributing 2 O
overall
Charges are balanced by the difference between ADP and ATP. In the cellular environment, all three hydroxy groups
of ADP dissociate into O
-
and H
+
, giving ADP
3
, and this ion tends to exist in an ionic bond with Mg
2+
, giving
ADPMg
-
. ATP behaves identically except that it has four hydroxy groups, giving ATPMg
2
. When these differences
along with the true charges on the two phosphate groups are considered together, the net charges of 4 on each side
are balanced.
Glycolysis
For simple fermentations, the
metabolism of one molecule of glucose
to two molecules of pyruvate has a net
yield of two molecules of ATP. Most
cells will then carry out further
reactions to 'repay' the used NAD
+
and
produce a final product of ethanol or
lactic acid. Many bacteria use
inorganic compounds as hydrogen
acceptors to regenerate the NAD
+
.
Cells performing aerobic respiration
synthesize much more ATP, but not as
part of glycolysis. These further aerobic reactions use pyruvate and NADH + H
+
from glycolysis. Eukaryotic aerobic
respiration produces approximately 34 additional molecules of ATP for each glucose molecule, however most of
these are produced by a vastly different mechanism to the substrate-level phosphorylation in glycolysis.
The lower-energy production, per glucose, of anaerobic respiration relative to aerobic respiration, results in greater
flux through the pathway under hypoxic (low-oxygen) conditions, unless alternative sources of anaerobically
oxidizable substrates, such as fatty acids, are found.
Metabolism of common monosaccharides, including glycolysis, gluconeogenesis, glycogenesis and glycogenolysis
Glycolysis
202
Elucidation of the pathway
In 1860, Louis Pasteur discovered that microorganisms are responsible for fermentation. In 1897, Eduard Buchner
found that extracts of certain cells can cause fermentation. In 1905, Arthur Harden and William Young along with
Nick Sheppard determined that a heat-sensitive high-molecular-weight subcellular fraction (the enzymes) and a
heat-insensitive low-molecular-weight cytoplasm fraction (ADP, ATP and NAD
+
and other cofactors) are required
together for fermentation to proceed. The details of the pathway were eventually determined by 1940, with a major
input from Otto Meyerhof and some years later by Luis Leloir. The biggest difficulties in determining the intricacies
of the pathway were due to the very short lifetime and low steady-state concentrations of the intermediates of the fast
glycolytic reactions.
Sequence of reactions
Preparatory phase
The first five steps are regarded as the preparatory (or investment) phase, since they consume energy to convert the
glucose into two three-carbon sugar phosphates
[5]
(G3P).
The first step in glycolysis is phosphorylation of glucose by a family of
enzymes called hexokinases to form glucose 6-phosphate (G6P). This reaction
consumes ATP, but it acts to keep the glucose concentration low, promoting
continuous transport of glucose into the cell through the plasma membrane
transporters. In addition, it blocks the glucose from leaking out the cell
lacks transporters for G6P, and free diffusion out of the cell is prevented due
to the charged nature of G6P. Glucose may alternatively be formed from the
phosphorolysis or hydrolysis of intracellular starch or glycogen.
In animals, an isozyme of hexokinase called glucokinase is also used in the
liver, which has a much lower affinity for glucose (K
m
in the vicinity of
normal glycemia), and differs in regulatory properties. The different substrate
affinity and alternate regulation of this enzyme are a reflection of the role of
the liver in maintaining blood sugar levels.
Cofactors: Mg
2+
D-Glucose (Glc) Hexokinase
(HK)
a
transferase
-D-Glucose-6-phosphate
(G6P)
ATP
H
+
+
ADP
G6P is then rearranged into fructose 6-phosphate (F6P) by glucose
phosphate isomerase. Fructose can also enter the glycolytic
pathway by phosphorylation at this point.
The change in structure is an isomerization, in which the G6P has
been converted to F6P. The reaction requires an enzyme,
phosphohexose isomerase, to proceed. This reaction is freely
reversible under normal cell conditions. However, it is often driven
forward because of a low concentration of F6P, which is constantly
consumed during the next step of glycolysis. Under conditions of
high F6P concentration, this reaction readily runs in reverse. This
phenomenon can be explained through Le Chatelier's Principle.
Isomerization to a keto sugar is necessary for carbanion
stabilization in the fourth reaction step (below).
-D-Glucose
6-phosphate (G6P)
Phosphoglucose
isomerase
an isomerase
-D-Fructose 6-phosphate
(F6P)
Glycolysis
203
The energy expenditure of another ATP in this step
is justified in 2 ways: The glycolytic process (up to
this step) is now irreversible, and the energy supplied
destabilizes the molecule. Because the reaction
catalyzed by Phosphofructokinase 1 (PFK-1) is
coupled to the hydrolysis of ATP, an energetically
favorable step, it is, in essence, irreversible, and a
different pathway must be used to do the reverse
conversion during gluconeogenesis. This makes the
reaction a key regulatory point (see below). This is
also the rate-limiting step.
Furthermore, the second phosphorylation event is
necessary to allow the formation of two charged
groups (rather than only one) in the subsequent step
of glycolysis, ensuring the prevention of free
diffusion of substrates out of the cell.
The same reaction can also be catalyzed by
pyrophosphate-dependent phosphofructokinase (PFP
or PPi-PFK), which is found in most plants, some
bacteria, archea, and protists, but not in animals. This
enzyme uses pyrophosphate (PPi) as a phosphate
donor instead of ATP. It is a reversible reaction,
increasing the flexibility of glycolytic metabolism.
[6]
A rarer ADP-dependent PFK enzyme variant has
been identified in archaean species.
[7]
-D-Fructose 6-phosphate
(F6P)
phosphofructokinase
(PFK-1)
a transferase
-D-Fructose 1,6-bisphosphate
(F1,6BP)
ATP
H
+
+
ADP
Cofactors: Mg
2+
Destabilizing the molecule in the
previous reaction allows the hexose
ring to be split by aldolase into two
triose sugars, dihydroxyacetone
phosphate, a ketone, and
glyceraldehyde 3-phosphate, an
aldehyde. There are two classes of
aldolases: class I aldolases, present in
animals and plants, and class II
aldolases, present in fungi and bacteria;
the two classes use different
mechanisms in cleaving the ketose
ring.
Electrons delocalized in the
carbon-carbon bond cleavage associate
with the alcohol group. The resulting
carbanion is stabilized by the structure
of the carbanion itself via resonance
charge distribution and by the presence
of a charged ion prosthetic group.
-D-Fructose 1,6-bisphosphate
(F1,6BP)
fructose-bisphosphate
aldolase (ALDO)
a lyase
D-glyceraldehyde
3-phosphate
(GADP)
Dihydroxyacetone
phosphate (DHAP)
+
Glycolysis
204
Triosephosphate isomerase rapidly interconverts dihydroxyacetone
phosphate with glyceraldehyde 3-phosphate (GADP) that proceeds further
into glycolysis. This is advantageous, as it directs dihydroxyacetone
phosphate down the same pathway as glyceraldehyde 3-phosphate,
simplifying regulation.
Dihydroxyacetone
phosphate (DHAP)
triosephosphate
isomerase (TPI)
an isomerase
D-glyceraldehyde
3-phosphate (GADP)
Pay-off phase
The second half of glycolysis is known as the pay-off phase, characterised by a net gain of the energy-rich molecules
ATP and NADH.
[5]
Since glucose leads to two triose sugars in the preparatory phase, each reaction in the pay-off
phase occurs twice per glucose molecule. This yields 2 NADH molecules and 4 ATP molecules, leading to a net gain
of 2 NADH molecules and 2 ATP molecules from the glycolytic pathway per glucose.
The triose sugars are dehydrogenated and inorganic phosphate is
added to them, forming 1,3-bisphosphoglycerate.
The hydrogen is used to reduce two molecules of NAD
+
, a hydrogen
carrier, to give NADH + H
+
for each triose.
Hydrogen atom balance and charge balance are both maintained
because the phosphate (P
i
) group actually exists in the form of a
hydrogen phosphate anion (HPO
4
2-
),
[]
which dissociates to
contribute the extra H
+
ion and gives a net charge of -3 on both
sides.
glyceraldehyde
3-phosphate
(GADP)
glyceraldehyde
phosphate
dehydrogenase
(GAPDH)
an oxidoreductase
D-1,3-bisphosphoglycerate
(1,3BPG)
NAD
+
+
P
i
NADH +
H
+
This step is the enzymatic transfer of a phosphate group from
1,3-bisphosphoglycerate to ADP by phosphoglycerate kinase, forming ATP
and 3-phosphoglycerate. At this step, glycolysis has reached the break-even
point: 2 molecules of ATP were consumed, and 2 new molecules have now
been synthesized. This step, one of the two substrate-level phosphorylation
steps, requires ADP; thus, when the cell has plenty of ATP (and little ADP),
this reaction does not occur. Because ATP decays relatively quickly when it
is not metabolized, this is an important regulatory point in the glycolytic
pathway..
ADP actually exists as ADPMg
-
, and ATP as ATPMg
2-
, balancing the
charges at -5 both sides.
Cofactors: Mg
2+
1,3-bisphosphoglycerate
(1,3-BPG)
phosphoglycerate
kinase (PGK)
a transferase
3-phosphoglycerate
(3-P-G)
ADP ATP
phosphoglycerate
kinase (PGK)
Glycolysis
205
Phosphoglycerate mutase now forms
2-phosphoglycerate.
3-phosphoglycerate
(3PG)
phosphoglycerate mutase
(PGM)
a mutase
2-phosphoglycerate
(2PG)
Enolase next forms phosphoenolpyruvate from 2-phosphoglycerate.
Cofactors: 2 Mg
2+
: one "conformational" ion to coordinate with the
carboxylate group of the substrate, and one "catalytic" ion that participates
in the dehydration.
2-phosphoglycerate
(2PG)
enolase
(ENO)
a lyase
phosphoenolpyruvate
(PEP)
H
2
O
enolase
(ENO)
A final substrate-level phosphorylation now forms a molecule of pyruvate and a
molecule of ATP by means of the enzyme pyruvate kinase. This serves as an additional
regulatory step, similar to the phosphoglycerate kinase step.
Cofactors: Mg
2+
phosphoenolpyruvate
(PEP)
pyruvate
kinase (PK)
a transferase
pyruvate
(Pyr)
ADP
+ H
+
ATP
Regulation
Glycolysis is regulated by slowing down or speeding up certain steps in the glycolysis pathway. This is
accomplished by inhibiting or activating the enzymes that are involved. The steps that are regulated may be
determined by calculating the change in free energy, G, for each step. If a step's products and reactants are in
equilibrium, then the step is assumed not to be regulated. Since the change in free energy is zero for a system at
equilibrium, any step with a free energy change near zero is not being regulated. If a step is being regulated, then
that step's enzyme is not converting reactants into products as fast as it could, resulting in a build-up of reactants,
which would be converted to products if the enzyme were operating faster. Since the reaction is thermodynamically
favorable, the change in free energy for the step will be negative. A step with a large negative change in free energy
is assumed to be regulated.
Glycolysis
206
Free energy changes
Concentrations of metabolites in erythrocytes
[8]
Compound Concentration / mM
glucose 5.0
glucose-6-phosphate 0.083
fructose-6-phosphate 0.014
fructose-1,6-bisphosphate 0.031
dihydroxyacetone phosphate 0.14
glyceraldehyde-3-phosphate 0.019
1,3-bisphosphoglycerate 0.001
2,3-bisphosphoglycerate 4.0
3-phosphoglycerate 0.12
2-phosphoglycerate 0.03
phosphoenolpyruvate 0.023
pyruvate 0.051
ATP 1.85
ADP 0.14
P
i
1.0
The change in free energy for each step of glycolysis estimated from the
concentration of metabolites in an erythrocyte.
The change in free energy, G, for each step in the glycolysis pathway can be calculated using G = G' + RTln Q,
where Q is the reaction quotient. This requires knowing the concentrations of the metabolites. All of these values are
available for erythrocytes, with the exception of the concentrations of NAD
+
and NADH. The ratio of NAD
+
to
NADH in the cytoplasm is approximately 1000, which makes the oxidation of glyceraldehyde-3-phosphate (step 6)
more favourable.
Using the measured concentrations of each step, and the standard free energy changes, the actual free energy change
can be calculated. (Neglecting this is very common - the delta G of ATP hydrolysis in cells is not the standard free
energy change of ATP hydrolysis quoted in textbooks).
Glycolysis
207
Change in free energy for each step of glycolysis
[9]
Step Reaction G' / (kJ/mol) G / (kJ/mol)
1
glucose + ATP
4-
glucose-6-phosphate
2-
+ ADP
3-
+ H
+ -16.7 -34
2
glucose-6-phosphate
2-
fructose-6-phosphate
2- 1.67 -2.9
3
fructose-6-phosphate
2-
+ ATP
4-
fructose-1,6-bisphosphate
4-
+ ADP
3-
+ H
+ -14.2 -19
4
fructose-1,6-bisphosphate
4-
dihydroxyacetone phosphate
2-
+ glyceraldehyde-3-phosphate
2- 23.9 -0.23
5
dihydroxyacetone phosphate
2-
glyceraldehyde-3-phosphate
2- 7.56 2.4
6
glyceraldehyde-3-phosphate
2-
+ P
i
2-
+ NAD
+
1,3-bisphosphoglycerate
4-
+ NADH + H
+ 6.30 -1.29
7
1,3-bisphosphoglycerate
4-
+ ADP
3-
3-phosphoglycerate
3-
+ ATP
4- -18.9 0.09
8
3-phosphoglycerate
3-
2-phosphoglycerate
3- 4.4 0.83
9
2-phosphoglycerate
3-
phosphoenolpyruvate
3-
+ H
2
O
1.8 1.1
10
phosphoenolpyruvate
3-
+ ADP
3-
+ H
+
pyruvate
-
+ ATP
4- -31.7 -23.0
From measuring the physiological concentrations of metabolites in an erythrocyte it seems that about seven of the
steps in glycolysis are in equilibrium for that cell type. Three of the steps the ones with large negative free energy
changes are not in equilibrium and are referred to as irreversible; such steps are often subject to regulation.
Step 5 in the figure is shown behind the other steps, because that step is a side-reaction that can decrease or increase
the concentration of the intermediate glyceraldehyde-3-phosphate. That compound is converted to dihydroxyacetone
phosphate by the enzyme triose phosphate isomerase, which is a catalytically perfect enzyme; its rate is so fast that
the reaction can be assumed to be in equilibrium. The fact that G is not zero indicates that the actual concentrations
in the erythrocyte are not accurately known.
Biochemical logic
The existence of more than one point of regulation indicates that intermediates between those points enter and leave
the glycolysis pathway by other processes. For example, in the first regulated step, hexokinase converts glucose into
glucose-6-phosphate. Instead of continuing through the glycolysis pathway, this intermediate can be converted into
glucose storage molecules, such as glycogen or starch. The reverse reaction, breaking down, e.g., glycogen, produces
mainly glucose-6-phosphate; very little free glucose is formed in the reaction. The glucose-6-phosphate so produced
can enter glycolysis after the first control point.
In the second regulated step (the third step of glycolysis), phosphofructokinase converts fructose-6-phosphate into
fructose-1,6-bisphosphate, which then is converted into glyceraldehyde-3-phosphate and dihydroxyacetone
phosphate. The dihydroxyacetone phosphate can be removed from glycolysis by conversion into
glycerol-3-phosphate, which can be used to form triglycerides.
[10]
On the converse, triglycerides can be broken down
into fatty acids and glycerol; the latter, in turn, can be converted into dihydroxyacetone phosphate, which can enter
glycolysis after the second control point.
Glycolysis
208
Regulation
The three regulated enzymes are hexokinase, phosphofructokinase, and pyruvate kinase.
The flux through the glycolytic pathway is adjusted in response to conditions both inside and outside the cell. The
rate in liver is regulated to meet major cellular needs: (1) the production of ATP, (2) the provision of building blocks
for biosynthetic reactions, and (3) to lower blood glucose, one of the major functions of the liver. When blood sugar
falls, glycolysis is halted in the liver to allow the reverse process, gluconeogenesis. In glycolysis, the reactions
catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase are effectively irreversible in most organisms. In
metabolic pathways, such enzymes are potential sites of control, and all three enzymes serve this purpose in
glycolysis.
Hexokinase
Yeast hexokinase B. PDB 1IG8
[11]
.
In animals, regulation of blood glucose levels by the pancreas in
conjunction with the liver is a vital part of homeostasis. In liver cells,
extra G6P (glucose-6-phosphate) may be converted to G1P for
conversion to glycogen, or it is alternatively converted by glycolysis to
acetyl-CoA and then citrate. Excess citrate is exported to the cytosol,
where ATP citrate lyase will regenerate acetyl-CoA and OAA. The
acetyl-CoA is then used for fatty acid synthesis and cholesterol
synthesis, two important ways of utilizing excess glucose when its
concentration is high in blood. Liver contains both hexokinase and
glucokinase; both catalyse the phosphorylation of glucose to G6P but
the latter is not inhibited by G6P. Thus, glucokinase allows glucose to be converted into glycogen, fatty acids, and
cholesterol even as G6P accumulates in hepatocytes.
[12]
This is important when blood glucose levels are high.
During hypoglycemia, the glycogen can be converted back to G6P and then converted to glucose by the
liver-specific enzyme glucose 6-phosphatase and released into the blood without taking up the low concentration of
glucose it releases. This reverse reaction is an important role of liver cells to maintain blood sugars levels during
fasting. This is critical for brain function, since the brain utilizes glucose as an energy source under most conditions.
Phosphofructokinase
Bacillus stearothermophilus phosphofructokinase.
PDB 6PFK
[13]
.
Phosphofructokinase is an important control point in the glycolytic
pathway, since it is one of the irreversible steps and has key allosteric
effectors, AMP and fructose 2,6-bisphosphate (F2,6BP).
Fructose 2,6-bisphosphate (F2,6BP) is a very potent activator of
phosphofructokinase (PFK-1), which is synthesised when F6P is
phosphorylated by a second phosphofructokinase (PFK2). In liver,
when blood sugar is low and glucagon elevates cAMP, PFK2 is
phosphorylated by protein kinase A. The phosphorylation inactivates
PFK2, and another domain on this protein becomes active as fructose
2,6-bisphosphatase, which converts F2,6BP back to F6P. Both
glucagon and epinephrine cause high levels of cAMP in the liver. The
result of lower levels of liver fructose-2,6-bisphosphate is a decrease in
activity of phosphofructokinase and an increase in activity of fructose 1,6-bisphosphatase, so that gluconeogenesis
(in essence, "glycolysis in reverse") is favored. This is consistent with the role of the liver in such situations, since
the response of the liver to these hormones is to release glucose to the blood.
Glycolysis
209
ATP competes with AMP for the allosteric effector site on the PFK enzyme. ATP concentrations in cells are much
higher than those of AMP, typically 100-fold higher,
[14]
but the concentration of ATP does not change more than
about 10% under physiological conditions, whereas a 10% drop in ATP results in a 6-fold increase in AMP.
[15]
Thus,
the relevance of ATP as an allosteric effector is questionable. An increase in AMP is a consequence of a decrease in
energy charge in the cell.
Citrate inhibits phosphofructokinase when tested in vitro by enhancing the inhibitory effect of ATP. However, it is
doubtful that this is a meaningful effect in vivo, because citrate in the cytosol is utilized mainly for conversion to
acetyl-CoA for fatty acid and cholesterol synthesis.
Pyruvate kinase
Yeast pyruvate kinase. PDB 1A3W
[16]
.
This enzyme catalyzes the last step of glycolysis, in which pyruvate
and ATP are formed. Regulation of this enzyme is discussed in the
main topic, pyruvate kinase.
Post-glycolysis processes
The overall process of glycolysis is:
glucose + 2 NAD
+
+ 2 ADP + 2 P
i
2 pyruvate + 2 NADH + 2
H
+
+ 2 ATP + 2 H
2
O
If glycolysis were to continue indefinitely, all of the NAD
+
would be
used up, and glycolysis would stop. To allow glycolysis to continue,
organisms must be able to oxidize NADH back to NAD
+
.
Fermentation
One method of doing this is to simply have the pyruvate do the oxidation; in this process, the pyruvate is converted
to lactate (the conjugate base of lactic acid) in a process called lactic acid fermentation:
pyruvate + NADH + H
+
lactate + NAD
+
This process occurs in the bacteria involved in making yogurt (the lactic acid causes the milk to curdle). This process
also occurs in animals under hypoxic (or partially anaerobic) conditions, found, for example, in overworked muscles
that are starved of oxygen, or in infarcted heart muscle cells. In many tissues, this is a cellular last resort for energy;
most animal tissue cannot tolerate anaerobic conditions for an extended period of time.
Some organisms, such as yeast, convert NADH back to NAD
+
in a process called ethanol fermentation. In this
process, the pyruvate is converted first to acetaldehyde and carbon dioxide, then to ethanol.
Lactic acid fermentation and ethanol fermentation can occur in the absence of oxygen. This anaerobic fermentation
allows many single-cell organisms to use glycolysis as their only energy source.
Anaerobic respiration
In the above two examples of fermentation, NADH is oxidized by transferring two electrons to pyruvate. However,
anaerobic bacteria use a wide variety of compounds as the terminal electron acceptors in cellular respiration:
nitrogenous compounds, such as nitrates and nitrites; sulfur compounds, such as sulfates, sulfites, sulfur dioxide, and
elemental sulfur; carbon dioxide; iron compounds; manganese compounds; cobalt compounds; and uranium
compounds.
Glycolysis
210
Aerobic respiration
In aerobic organisms, a complex mechanism has been developed to use the oxygen in air as the final electron
acceptor.
First, pyruvate is converted to acetyl-CoA and CO
2
within the mitochondria in a process called pyruvate
decarboxylation.
Second, the acetyl-CoA enters the citric acid cycle, also known as Krebs Cycle, where it is fully oxidized to
carbon dioxide and water, producing yet more NADH.
Third, the NADH is oxidized to NAD
+
by the electron transport chain, using oxygen as the final electron
acceptor. This process creates a hydrogen ion gradient across the inner membrane of the mitochondria.
Fourth, the proton gradient is used to produce about 2.5 ATP for every NADH oxidized in a process called
oxidative phosphorylation.
Intermediates for other pathways
This article concentrates on the catabolic role of glycolysis with regard to converting potential chemical energy to
usable chemical energy during the oxidation of glucose to pyruvate. Many of the metabolites in the glycolytic
pathway are also used by anabolic pathways, and, as a consequence, flux through the pathway is critical to maintain
a supply of carbon skeletons for biosynthesis.
In addition, not all carbon entering the pathway leaves as pyruvate and may be extracted at earlier stages to provide
carbon compounds for other pathways.
These metabolic pathways are all strongly reliant on glycolysis as a source of metabolites: and many more.
Gluconeogenesis
Lipid metabolism
Pentose phosphate pathway
Citric acid cycle, which in turn leads to:
Amino acid synthesis
Nucleotide synthesis
Tetrapyrrole synthesis
From an anabolic metabolism perspective, the NADH has a role to drive synthetic reactions, doing so by directly or
indirectly reducing the pool of NADP+ in the cell to NADPH, which is another important reducing agent for
biosynthetic pathways in a cell.
Glycolysis in disease
Genetic diseases
Glycolytic mutations are generally rare due to importance of the metabolic pathway, this means that the majority of
occurring mutations result in an inability for the cell to respire, and therefore cause the death of the cell at an early
stage. However, some mutations are seen with one notable example being Pyruvate kinase deficiency, leading to
chronic hemolytic anemia.
Cancer
Malignant rapidly growing tumor cells typically have glycolytic rates that are up to 200 times higher than those of
their normal tissues of origin. This phenomenon was first described in 1930 by Otto Warburg and is referred to as the
Warburg effect. The Warburg hypothesis claims that cancer is primarily caused by dysfunctionality in mitochondrial
metabolism, rather than because of uncontrolled growth of cells. A number of theories have been advanced to
explain the Warburg effect. One such theory suggests that the increased glycolysis is a normal protective process of
Glycolysis
211
the body and that malignant change could be primarily caused by energy metabolism.
[17]
This high glycolysis rate has important medical applications, as high aerobic glycolysis by malignant tumors is
utilized clinically to diagnose and monitor treatment responses of cancers by imaging uptake of
2-
18
F-2-deoxyglucose (FDG) (a radioactive modified hexokinase substrate) with positron emission tomography
(PET).
[18][19]
There is ongoing research to affect mitochondrial metabolism and treat cancer by reducing glycolysis and thus
starving cancerous cells in various new ways, including a ketogenic diet.
Alzheimer's disease
Disfunctioning glycolysis or glucose metabolism in fronto-temporo-parietal and cingulate cortices has been
associated with Alzheimer's disease,
[]
probably due to the decreased amyloid (1-42) (A42) and increased tau,
phosphorylated tau in cerebrospinal fluid (CSF)
[]
Interactive pathway map
Click on genes, proteins and metabolites below to link to respective articles.
[20]
Glycolysis
212
Glycolysis
213
Glycolysis and Gluconeogenesis edit
[21]
[1] [1] Webster's New International Dictionary of the English Language, 2nd ed. (1937) Merriam Company, Springfield, Mass.
[3] Romano AH, Conway T. (1996) Evolution of carbohydrate metabolic pathways. Res Microbiol. 147(67):44855 PMID 9084754
[4] [4] Kim BH, Gadd GM. (2011) Bacterial Physiology and Metabolism, 3rd edition.
[5] Glycolysis Animation and Notes (http:/ / pharmaxchange. info/ press/ 2011/ 09/ glycolysis-animation-and-notes/ )
[11] http:/ / www.rcsb. org/ pdb/ explore/ explore.do?structureId=1IG8
[12] Voet D., and Voet J. G. (2004). Biochemistry 3rd Edition (New York, John Wiley & Sons, Inc.)
[13] http:/ / www.rcsb. org/ pdb/ explore/ explore.do?structureId=6PFK
[14] [14] Beis I., and Newsholme E. A. (1975). The contents of adenine nucleotides, phosphagens and some glycolytic intermediates in resting
muscles from vertebrates and invertebrates. Biochem J 152, 23-32.
[15] Voet D., and Voet J. G. (2004). Biochemistry 3rd Edition (New York, John Wiley & Sons, Inc.).
[16] http:/ / www.rcsb. org/ pdb/ explore/ explore.do?structureId=1A3W
[20] [20] The interactive pathway map can be edited at WikiPathways:
[21] http:/ / www.wikipathways. org/ index.php/ Pathway:WP534
Alternative nomenclature
Some of the metabolites in glycolysis have alternative names and nomenclature. In part, this is because some of them
are common to other pathways, such as the Calvin cycle.
This article Alternative names Alternative nomenclature
1 glucose Glc dextrose
3 fructose 6-phosphate F6P
4 fructose 1,6-bisphosphate F1,6BP fructose 1,6-diphosphate FBP, FDP, F1,6DP
5 dihydroxyacetone phosphate DHAP glycerone phosphate
6 glyceraldehyde 3-phosphate GADP 3-phosphoglyceraldehyde PGAL, G3P, GALP,GAP,TP
7 1,3-bisphosphoglycerate 1,3BPG glycerate
1,3-bisphosphate,
glycerate 1,3-diphosphate,
1,3-diphosphoglycerate
PGAP, BPG, DPG
8 3-phosphoglycerate 3PG glycerate 3-phosphate PGA, GP
9 2-phosphoglycerate 2PG glycerate 2-phosphate
10 phosphoenolpyruvate PEP
11 pyruvate Pyr pyruvic acid
References
External links
A Detailed Glycolysis Animation provided by [[IUBMB (http:/ / www. iubmb-nicholson. org/ swf/ glycolysis.
swf)]] ( Adobe Flash (http:/ / get. adobe. com/ flashplayer/ ) Required)
The Glycolytic enzymes in Glycolysis (http:/ / nist. rcsb. org/ pdb/ molecules/ pdb50_1. html) at RCSB PDB
Glycolytic cycle with animations (http:/ / www. wdv. com/ CellWorld/ Biochemistry/ Glycolytic) at wdv.com
Metabolism, Cellular Respiration and Photosynthesis - The Virtual Library of Biochemistry and Cell Biology
(http:/ / www. biochemweb. org/ metabolism. shtml) at biochemweb.org
notes on glycolysis (http:/ / www. rahulgladwin. com/ blog/ 2007/ 01/ notes-on-glycolysis. html) at
rahulgladwin.com
The chemical logic behind glycolysis (http:/ / www2. ufp. pt/ ~pedros/ bq/ glycolysis. htm) at ufp.pt
Glycolysis
214
Expasy biochemical pathways poster (http:/ / www. expasy. org/ tools/ pathways/ boehringer_legends. html) at
ExPASy
Mnemonic at medicalmnemonics.com 317 5468 (http:/ / www. medicalmnemonics. com/ cgi-bin/ lookup.
cfm?id1=317& id2=5468& id3=& id4=)
Gluconeogenesis
Gluconeogenesis (abbreviated GNG) is a metabolic pathway that results in the generation of glucose from
non-carbohydrate carbon substrates such as pyruvate, lactate, glycerol, glucogenic amino acids, and odd-chain fatty
acid.
It is one of the two main mechanisms humans and many other animals use to keep blood glucose levels from
dropping too low (hypoglycemia). The other means of maintaining blood glucose levels is through the degradation of
glycogen (glycogenolysis).
[1]
Gluconeogenesis is a ubiquitous process, present in plants, animals, fungi, bacteria, and other microorganisms.
[2]
In
vertebrates, gluconeogenesis takes place mainly in the liver and, to a lesser extent, in the cortex of kidneys. In
ruminants, this tends to be a continuous process.
[3]
In many other animals, the process occurs during periods of
fasting, starvation, low-carbohydrate diets, or intense exercise. The process is highly endergonic until ATP or GTP
are utilized, effectively making the process exergonic. For example, the pathway leading from pyruvate to
glucose-6-phosphate requires 4 molecules of ATP and 2 molecules of GTP. Gluconeogenesis is often associated with
ketosis. Gluconeogenesis is also a target of therapy for type II diabetes, such as metformin, which inhibits glucose
formation and stimulates glucose uptake by cells.
[4]
In ruminants, because metabolizable dietary carbohydrates tend
to be metabolized by rumen organisms, gluconeogenesis occurs regardless of fasting, low-carbohydrate diets,
exercise, etc.
[5]
Precursors
Catabolism of proteinogenic amino acids. Amino acids are classified according the
abilities of their products to enter gluconeogenesis:
[6]
Glucogenic amino acids have
this abilityKetogenic amino acids do not. These products may still be used for
ketogenesis or lipid synthesis.Some amino acids are catabolized into both
glucogenic and ketogenic products.
In humans the main gluconeogenic
precursors are lactate, glycerol (which is a
part of the triacylglycerol molecule), alanine
and glutamine. Altogether, they account for
over 90% of the overall gluconeogenesis.
[]
Other glucogenic amino acid as well as all
citric acid cycle intermediates, the latter
through conversion to oxaloacetate, can also
function as substrates for gluconeogenesis.
[]
In ruminants, propionate is the principal
gluconeogenic substrate.
[5][7]
Lactate is transported back to the liver
where it is converted into pyruvate by the
Cori cycle using the enzyme lactate
dehydrogenase. Pyruvate, the first
designated substrate of the gluconeogenic
pathway, can then be used to generate
glucose.
[]
Transamination or deamination of amino acids facilitates entering of their carbon skeleton into the cycle
directly (as pyruvate or oxaloacetate), or indirectly via the citric acid cycle.
Gluconeogenesis
215
Whether even-chain fatty acids can be converted into glucose in animals has been a longstanding question in
biochemistry.
[]
It is known that odd-chain fatty acids can be oxidized to yield propionyl CoA, a precursor for
succinyl CoA, which can be converted to pyruvate and enter into gluconeogenesis. In plants, specifically seedlings,
the glyoxylate cycle can be used to convert fatty acids (acetate) into the primary carbon source of the organism. The
glyoxylate cycle produces four-carbon dicarboxylic acids that can enter gluconeogenesis.
[]
In 1995, researchers identified the glyoxylate cycle in nematodes.
[]
In addition, the glyoxylate enzymes malate
synthase and isocitrate lyase have been found in animal tissues.
[]
Genes coding for malate synthase gene have been
identified in other [metazoans] including arthropods, echinoderms, and even some vertebrates. Mammals found to
possess these genes include monotremes (platypus) and marsupials (opossum) but not placental mammals. Genes for
isocitrate lyase are found only in nematodes, in which, it is apparent, they originated in horizontal gene transfer from
bacteria.
The existence of glyoxylate cycles in humans has not been established, and it is widely held that fatty acids cannot
be converted to glucose in humans directly. However, carbon-14 has been shown to end up in glucose when it is
supplied in fatty acids.
[]
Despite these findings, it is considered unlikely that the 2-carbon acetyl-CoA derived from
the oxidation of fatty acids would produce a net yield of glucose via the citric acid cycle.
[]
Location
In mammals, gluconeogenesis is restricted to the liver,
[]
the kidney
[]
and the intestine.
[]
However these organs use
somewhat different gluconeogenic precursors. Liver uses primarily lactate and alanine while kidney uses lactate and
glutamine.
[]
Propionate is the principal substrate for gluconeogenesis in the ruminant liver, and the ruminant liver
may make increased use of gluconeogenic amino acids, e.g. alanine, when glucose demand is increased.
[8]
The
capacity of liver cells to use lactate for gluconeogenesis declines from the preruminant stage to the ruminant stage in
calves and lambs.
[9]
In sheep kidney tissue, very high rates of gluconeogenesis from propionate have been
observed.
[10]
The intestine uses mostly glutamine and glycerol.
[]
In all species, the formation of oxaloacetate from pyruvate and TCA cycle intermediates is restricted to the
mitochondrion, and the enzymes that convert Phosphoenolpyruvic acid (PEP) to glucose are found in the cytosol.
[]
The location of the enzyme that links these two parts of gluconeogenesis by converting oxaloacetate to PEP, PEP
carboxykinase, is variable by species: it can be found entirely within the mitochondria, entirely within the cytosol, or
dispersed evenly between the two, as it is in humans.
[]
Transport of PEP across the mitochondrial membrane is
accomplished by dedicated transport proteins; however no such proteins exist for oxaloacetate.
[]
Therefore, in
species that lack intra-mitochondrial PEP, oxaloacetate must be converted into malate or asparate, exported from the
mitochondrion, and converted back into oxaloacetate in order to allow gluconeogenesis to continue.
[]
Gluconeogenesis
216
Gluconeogenesis pathway with key molecules
and enzymes. Many steps are the opposite of
those found in the glycolysis.
Pathway
Gluconeogenesis is a pathway consisting of a series of eleven
enzyme-catalyzed reactions. The pathway may begin in the mitochondria
or cytoplasm, this being dependent on the substrate being used. Many of
the reactions are the reversible steps found in glycolysis.
Gluconeogenesis begins in the mitochondria with the formation of
oxaloacetate by the carboxylation of pyruvate. This reaction also
requires one molecule of ATP, and is catalyzed by pyruvate
carboxylase. This enzyme is stimulated by high levels of acetyl-CoA
(produced in -oxidation in the liver) and inhibited by high levels of
ADP.
Oxaloacetate is reduced to malate using NADH, a step required for its
transportation out of the mitochondria.
Malate is oxidized to oxaloacetate using NAD
+
in the cytosol, where
the remaining steps of gluconeogenesis take place.
Oxaloacetate is decarboxylated and then phosphorylated to form
phosphoenolpyruvate using the enzyme phosphoenolpyruvate
carboxykinase. A molecule of GTP is hydrolyzed to GDP during this
reaction.
The next steps in the reaction are the same as reversed glycolysis.
However, fructose-1,6-bisphosphatase converts
fructose-1,6-bisphosphate to fructose 6-phosphate, using one water
molecule and releasing one phosphate. This is also the rate-limiting
step of gluconeogenesis.
Glucose-6-phosphate is formed from fructose 6-phosphate by
phosphoglucoisomerase. Glucose-6-phosphate can be used in other metabolic pathways or dephosphorylated to
free glucose. Whereas free glucose can easily diffuse in and out of the cell, the phosphorylated form
(glucose-6-phosphate) is locked in the cell, a mechanism by which intracellular glucose levels are controlled by
cells.
The final reaction of gluconeogenesis, the formation of glucose, occurs in the lumen of the endoplasmic
reticulum, where glucose-6-phosphate is hydrolyzed by glucose-6-phosphatase to produce glucose. Glucose is
shuttled into the cytoplasm by glucose transporters located in the endoplasmic reticulum's membrane.
Metabolism of common monosaccharides, including glycolysis, gluconeogenesis, glycogenesis and glycogenolysis
Gluconeogenesis
217
Regulation
While most steps in gluconeogenesis are the reverse of those found in glycolysis, three regulated and strongly
exergonic reactions are replaced with more kinetically favorable reactions. Hexokinase/glucokinase,
phosphofructokinase, and pyruvate kinase enzymes of glycolysis are replaced with glucose-6-phosphatase,
fructose-1,6-bisphosphatase, and PEP carboxykinase. This system of reciprocal control allow glycolysis and
gluconeogenesis to inhibit each other and prevent the formation of a futile cycle.
The majority of the enzymes responsible for gluconeogenesis are found in the cytoplasm; the exceptions are
mitochondrial pyruvate carboxylase and, in animals, phosphoenolpyruvate carboxykinase. The latter exists as an
isozyme located in both the mitochondrion and the cytosol.
[11]
The rate of gluconeogenesis is ultimately controlled
by the action of a key enzyme, fructose-1,6-bisphosphatase, which is also regulated through signal transduction by
cAMP and its phosphorylation.
Most factors that regulate the activity of the gluconeogenesis pathway do so by inhibiting the activity or expression
of key enzymes. However, both acetyl CoA and citrate activate gluconeogenesis enzymes (pyruvate carboxylase and
fructose-1,6-bisphosphatase, respectively). Due to the reciprocal control of the cycle, acetyl-CoA and citrate also
have inhibitory roles in the activity of pyruvate kinase.
Global control of gluconeogenesis is mediated by glucagon (released when blood glucose is low); it triggers
phosphorylation of enzymes and regulatory proteins by Protein Kinase A (a cyclic AMP regulated kinase) resulting
in inhibition of glycolysis and stimulation of gluconeogenesis. Recent studies have shown that the absence of hepatic
glucose production has no major effect on the control of fasting plasma glucose concentration. Compensatory
induction of gluconeogenesis occurs in the kidneys and intestine, driven by glucagon, glucocorticoids, and acidosis.
[]
Gluconeogenesis
218
References
[3] [3] Young, J. W. 1977. Gluconeogenesis in cattle: significance and methodology. J. Dairy Sci. 60: 1-15.
[5] [5] Beitz, D. C. 2004. Carbohydrate metabolism. In: Reese, W. O. Dukes' physiology of domestic animals. 12th ed. Cornell Univ. Press. pp.
501-515.
[6] [6] Chapter 20 (Amino Acid Degradation and Synthesis) in:
[7] [7] Van Soest, P. J. 1994. Nutritional ecology of the ruminant. 2nd Ed. Cornell Univ. Press. 476 pp.
[8] [8] Overton, T. R., J. K. Drackley, C. J. Ottemann-Abbamonte, A. D. Beaulieu, L. S. Emmert and J. H. Clark. 1999. Substrate utilization for
hepatic gluconeogenesis is altered by increased glucose demand in ruminants. J. Anim. Sci. 77: 1940-1951.
[9] [9] Donkin, S. S. and L. E. Armentano. 1995. Insulin and glucagon regulation of gluconeogenesis in preruminating and ruminating bovine. J.
Anim. Sci. 73: 546-551.
[10] [10] Sasaki, S., K. Ambo, M. Muramatsu and T. Tsuda. 1975. Gluconeogenesis in the kidney-cortex slices of normal fed and starved sheep.
Tohoku J. Agr. Res. 26: 20-29.
[11] Chakravarty, K., Cassuto, H., Resef, L., & Hanson, R.W. (2005) Factors that control the tissue-specific transcription of the gene for
phosphoenolpyruvate carboxykinase-C. Critical Reviews of Biochemistry and Molecular Biology, 40(3), 129-154.
External links
Overview at indstate.edu (http:/ / themedicalbiochemistrypage. org/ gluconeogenesis. html)
Interactive diagram at uakron.edu (http:/ / ull. chemistry. uakron. edu/ Pathways/ gluconeogenesis/ index. html#)
The chemical logic behind gluconeogenesis (http:/ / homepage. ufp. pt/ pedros/ bq/ gng. htm)
Glycogen
Schematic 2-D cross-sectional view of glycogen. A core protein of
glycogenin is surrounded by branches of glucose units. The entire
globular granule may contain approximately 30,000 glucose units.
[1]
Glycogen is a multibranched polysaccharide that
serves as a form of energy storage in animals
[2]
and
fungi. In humans, glycogen is made and stored
primarily in the cells of the liver and the muscles,
and functions as the secondary long-term energy
storage (with the primary energy stores being fats
held in adipose tissue).
Glycogen is the analogue of starch, a glucose
polymer in plants, and is sometimes referred to as
animal starch, having a similar structure to
amylopectin but more extensively branched and
compact than starch. Glycogen is found in the form
of granules in the cytosol/cytoplasm in many cell
types, and plays an important role in the glucose
cycle. Glycogen forms an energy reserve that can be
quickly mobilized to meet a sudden need for glucose,
but one that is less compact than the energy reserves
of triglycerides (lipids).
Polysaccharide represents the main storage form of
glucose in the body. Found in the liver and muscles, muscle glycogen is converted into glucose by muscle cells, and
liver glycogen converts to glucose for use throughout the body including the Central Nervous System.
Glycogen
219
A view of the atomic structure of a single branched strand of glucose
units in a glycogen molecule.
In the liver cells (hepatocyte)s, glycogen can
compose up to eight percent of the fresh weight
(100120g in an adult) soon after a meal.
[3]
Only the
glycogen stored in the liver can be made accessible
to other organs. In the muscles, glycogen is found in
a low concentration (one to two percent of the
muscle mass). The amount of glycogen stored in the
bodyespecially within the muscles, liver, and red
blood cells
[4][5][6]
mostly depends on physical
training, basal metabolic rate, and eating habits such
as intermittent fasting. Small amounts of glycogen
are found in the kidneys, and even smaller amounts
in certain glial cells in the brain and white blood
cells. The uterus also stores glycogen during
pregnancy to nourish the embryo.
[7]
Structure
Schematic of glycogen structure
Glycogen is a branched biopolymer
consisting of linear chains of glucose
residues with further chains branching
off every ten glucoses or so. Glucoses
are linked together linearly by (14)
glycosidic bonds from one glucose to
the next. Branches are linked to the
chains they are branching off from by
(16) glycosidic bonds between the
first glucose of the new branch and a
glucose on the stem chain.
[8]
Due to the way that glycogen is synthesised, every glycogen granule has at its core a glycogenin protein.
[9]
Function
Liver
As a meal containing carbohydrates is eaten and digested, blood glucose levels rise, and the pancreas secretes
insulin. Blood glucose from the portal vein enters liver cells (hepatocytes). Insulin acts on the hepatocytes to
stimulate the action of several enzymes, including glycogen synthase. Glucose molecules are added to the chains of
glycogen as long as both insulin and glucose remain plentiful. In this postprandial or "fed" state, the liver takes in
more glucose from the blood than it releases.
After a meal has been digested and glucose levels begin to fall, insulin secretion is reduced, and glycogen synthesis
stops. When it is needed for energy, glycogen is broken down and converted again to glucose. Glycogen
phosphorylase is the primary enzyme of glycogen breakdown. For the next 812 hours, glucose derived from liver
Glycogen
220
glycogen will be the primary source of blood glucose to be used by the rest of the body for fuel.
Glucagon is another hormone produced by the pancreas, which in many respects serves as a counter-signal to insulin.
In response to insulin level below normal (when blood levels of glucose begin to fall below the normal range),
glucagon is secreted in increasing amounts to stimulate glycogenolysis and gluconeogenesis pathways.
Muscle
Muscle cell glycogen appears to function as an immediate reserve source of available glucose for muscle cells. Other
cells that contain small amounts use it locally as well. Muscle cells lack the enzyme glucose-6-phosphatase, which is
required to pass glucose into the blood, so the glycogen they store is destined for internal use and is not shared with
other cells. (This is in contrast to liver cells, which, on demand, readily do break down their stored glycogen into
glucose and send it through the blood stream as fuel for the brain or muscles). Glycogen is also a suitable storage
substance due to its insolubility in water, which means it does not affect the osmotistic levels and pressure of a cell.
History
Glycogen was discovered by Claude Bernard. His experiments showed that the liver contained a substance that could
give rise to reducing sugar by the action of a "ferment" in the liver. By 1857 he described the isolation of a substance
that he called "la matire glycogne", or "sugar-forming substance". Soon after the discovery of glycogen in the
liver, A. Sanson found that muscular tissue also contains glycogen. The empirical formula for glycogen of
(C
6
H
10
O
5
)
n
was established by Kekule in 1858.
[10]
Metabolism
Synthesis
Glycogen synthesis is, unlike its breakdown, endergonic. This means that glycogen synthesis requires the input of
energy. Energy for glycogen synthesis comes from UTP, which reacts with glucose-1-phosphate, forming
UDP-glucose, in a reaction catalysed by UDP-glucose pyrophosphorylase. Glycogen is synthesized from monomers
of UDP-glucose by the enzyme glycogen synthase, which progressively lengthens the glycogen chain with (14)
bonded glucose. As glycogen synthase can lengthen only an existing chain, the protein glycogenin is needed to
initiate the synthesis of glycogen. The glycogen-branching enzyme, amylo (1 4) to (1 6) transglycosylase,
catalyzes the transfer of a terminal fragment of 6-7 glucose residues from a nonreducing end to the C-6 hydroxyl
group of a glucose residue deeper into the interior of the glycogen molecule. The branching enzyme can act upon
only a branch having at least 11 residues, and the enzyme may transfer to the same glucose chain or adjacent glucose
chains.
Breakdown
Action of Glycogen Phosphorylase on Glycogen
Glycogen is cleaved from the nonreducing ends of the chain by the
enzyme glycogen phosphorylase to produce monomers of
glucose-1-phosphate, which is then converted to glucose 6-phosphate
by phosphoglucomutase. A special debranching enzyme is needed to
remove the alpha(1-6) branches in branched glycogen and reshape the
chain into linear polymer. The G6P monomers produced have three possible fates:
G6P can continue on the glycolysis pathway and be used as fuel.
G6P can enter the pentose phosphate pathway via the enzyme glucose-6-phosphate dehydrogenase to produce
NADPH and 5-carbon sugars.
Glycogen
221
In the liver and kidney, G6P can be dephosphorylated back to glucose by the enzyme glucose 6-phosphatase. This
is the final step in the gluconeogenesis pathway.
Clinical relevance
Disorders of glycogen metabolism
The most common disease in which glycogen metabolism becomes abnormal is diabetes, in which, because of
abnormal amounts of insulin, liver glycogen can be abnormally accumulated or depleted. Restoration of normal
glucose metabolism usually normalizes glycogen metabolism as well.
In hypoglycemia caused by excessive insulin, liver glycogen levels are high, but the high insulin level prevents the
glycogenolysis necessary to maintain normal blood sugar levels. Glucagon is a common treatment for this type of
hypoglycemia.
Various inborn errors of metabolism are caused by deficiencies of enzymes necessary for glycogen synthesis or
breakdown. These are collectively referred to as glycogen storage diseases.
Glycogen depletion and endurance exercise
Long-distance athletes such as marathon runners, cross-country skiers, and cyclists often experience glycogen
depletion, where almost all of the athlete's glycogen stores are depleted after long periods of exertion without enough
energy consumption. This phenomenon is referred to as "hitting the wall".
Glycogen depletion can be forestalled in three possible ways. First, during exercise carbohydrates with the highest
possible rate of conversion to blood glucose per time (high glycemic Index) are ingested continuously. The best
possible outcome of this strategy replaces about 35% of glucose consumed at heart rates above about 80% of
maximum. Second, through training, the body can be conditioned to burn fat earlier, faster, and more
efficiently
[citation needed]
, sparing carbohydrate use from all sources. Third, by consuming foods low on the glycemic
Index for 1218 hours before the event, the liver and muscles will store the resulting slow but steady stream of
glucose as glycogen, instead of fat. This process is known as carbohydrate loading.
When experiencing glycogen debt, athletes often experience extreme fatigue to the point that it is difficult to move.
As a reference, the very best professional cyclists in the world will usually finish a 4-5hr stage race right at the limit
of glycogen depletion using the first 3 strategies.
A study published in the Journal of Applied Physiology (online May 8, 2008) suggests that, when athletes ingest
both carbohydrate and caffeine following exhaustive exercise, their glycogen is replenished more
rapidly.Wikipedia:Identifying reliable sources (medicine)
[11][12]
References
External links
Glycogen detection using Periodic Acid Schiff Staining (http:/ / www. histochem. net/ protocol periodic acid
schiff. htm)
Glycogen storage disease - McArdle's Disease Website (http:/ / mcardlesdisease. org)
Glycogen (http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2011/ MB_cgi?mode=& term=Glycogen) at the US National
Library of Medicine Medical Subject Headings (MeSH)
Pentose phosphate pathway
222
Pentose phosphate pathway
The Pentose Phosphate Pathway
The pentose phosphate pathway (also called the
phosphogluconate pathway and the hexose monophosphate
shunt) is a process that generates NADPH and pentoses
(5-carbon sugars). There are two distinct phases in the pathway.
The first is the oxidative phase, in which NADPH is generated,
and the second is the non-oxidative synthesis of 5-carbon
sugars. This pathway is an alternative to glycolysis. While it
does involve oxidation of glucose, its primary role is anabolic
rather than catabolic. For most organisms, it takes place in the
cytosol; in plants, most steps take place in plastids.
[1]
Outcome
The primary results of the Pathway are:
The generation of reducing equivalents, in the form of
NADPH, used in reductive biosynthesis reactions within
cells. (e.g. fatty acid synthesis)
Production of ribose-5-phosphate (R5P), used in the
synthesis of nucleotides and nucleic acids.
Production of erythrose-4-phosphate (E4P), used in the
synthesis of aromatic amino acids.
Aromatic amino acids, in turn, are precursors for many
biosynthetic pathways, notably including the lignin in wood.
Dietary pentose sugars derived from the digestion of nucleic
acids may be metabolized through the pentose phosphate pathway, and the carbon skeletons of dietary carbohydrates
may be converted into glycolytic/gluconeogenic intermediates.
In mammals, the PPP occurs exclusively in the cytoplasm, and is found to be most active in the liver, mammary
gland and adrenal cortex in the human. The PPP is one of the three main ways the body creates molecules with
reducing power, accounting for approximately 60% of NADPH production in humans.
One of the uses of NADPH in the cell is to prevent oxidative stress. It reduces glutathione via glutathione reductase,
which converts reactive H
2
O
2
into H
2
O by glutathione peroxidase. If absent, the H
2
O
2
would be converted to
hydroxyl free radicals by Fenton chemistry, which can attack the cell. Erythrocytes, for example, generate a large
amount of NADPH through the pentose phosphate pathway to use in the reduction of glutathione.
Hydrogen peroxide is also generated for phagocytes in a process often referred to as a respiratory burst.
[2]
Pentose phosphate pathway
223
Phases
Oxidative phase
In this phase, two molecules of NADP
+
are reduced to NADPH, utilizing the energy from the conversion of
glucose-6-phosphate into ribulose 5-phosphate.
Oxidative phase of pentose phosphate pathway. glucose-6-phosphate (1), 6-phosphoglucono--lactone (2), 6-phosphogluconate (3),
ribulose 5-phosphate (4).
The entire set of reactions can be summarized as follows:
Reactants Products Enzyme Description
Glucose 6-phosphate +
NADP+

6-phosphoglucono--lactone +
NADPH
glucose 6-phosphate
dehydrogenase
Dehydrogenation. The hemiacetal hydroxyl group
located on carbon 1 of glucose 6-phosphate is
converted into a carbonyl group, generating a
lactone, and, in the process, NADPH is generated.
6-phosphoglucono--lactone
+ H
2
O
6-phosphogluconate + H
+ 6-phosphogluconolactonase Hydrolysis
6-phosphogluconate +
NADP
+
ribulose 5-phosphate +
NADPH + CO
2
6-phosphogluconate
dehydrogenase
Oxidative decarboxylation. NADP
+
is the electron
acceptor, generating another molecule of NADPH,
a CO
2
, and ribulose 5-phosphate.
The overall reaction for this process is:
Glucose 6-phosphate + 2 NADP
+
+ H
2
O ribulose 5-phosphate + 2 NADPH + 2 H
+
+ CO
2
Pentose phosphate pathway
224
Non-oxidative phase
The pentose phosphate pathway's nonoxidative phase
Reactants Products Enzymes
ribulose 5-phosphate ribose 5-phosphate Ribulose 5-Phosphate Isomerase
ribulose 5-phosphate xylulose 5-phosphate Ribulose 5-Phosphate
3-Epimerase
xylulose 5-phosphate + ribose 5-phosphate glyceraldehyde 3-phosphate + sedoheptulose
7-phosphate
transketolase
sedoheptulose 7-phosphate + glyceraldehyde
3-phosphate
erythrose 4-phosphate + fructose 6-phosphate transaldolase
xylulose 5-phosphate + erythrose 4-phosphate glyceraldehyde 3-phosphate + fructose 6-phosphate transketolase
Net reaction: 3 ribulose-5-phosphate 1 ribose-5-phosphate + 2 xylulose-5-phosphate 2 fructose-6-phosphate +
glyceraldehyde-3-phosphate
Regulation
Glucose-6-phosphate dehydrogenase is the rate-controlling enzyme of this pathway. It is allosterically stimulated by
NADP
+
. The ratio of NADPH:NADP
+
is normally about 100:1 in liver cytosol. This makes the cytosol a
highly-reducing environment. An NADPH-utilizing pathway forms NADP
+
, which stimulates Glucose-6-phosphate
dehydrogenase to produce more NADPH. This step is also inhibited by acetyl CoA.
Erythrocytes and the pentose phosphate pathway
Several deficiencies in the level of activity (not function) of glucose-6-phosphate dehydrogenase have been observed
to be associated with resistance to the malarial parasite Plasmodium falciparum among individuals of Mediterranean
and African descent. The basis for this resistance may be a weakening of the red cell membrane (the erythrocyte is
the host cell for the parasite) such that it cannot sustain the parasitic life cycle long enough for productive growth.
[3]
Pentose phosphate pathway
225
References
External links
The chemical logic behind the pentose phosphate pathway (http:/ / www2. ufp. pt/ ~pedros/ bq/ ppp. htm)
Pentose Phosphate Pathway (http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2011/ MB_cgi?mode=& term=Pentose+
Phosphate+ Pathway) at the US National Library of Medicine Medical Subject Headings (MeSH)
Pentose phosphate pathway Map - Homo sapiens (http:/ / www. genome. jp/ dbget-bin/
www_bget?path:hsa00030)
226
Citric acid cycle
Citric acid cycle
Overview of the citric acid cycle (click to enlarge)
The citric acid cycle also known as
the tricarboxylic acid cycle (TCA
cycle), or the Krebs cycle.
[][]
is a
series of chemical reactions used by all
aerobic organisms to generate energy
through the oxidization of acetate
derived from carbohydrates, fats and
proteins into carbon dioxide. In
addition, the cycle provides precursors
including certain amino acids as well
as the reducing agent NADH that is
used in numerous biochemical
reactions. Its central importance to
many biochemical pathways suggests
that it was one of the earliest
established components of cellular
metabolism and may have originated
abiogenically.
[]
The name of this metabolic pathway is derived from citric acid (a type of tricarboxylic acid) that is first consumed
and then regenerated by this sequence of reactions to complete the cycle. In addition, the cycle consumes acetate (in
the form of acetyl-CoA) and water, reduces NAD
+
to NADH, and produces carbon dioxide. The NADH generated
by the TCA cycle is fed into the oxidative phosphorylation pathway. The net result of these two closely linked
pathways is the oxidation of nutrients to produce usable energy in the form of ATP.
In eukaryotic cells, the citric acid cycle occurs in the matrix of the mitochondrion. Bacteria also use the TCA cycle
to generate energy, but since they lack mitochondria, the reaction sequence is performed in the cytosol with the
proton gradient for ATP production being across the plasma membrane rather than the inner membrane of the
mitochondrion.
Several of the components and reactions of the citric acid cycle were established in the 1930s by the research of the
Nobel laureate Albert Szent-Gyrgyi, for which he received the Nobel Prize in 1937 for his discoveries pertaining to
fumaric acid, a key component of the cycle.
[]
The citric acid cycle itself was finally identified in 1937 by Hans Adolf Krebs whilst at the University of Sheffield,
for which he received the Nobel Prize for Physiology or Medicine in 1953.
[]
Citric acid cycle
227
Evolution
Components of the TCA cycle were derived from anaerobic bacteria, and the TCA cycle itself may have evolved
more than once.
[]
Theoretically there are several alternatives to the TCA cycle, however the TCA cycle appears to be
the most efficient. If several TCA alternatives had independently evolved, they all appear to have converged onto the
canonical TCA cycle.
[][]
Overview
The citric acid cycle is a key component of the metabolic pathway by which all aerobic organisms generate energy.
Through catabolism of sugars, fats, and proteins, a two carbon organic product acetate in the form of acetyl-CoA is
produced. Acetyl-CoA along with two equivalents of water (H
2
O) is consumed by the citric acid cycle producing
two equivalents of carbon dioxide (CO
2
) and one equivalent of HS-CoA. In addition, one complete turn of the cycle
converts three equivalents of nicotinamide adenine dinucleotide (NAD
+
) into three equivalents of reduced NAD
+
(NADH), one equivalent of ubiquinone (Q) into one equivalent of reduced ubiquinone (QH
2
), and one equivalent
each of guanosine diphosphate (GDP) and inorganic phosphate (P
i
) into one equivalent of guanosine triphosphate
(GTP). The NADH and QH
2
generated by the citric acid cycle are in turn used by the oxidative phosphorylation
pathway to generate energy-rich adenosine triphosphate (ATP).
One of the primary sources of acetyl-CoA is sugars that are broken down by glycolysis to produce pyruvate that in
turn is decarboxylated by the enzyme pyruvate dehydrogenase generating acetyl-CoA according to the following
reaction scheme:
CH
3
C(=O)C(=O)O

(pyruvate) + HSCoA + NAD
+
CH
3
C(=O)SCoA (acetyl-CoA) + NADH + CO
2
The product of this reaction, acetyl-CoA, is the starting point for the citric acid cycle. Below is a schematic outline of
the cycle:
The citric acid cycle begins with the transfer of a two-carbon acetyl group from acetyl-CoA to the four-carbon
acceptor compound (oxaloacetate) to form a six-carbon compound (citrate).
The citrate then goes through a series of chemical transformations, losing two carboxyl groups as CO
2
. The
carbons lost as CO
2
originate from what was oxaloacetate, not directly from acetyl-CoA. The carbons donated by
acetyl-CoA become part of the oxaloacetate carbon backbone after the first turn of the citric acid cycle. Loss of
the acetyl-CoA-donated carbons as CO
2
requires several turns of the citric acid cycle. However, because of the
role of the citric acid cycle in anabolism, they may not be lost, since many TCA cycle intermediates are also used
as precursors for the biosynthesis of other molecules.
[]
Most of the energy made available by the oxidative steps of the cycle is transferred as energy-rich electrons to
NAD
+
, forming NADH. For each acetyl group that enters the citric acid cycle, three molecules of NADH are
produced.
Electrons are also transferred to the electron acceptor Q, forming QH
2
.
At the end of each cycle, the four-carbon oxaloacetate has been regenerated, and the cycle continues.
Steps
Two carbon atoms are oxidized to CO
2
, the energy from these reactions being transferred to other metabolic
processes by GTP (or ATP), and as electrons in NADH and QH
2
. The NADH generated in the TCA cycle may later
donate its electrons in oxidative phosphorylation to drive ATP synthesis; FADH
2
is covalently attached to succinate
dehydrogenase, an enzyme functioning both in the TCA cycle and the mitochondrial electron transport chain in
oxidative phosphorylation. FADH
2
, therefore, facilitates transfer of electrons to coenzyme Q, which is the final
electron acceptor of the reaction catalyzed by the Succinate:ubiquinone oxidoreductase complex, also acting as an
intermediate in the electron transport chain.
[]
The citric acid cycle is continuously supplied with new carbon in the form of acetyl-CoA, entering at step 1 below.
[]
Citric acid cycle
228
Substrates Products Enzyme Reaction type Comment
1 Oxaloacetate +
Acetyl CoA +
H
2
O
Citrate +
CoA-SH
Citrate synthase Aldol condensation irreversible,
extends the 4C oxaloacetate to a 6C molecule
2 Citrate cis-Aconitate +
H
2
O
Aconitase Dehydration reversible isomerisation
3 cis-Aconitate +
H
2
O
Isocitrate Hydration
4
Isocitrate +
NAD
+
Oxalosuccinate
+
NADH + H
+
Isocitrate
dehydrogenase
Oxidation generates NADH (equivalent of 2.5 ATP)
5 Oxalosuccinate -Ketoglutarate
+
CO
2
Decarboxylation rate-limiting, irreversible stage,
generates a 5C molecule
6
-Ketoglutarate
+
NAD
+
+
CoA-SH
Succinyl-CoA +
NADH + H
+
+
CO
2
-Ketoglutarate
dehydrogenase
Oxidative
decarboxylation
irreversible stage,
generates NADH (equivalent of 2.5 ATP),
regenerates the 4C chain (CoA excluded)
7 Succinyl-CoA +
GDP + P
i
Succinate +
CoA-SH +
GTP
Succinyl-CoA
synthetase
substrate-level
phosphorylation
or ADPATP instead of GDPGTP,
[]
generates 1 ATP or equivalent
Condensation reaction of GDP + P
i
and hydrolysis of
Succinyl-CoA involve the H
2
O needed for balanced
equation.
8 Succinate +
ubiquinone (Q)
Fumarate +
ubiquinol (QH
2
)
Succinate
dehydrogenase
Oxidation
uses FAD as a prosthetic group (FADFADH
2
in the
first step of the reaction) in the enzyme,
[]
generates the equivalent of 1.5 ATP
9 Fumarate +
H
2
O
L-Malate Fumarase Hydration
10
L-Malate +
NAD
+
Oxaloacetate +
NADH + H
+
Malate
dehydrogenase
Oxidation reversible (in fact, equilibrium favors malate), generates
NADH (equivalent of 2.5 ATP)
Mitochondria in animals, including humans, possess two succinyl-CoA synthetases: one that produces GTP from
GDP, and another that produces ATP from ADP.
[]
Plants have the type that produces ATP (ADP-forming
succinyl-CoA synthetase).
[]
Several of the enzymes in the cycle may be loosely associated in a multienzyme protein
complex within the mitochondrial matrix.
[]
The GTP that is formed by GDP-forming succinyl-CoA synthetase may be utilized by nucleoside-diphosphate kinase
to form ATP (the catalyzed reaction is GTP + ADP GDP + ATP).
[]
Citric acid cycle
229
Products
Products of the first turn of the cycle are: one GTP (or ATP), three NADH, one QH
2
, two CO
2
.
Because two acetyl-CoA molecules are produced from each glucose molecule, two cycles are required per glucose
molecule. Therefore, at the end of two cycles, the products are: two GTP, six NADH, two QH
2
, and four CO
2
Description Reactants Products
The sum of all reactions in the citric acid cycle is:
Acetyl-CoA + 3 NAD
+
+ Q +
GDP + P
i
+ 3 H
2
O
CoA-SH + 3 NADH + 3
H
+
+ QH
2
+ GTP + 2 CO
2
Combining the reactions occurring during the pyruvate oxidation with those
occurring during the citric acid cycle, the following overall pyruvate oxidation
reaction is obtained:
Pyruvate ion + 4 NAD
+
+ Q +
GDP + P
i
+ 2 H
2
O
4 NADH + 4 H
+
+ QH
2
+
GTP + 3 CO
2
Combining the above reaction with the ones occurring in the course of glycolysis,
the following overall glucose oxidation reaction (excluding reactions in the
respiratory chain) is obtained:
Glucose + 10 NAD
+
+ 2 Q + 2
ADP + 2 GDP + 4 P
i
+ 2 H
2
O
10 NADH + 10 H
+
+ 2
QH
2
+ 2 ATP + 2 GTP + 6
CO
2
The above reactions are balanced if P
i
represents the H
2
PO
4
-
ion, ADP and GDP the ADP
2-
and GDP
2-
ions,
respectively, and ATP and GTP the ATP
3-
and GTP
3-
ions, respectively.
The total number of ATP obtained after complete oxidation of one glucose in glycolysis, citric acid cycle, and
oxidative phosphorylation is estimated to be between 30 and 38.
[]
Efficiency
The theoretical maximum yield of ATP through oxidation of one molecule of glucose in glycolysis, citric acid cycle,
and oxidative phosphorylation is 38 (assuming 3 molar equivalents of ATP per equivalent NADH and 2 ATP per
FADH
2
). In eukaryotes, two equivalents of NADH are generated in glycolysis, which occurs in the cytoplasm.
Transport of these two equivalents into the mitochondria consumes two equivalents of ATP, thus reducing the net
production of ATP to 36. Furthermore, inefficiencies in oxidative phosphorylation due to leakage of protons across
of the mitochondrial membrane and slippage of the ATP synthase/proton pump commonly reduces the ATP yield
from NADH and FADH
2
to less than the theoretical maximum yield.
[]
The observed yields are, therefore, closer to
~2.5 ATP per NADH and ~1.5 ATP per FADH
2
, further reducing the total net production of ATP to approximately
30.
[]
An assessment of the total ATP yield with newly revised proton-to-ATP ratios provides an estimate of 29.85
ATP per glucose molecule.
[]
Regulation
The regulation of the TCA cycle is largely determined by product inhibition and substrate availability. NADH, a
product of all dehydrogenases in the TCA cycle with the exception of succinate dehydrogenase, inhibits pyruvate
dehydrogenase, isocitrate dehydrogenase, -ketoglutarate dehydrogenase, and also citrate synthase. Acetyl-coA
inhibits pyruvate dehydrogenase, while succinyl-CoA inhibits alpha-ketoglutarate dehydrogenase and citrate
synthase. When tested in vitro with TCA enzymes, ATP inhibits citrate synthase and -ketoglutarate dehydrogenase;
however, ATP levels do not change more than 10% in vivo between rest and vigorous exercise. There is no known
allosteric mechanism that can account for large changes in reaction rate from an allosteric effector whose
concentration changes less than 10%.
[1]
Calcium is used as a regulator. It activates pyruvate dehydrogenase phosphatase which in turn activates the pyruvate
dehydrogenase complex. Calcium also activates isocitrate dehydrogenase and -ketoglutarate dehydrogenase.
[]
This
increases the reaction rate of many of the steps in the cycle, and therefore increases flux throughout the pathway.
Citric acid cycle
230
Citrate is used for feedback inhibition, as it inhibits phosphofructokinase, an enzyme involved in glycolysis that
catalyses formation of fructose 1,6-bisphosphate,a precursor of pyruvate. This prevents a constant high rate of flux
when there is an accumulation of citrate and a decrease in substrate for the enzyme.
Recent work has demonstrated an important link between intermediates of the citric acid cycle and the regulation of
hypoxia-inducible factors (HIF). HIF plays a role in the regulation of oxygen homeostasis, and is a transcription
factor that targets angiogenesis, vascular remodeling, glucose utilization, iron transport and apoptosis. HIF is
synthesized consititutively, and hydroxylation of at least one of two critical proline residues mediates their
interaction with the von Hippel Lindau E3 ubiquitin ligase complex, which targets them for rapid degradation. This
reaction is catalysed by prolyl 4-hydroxylases. Fumarate and succinate have been identified as potent inhibitors of
prolyl hydroxylases, thus leading to the stabilisation of HIF.
[]
Major metabolic pathways converging on the TCA cycle
Several catabolic pathways converge on the TCA cycle. Reactions that form intermediates of the TCA cycle in order
to replenish them (especially during the scarcity of the intermediates) are called anaplerotic reactions.
The citric acid cycle is the third step in carbohydrate catabolism (the breakdown of sugars). Glycolysis breaks
glucose (a six-carbon-molecule) down into pyruvate (a three-carbon molecule). In eukaryotes, pyruvate moves into
the mitochondria. It is converted into acetyl-CoA by decarboxylation and enters the citric acid cycle.
In protein catabolism, proteins are broken down by proteases into their constituent amino acids. The carbon
backbone of these amino acids can become a source of energy by being converted to acetyl-CoA and entering into
the citric acid cycle.
In fat catabolism, triglycerides are hydrolyzed to break them into fatty acids and glycerol. In the liver the glycerol
can be converted into glucose via dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by way of
gluconeogenesis. In many tissues, especially heart tissue, fatty acids are broken down through a process known as
beta oxidation, which results in acetyl-CoA, which can be used in the citric acid cycle. Beta oxidation of fatty acids
with an odd number of methylene bridges produces propionyl CoA, which is then converted into succinyl-CoA and
fed into the citric acid cycle.
[]
The total energy gained from the complete breakdown of one molecule of glucose by glycolysis, the citric acid cycle,
and oxidative phosphorylation equals about 30 ATP molecules, in eukaryotes. The citric acid cycle is called an
amphibolic pathway because it participates in both catabolism and anabolism.
Interactive pathway map
Click on genes, proteins and metabolites below to link to respective articles.
[2]
Citric acid cycle
231
TCA Cycle edit
[3]
[2] [2] The interactive pathway map can be edited at WikiPathways:
[3] http:/ / www. wikipathways.org/ index.php/ Pathway:WP78
Citric acid cycle
232
References
External links
An animation of the citric acid cycle (http:/ / www. science. smith. edu/ departments/ Biology/ Bio231/ krebs.
html) at Smith College
Citric acid cycle variants (http:/ / biocyc. org/ META/ NEW-IMAGE?object=TCA-VARIANTS) at MetaCyc
Pathways connected to the citric acid cycle (http:/ / www. genome. ad. jp/ kegg/ pathway/ map/ map00020. html)
at Kyoto Encyclopedia of Genes and Genomes
Introduction at Khan Academy (https:/ / www. khanacademy. org/ science/ biology/ cellular-respiration/ v/
krebs---citric-acid-cycle)
233
Oxidative phosphorylation
Oxidative phosphorylation
The electron transport chain in the mitochondrion is the site of oxidative phosphorylation
in eukaryotes. The NADH and succinate generated in the citric acid cycle are oxidized,
releasing energy to power the ATP synthase.
Oxidative phosphorylation (or
OXPHOS in short) is the metabolic
pathway in which the mitochondria in
cells use their structure, enzymes, and
energy released by the oxidation of
nutrients to reform ATP. Although the
many forms of life on earth use a range
of different nutrients, ATP is the
molecule that supplies energy to
metabolism. Almost all aerobic
organisms carry out oxidative
phosphorylation. This pathway is
probably so pervasive because it is a
highly efficient way of releasing
energy, compared to alternative
fermentation processes such as
anaerobic glycolysis.
During oxidative phosphorylation,
electrons are transferred from electron
donors to electron acceptors such as
oxygen, in redox reactions. These
redox reactions release energy, which is used to form ATP. In eukaryotes, these redox reactions are carried out by a
series of protein complexes within the cell's intermembrane wall mitochondria, whereas, in prokaryotes, these
proteins are located in the cells' intermembrane space. These linked sets of proteins are called electron transport
chains. In eukaryotes, five main protein complexes are involved, whereas in prokaryotes many different enzymes are
present, using a variety of electron donors and acceptors.
The energy released by electrons flowing through this electron transport chain is used to transport protons across the
inner mitochondrial membrane, in a process called electron transport. This generates potential energy in the form of
a pH gradient and an electrical potential across this membrane. This store of energy is tapped by allowing protons to
flow back across the membrane and down this gradient, through a large enzyme called ATP synthase; this process is
known as chemiosmosis. This enzyme uses this energy to generate ATP from adenosine diphosphate (ADP), in a
phosphorylation reaction. This reaction is driven by the proton flow, which forces the rotation of a part of the
enzyme; the ATP synthase is a rotary mechanical motor.
Although oxidative phosphorylation is a vital part of metabolism, it produces reactive oxygen species such as
superoxide and hydrogen peroxide, which lead to propagation of free radicals, damaging cells and contributing to
disease and, possibly, aging (senescence). The enzymes carrying out this metabolic pathway are also the target of
many drugs and poisons that inhibit their activities.
Oxidative phosphorylation
234
Overview of energy transfer by chemiosmosis
Oxidative phosphorylation works by using energy-releasing chemical reactions to drive energy-requiring reactions:
The two sets of reactions are said to be coupled. This means one cannot occur without the other. The flow of
electrons through the electron transport chain, from electron donors such as NADH to electron acceptors such as
oxygen, is an exergonic process it releases energy, whereas the synthesis of ATP is an endergonic process, which
requires an input of energy. Both the electron transport chain and the ATP synthase are embedded in a membrane,
and energy is transferred from electron transport chain to the ATP synthase by movements of protons across this
membrane, in a process called chemiosmosis.
[1]
In practice, this is like a simple electric circuit, with a current of
protons being driven from the negative N-side of the membrane to the positive P-side by the proton-pumping
enzymes of the electron transport chain. These enzymes are like a battery, as they perform work to drive current
through the circuit. The movement of protons creates an electrochemical gradient across the membrane, which is
often called the proton-motive force. It has two components: a difference in proton concentration (a H+ gradient,
pH) and a difference in electric potential, with the N-side having a negative charge.
[]
ATP synthase releases this stored energy by completing the circuit and allowing protons to flow down the
electrochemical gradient, back to the N-side of the membrane.
[]
This kinetic energy drives the rotation of part of the
enzymes structure and couples this motion to the synthesis of ATP.
The two components of the proton-motive force are thermodynamically equivalent: In mitochondria, the largest part
of energy is provided by the potential; in alkaliphile bacteria the electrical energy even has to compensate for a
counteracting inverse pH difference. Inversely, chloroplasts operate mainly on pH. However, they also require a
small membrane potential for the kinetics of ATP synthesis. At least in the case of the fusobacterium P. modestum it
drives the counter-rotation of subunits a and c of the F
O
motor of ATP synthase.
[]
The amount of energy released by oxidative phosphorylation is high, compared with the amount produced by
anaerobic fermentation. Glycolysis produces only 2 ATP molecules, but somewhere between 30 and 36 ATPs are
produced by the oxidative phosphorylation of the 10 NADH and 2 succinate molecules made by converting one
molecule of glucose to carbon dioxide and water,
[2]
while each cycle of beta oxidation of a fatty acid yields about 14
ATPs. These ATP yields are theoretical maximum values; in practice, some protons leak across the membrane,
lowering the yield of ATP.
[3]
Electron and proton transfer molecules
Reduction of coenzyme Q from its ubiquinone form (Q) to the
reduced ubiquinol form (QH
2
).
The electron transport chain carries both protons and
electrons, passing electrons from donors to acceptors,
and transporting protons across a membrane. These
processes use both soluble and protein-bound transfer
molecules. In mitochondria, electrons are transferred
within the intermembrane space by the water-soluble
electron transfer protein cytochrome c.
[4]
This carries
only electrons, and these are transferred by the
reduction and oxidation of an iron atom that the protein
holds within a heme group in its structure. Cytochrome
c is also found in some bacteria, where it is located
within the periplasmic space.
[5]
Within the inner mitochondrial membrane, the
lipid-soluble electron carrier coenzyme Q10 (Q) carries
Oxidative phosphorylation
235
both electrons and protons by a redox cycle.
[6]
This small benzoquinone molecule is very hydrophobic, so it diffuses
freely within the membrane. When Q accepts two electrons and two protons, it becomes reduced to the ubiquinol
form (QH
2
); when QH
2
releases two electrons and two protons, it becomes oxidized back to the ubiquinone (Q)
form. As a result, if two enzymes are arranged so that Q is reduced on one side of the membrane and QH
2
oxidized
on the other, ubiquinone will couple these reactions and shuttle protons across the membrane.
[7]
Some bacterial
electron transport chains use different quinones, such as menaquinone, in addition to ubiquinone.
[]
Within proteins, electrons are transferred between flavin cofactors,
[][]
ironsulfur clusters, and cytochromes. There
are several types of ironsulfur cluster. The simplest kind found in the electron transfer chain consists of two iron
atoms joined by two atoms of inorganic sulfur; these are called [2Fe2S] clusters. The second kind, called [4Fe4S],
contains a cube of four iron atoms and four sulfur atoms. Each iron atom in these clusters is coordinated by an
additional amino acid, usually by the sulfur atom of cysteine. Metal ion cofactors undergo redox reactions without
binding or releasing protons, so in the electron transport chain they serve solely to transport electrons through
proteins. Electrons move quite long distances through proteins by hopping along chains of these cofactors.
[8]
This
occurs by quantum tunnelling, which is rapid over distances of less than 1.410
9
m.
[9]
Eukaryotic electron transport chains
Many catabolic biochemical processes, such as glycolysis, the citric acid cycle, and beta oxidation, produce the
reduced coenzyme NADH. This coenzyme contains electrons that have a high transfer potential; in other words, they
will release a large amount of energy upon oxidation. However, the cell does not release this energy all at once, as
this would be an uncontrollable reaction. Instead, the electrons are removed from NADH and passed to oxygen
through a series of enzymes that each release a small amount of the energy. This set of enzymes, consisting of
complexes I through IV, is called the electron transport chain and is found in the inner membrane of the
mitochondrion. Succinate is also oxidized by the electron transport chain, but feeds into the pathway at a different
point.
In eukaryotes, the enzymes in this electron transport system use the energy released from the oxidation of NADH to
pump protons across the inner membrane of the mitochondrion. This causes protons to build up in the
intermembrane space, and generates an electrochemical gradient across the membrane. The energy stored in this
potential is then used by ATP synthase to produce ATP. Oxidative phosphorylation in the eukaryotic mitochondrion
is the best-understood example of this process. The mitochondrion is present in almost all eukaryotes, with the
exception of anaerobic protozoa such as Trichomonas vaginalis that instead reduce protons to hydrogen in a remnant
mitochondrion called a hydrogenosome.
[10]
Typical respiratory enzymes and substrates in eukaryotes.
Respiratory enzyme Redox pair Midpoint potential (Volts)
NADH dehydrogenase
NAD
+
/ NADH 0.32
[11]
Succinate dehydrogenase FMN or FAD / FMNH
2
or FADH
2 0.20
[11]
Cytochrome bc
1
complex Coenzyme Q10
ox
/ Coenzyme Q10
red +0.06
[11]
Cytochrome bc
1
complex Cytochrome b
ox
/ Cytochrome b
red +0.12
[11]
Complex IV Cytochrome c
ox
/ Cytochrome c
red +0.22
[11]
Complex IV Cytochrome a
ox
/ Cytochrome a
red +0.29
[11]
Complex IV
O
2
/ HO

+0.82
[11]
Conditions: pH = 7
[11]
Oxidative phosphorylation
236
NADH-coenzyme Q oxidoreductase (complex I)
Complex I or NADH-Q oxidoreductase. The abbreviations are discussed in the text. In all
diagrams of respiratory complexes in this article, the matrix is at the bottom, with the
intermembrane space above.
NADH-coenzyme Q oxidoreductase,
also known as NADH dehydrogenase
or complex I, is the first protein in the
electron transport chain.
[]
Complex I is
a giant enzyme with the mammalian
complex I having 46 subunits and a
molecular mass of about 1,000
kilodaltons (kDa).
[]
The structure is
known in detail only from a
bacterium;
[][12]
in most organisms the
complex resembles a boot with a large
"ball" poking out from the membrane
into the mitochondrion.
[13][14]
The
genes that encode the individual
proteins are contained in both the cell
nucleus and the mitochondrial genome,
as is the case for many enzymes
present in the mitochondrion.
The reaction that is catalyzed by this
enzyme is the two electron oxidation
of NADH by coenzyme Q10 or ubiquinone (represented as Q in the equation below), a lipid-soluble quinone that is
found in the mitochondrion membrane:
The start of the reaction, and indeed of the entire electron chain, is the binding of a NADH molecule to complex I
and the donation of two electrons. The electrons enter complex I via a prosthetic group attached to the complex,
flavin mononucleotide (FMN). The addition of electrons to FMN converts it to its reduced form, FMNH
2
. The
electrons are then transferred through a series of ironsulfur clusters: the second kind of prosthetic group present in
the complex.
[]
There are both [2Fe2S] and [4Fe4S] ironsulfur clusters in complex I.
As the electrons pass through this complex, four protons are pumped from the matrix into the intermembrane space.
Exactly how this occurs is unclear, but it seems to involve conformational changes in complex I that cause the
protein to bind protons on the N-side of the membrane and release them on the P-side of the membrane.
[15]
Finally,
the electrons are transferred from the chain of ironsulfur clusters to a ubiquinone molecule in the membrane.
[]
Reduction of ubiquinone also contributes to the generation of a proton gradient, as two protons are taken up from the
matrix as it is reduced to ubiquinol (QH
2
).
Oxidative phosphorylation
237
Succinate-Q oxidoreductase (complex II)
Complex II: Succinate-Q oxidoreductase.
Succinate-Q oxidoreductase, also known as complex II
or succinate dehydrogenase, is a second entry point to
the electron transport chain.
[16]
It is unusual because it
is the only enzyme that is part of both the citric acid
cycle and the electron transport chain. Complex II
consists of four protein subunits and contains a bound
flavin adenine dinucleotide (FAD) cofactor, ironsulfur
clusters, and a heme group that does not participate in
electron transfer to coenzyme Q, but is believed to be
important in decreasing production of reactive oxygen
species.
[17][18]
It oxidizes succinate to fumarate and
reduces ubiquinone. As this reaction releases less
energy than the oxidation of NADH, complex II does
not transport protons across the membrane and does not
contribute to the proton gradient.
In some eukaryotes, such as the parasitic worm Ascaris
suum, an enzyme similar to complex II, fumarate reductase (menaquinol:fumarate oxidoreductase, or QFR), operates
in reverse to oxidize ubiquinol and reduce fumarate. This allows the worm to survive in the anaerobic environment
of the large intestine, carrying out anaerobic oxidative phosphorylation with fumarate as the electron acceptor.
[19]
Another unconventional function of complex II is seen in the malaria parasite Plasmodium falciparum. Here, the
reversed action of complex II as an oxidase is important in regenerating ubiquinol, which the parasite uses in an
unusual form of pyrimidine biosynthesis.
[20]
Electron transfer flavoprotein-Q oxidoreductase
Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-Q oxidoreductase), also known as electron
transferring-flavoprotein dehydrogenase, is a third entry point to the electron transport chain. It is an enzyme that
accepts electrons from electron-transferring flavoprotein in the mitochondrial matrix, and uses these electrons to
reduce ubiquinone.
[21]
This enzyme contains a flavin and a [4Fe4S] cluster, but, unlike the other respiratory
complexes, it attaches to the surface of the membrane and does not cross the lipid bilayer.
[22]
In mammals, this metabolic pathway is important in beta oxidation of fatty acids and catabolism of amino acids and
choline, as it accepts electrons from multiple acetyl-CoA dehydrogenases.
[23][24]
In plants, ETF-Q oxidoreductase is
also important in the metabolic responses that allow survival in extended periods of darkness.
[25]
Oxidative phosphorylation
238
Q-cytochrome c oxidoreductase (complex III)
The two electron transfer steps in complex III: Q-cytochrome c oxidoreductase. After
each step, Q (in the upper part of the figure) leaves the enzyme.
Q-cytochrome c oxidoreductase is also
known as cytochrome c reductase,
cytochrome bc
1
complex, or simply
complex III.
[26][27]
In mammals, this
enzyme is a dimer, with each subunit
complex containing 11 protein
subunits, an [2Fe-2S] ironsulfur
cluster and three cytochromes: one
cytochrome c
1
and two b
cytochromes.
[28]
A cytochrome is a
kind of electron-transferring protein
that contains at least one heme group.
The iron atoms inside complex IIIs
heme groups alternate between a
reduced ferrous (+2) and oxidized ferric (+3) state as the electrons are transferred through the protein.
The reaction catalyzed by complex III is the oxidation of one molecule of ubiquinol and the reduction of two
molecules of cytochrome c, a heme protein loosely associated with the mitochondrion. Unlike coenzyme Q, which
carries two electrons, cytochrome c carries only one electron.
As only one of the electrons can be transferred from the QH
2
donor to a cytochrome c acceptor at a time, the reaction
mechanism of complex III is more elaborate than those of the other respiratory complexes, and occurs in two steps
called the Q cycle.
[29]
In the first step, the enzyme binds three substrates, first, QH
2
, which is then oxidized, with one
electron being passed to the second substrate, cytochrome c. The two protons released from QH
2
pass into the
intermembrane space. The third substrate is Q, which accepts the second electron from the QH
2
and is reduced to Q
.-
,
which is the ubisemiquinone free radical. The first two substrates are released, but this ubisemiquinone intermediate
remains bound. In the second step, a second molecule of QH
2
is bound and again passes its first electron to a
cytochrome c acceptor. The second electron is passed to the bound ubisemiquinone, reducing it to QH
2
as it gains
two protons from the mitochondrial matrix. This QH
2
is then released from the enzyme.
[30]
As coenzyme Q is reduced to ubiquinol on the inner side of the membrane and oxidized to ubiquinone on the other, a
net transfer of protons across the membrane occurs, adding to the proton gradient.
[]
The rather complex two-step
mechanism by which this occurs is important, as it increases the efficiency of proton transfer. If, instead of the Q
cycle, one molecule of QH
2
were used to directly reduce two molecules of cytochrome c, the efficiency would be
halved, with only one proton transferred per cytochrome c reduced.
[]
Oxidative phosphorylation
239
Cytochrome c oxidase (complex IV)
Complex IV: cytochrome c oxidase.
Cytochrome c oxidase, also known as complex IV, is the final protein
complex in the electron transport chain.
[31]
The mammalian enzyme
has an extremely complicated structure and contains 13 subunits, two
heme groups, as well as multiple metal ion cofactors in all three
atoms of copper, one of magnesium and one of zinc.
[32]
This enzyme mediates the final reaction in the electron transport chain
and transfers electrons to oxygen, while pumping protons across the
membrane.
[33]
The final electron acceptor oxygen, which is also called
the terminal electron acceptor, is reduced to water in this step. Both
the direct pumping of protons and the consumption of matrix protons
in the reduction of oxygen contribute to the proton gradient. The
reaction catalyzed is the oxidation of cytochrome c and the reduction
of oxygen:
Alternative reductases and oxidases
Many eukaryotic organisms have electron transport chains that differ from the much-studied mammalian enzymes
described above. For example, plants have alternative NADH oxidases, which oxidize NADH in the cytosol rather
than in the mitochondrial matrix, and pass these electrons to the ubiquinone pool.
[34]
These enzymes do not transport
protons, and, therefore, reduce ubiquinone without altering the electrochemical gradient across the inner
membrane.
[35]
Another example of a divergent electron transport chain is the alternative oxidase, which is found in plants, as well
as some fungi, protists, and possibly some animals.
[36][37]
This enzyme transfers electrons directly from ubiquinol to
oxygen.
[38]
The electron transport pathways produced by these alternative NADH and ubiquinone oxidases have lower ATP
yields than the full pathway. The advantages produced by a shortened pathway are not entirely clear. However, the
alternative oxidase is produced in response to stresses such as cold, reactive oxygen species, and infection by
pathogens, as well as other factors that inhibit the full electron transport chain.
[39][40]
Alternative pathways might,
therefore, enhance an organisms' resistance to injury, by reducing oxidative stress.
[41]
Organization of complexes
The original model for how the respiratory chain complexes are organized was that they diffuse freely and
independently in the mitochondrial membrane.
[]
However, recent data suggest that the complexes might form
higher-order structures called supercomplexes or "respirasomes."
[42]
In this model, the various complexes exist as
organized sets of interacting enzymes.
[43]
These associations might allow channeling of substrates between the
various enzyme complexes, increasing the rate and efficiency of electron transfer.
[44]
Within such mammalian
supercomplexes, some components would be present in higher amounts than others, with some data suggesting a
ratio between complexes I/II/III/IV and the ATP synthase of approximately 1:1:3:7:4.
[45]
However, the debate over
this supercomplex hypothesis is not completely resolved, as some data do not appear to fit with this model.
[][46]
Oxidative phosphorylation
240
Prokaryotic electron transport chains
In contrast to the general similarity in structure and function of the electron transport chains in eukaryotes, bacteria
and archaea possess a large variety of electron-transfer enzymes. These use an equally wide set of chemicals as
substrates.
[47]
In common with eukaryotes, prokaryotic electron transport uses the energy released from the
oxidation of a substrate to pump ions across a membrane and generate an electrochemical gradient. In the bacteria,
oxidative phosphorylation in Escherichia coli is understood in most detail, while archaeal systems are at present
poorly understood.
[48]
The main difference between eukaryotic and prokaryotic oxidative phosphorylation is that bacteria and archaea use
many different substances to donate or accept electrons. This allows prokaryotes to grow under a wide variety of
environmental conditions.
[]
In E. coli, for example, oxidative phosphorylation can be driven by a large number of
pairs of reducing agents and oxidizing agents, which are listed below. The midpoint potential of a chemical measures
how much energy is released when it is oxidized or reduced, with reducing agents having negative potentials and
oxidizing agents positive potentials.
Respiratory enzymes and substrates in E. coli.
[]
Respiratory enzyme Redox pair Midpoint potential (Volts)
Formate dehydrogenase Bicarbonate / Formate 0.43
Hydrogenase Proton / Hydrogen 0.42
NADH dehydrogenase
NAD
+
/ NADH
0.32
Glycerol-3-phosphate dehydrogenase DHAP / Gly-3-P 0.19
Pyruvate oxidase Acetate + Carbon dioxide / Pyruvate ?
Lactate dehydrogenase Pyruvate / Lactate 0.19
D-amino acid dehydrogenase 2-oxoacid + ammonia / D-amino acid ?
Glucose dehydrogenase Gluconate / Glucose 0.14
Succinate dehydrogenase Fumarate / Succinate +0.03
Ubiquinol oxidase Oxygen / Water +0.82
Nitrate reductase Nitrate / Nitrite +0.42
Nitrite reductase Nitrite / Ammonia +0.36
Dimethyl sulfoxide reductase DMSO / DMS +0.16
Trimethylamine N-oxide reductase TMAO / TMA +0.13
Fumarate reductase Fumarate / Succinate +0.03
As shown above, E. coli can grow with reducing agents such as formate, hydrogen, or lactate as electron donors, and
nitrate, DMSO, or oxygen as acceptors.
[]
The larger the difference in midpoint potential between an oxidizing and
reducing agent, the more energy is released when they react. Out of these compounds, the succinate/fumarate pair is
unusual, as its midpoint potential is close to zero. Succinate can therefore be oxidized to fumarate if a strong
oxidizing agent such as oxygen is available, or fumarate can be reduced to succinate using a strong reducing agent
such as formate. These alternative reactions are catalyzed by succinate dehydrogenase and fumarate reductase,
respectively.
[49]
Some prokaryotes use redox pairs that have only a small difference in midpoint potential. For example, nitrifying
bacteria such as Nitrobacter oxidize nitrite to nitrate, donating the electrons to oxygen. The small amount of energy
released in this reaction is enough to pump protons and generate ATP, but not enough to produce NADH or NADPH
directly for use in anabolism.
[50]
This problem is solved by using a nitrite oxidoreductase to produce enough
Oxidative phosphorylation
241
proton-motive force to run part of the electron transport chain in reverse, causing complex I to generate
NADH.
[51][52]
Prokaryotes control their use of these electron donors and acceptors by varying which enzymes are produced, in
response to environmental conditions.
[53]
This flexibility is possible because different oxidases and reductases use
the same ubiquinone pool. This allows many combinations of enzymes to function together, linked by the common
ubiquinol intermediate.
[]
These respiratory chains therefore have a modular design, with easily interchangeable sets
of enzyme systems.
In addition to this metabolic diversity, prokaryotes also possess a range of isozymes different enzymes that
catalyze the same reaction. For example, in E. coli, there are two different types of ubiquinol oxidase using oxygen
as an electron acceptor. Under highly aerobic conditions, the cell uses an oxidase with a low affinity for oxygen that
can transport two protons per electron. However, if levels of oxygen fall, they switch to an oxidase that transfers
only one proton per electron, but has a high affinity for oxygen.
[54]
ATP synthase (complex V)
ATP synthase, also called complex V, is the final enzyme in the oxidative phosphorylation pathway. This enzyme is
found in all forms of life and functions in the same way in both prokaryotes and eukaryotes.
[]
The enzyme uses the
energy stored in a proton gradient across a membrane to drive the synthesis of ATP from ADP and phosphate (P
i
).
Estimates of the number of protons required to synthesize one ATP have ranged from three to four,
[55][56]
with some
suggesting cells can vary this ratio, to suit different conditions.
[57]
This phosphorylation reaction is an equilibrium, which can be shifted by altering the proton-motive force. In the
absence of a proton-motive force, the ATP synthase reaction will run from right to left, hydrolyzing ATP and
pumping protons out of the matrix across the membrane. However, when the proton-motive force is high, the
reaction is forced to run in the opposite direction; it proceeds from left to right, allowing protons to flow down their
concentration gradient and turning ADP into ATP.
[]
Indeed, in the closely related vacuolar type H+-ATPases, the
hydrolysis reaction is used to acidify cellular compartments, by pumping protons and hydrolysing ATP.
[58]
ATP synthase is a massive protein complex with a mushroom-like shape. The mammalian enzyme complex contains
16 subunits and has a mass of approximately 600 kilodaltons.
[59]
The portion embedded within the membrane is
called F
O
and contains a ring of c subunits and the proton channel. The stalk and the ball-shaped headpiece is called
F
1
and is the site of ATP synthesis. The ball-shaped complex at the end of the F
1
portion contains six proteins of two
different kinds (three subunits and three subunits), whereas the "stalk" consists of one protein: the subunit, with
the tip of the stalk extending into the ball of and subunits.
[60]
Both the and subunits bind nucleotides, but
only the subunits catalyze the ATP synthesis reaction. Reaching along the side of the F
1
portion and back into the
membrane is a long rod-like subunit that anchors the and subunits into the base of the enzyme.
As protons cross the membrane through the channel in the base of ATP synthase, the F
O
proton-driven motor
rotates.
[61]
Rotation might be caused by changes in the ionization of amino acids in the ring of c subunits causing
electrostatic interactions that propel the ring of c subunits past the proton channel.
[62]
This rotating ring in turn drives
the rotation of the central axle (the subunit stalk) within the and subunits. The and subunits are prevented
from rotating themselves by the side-arm, which acts as a stator. This movement of the tip of the subunit within the
ball of and subunits provides the energy for the active sites in the subunits to undergo a cycle of movements
that produces and then releases ATP.
[]
Oxidative phosphorylation
242
Mechanism of ATP synthase. ATP is shown in red, ADP and
phosphate in pink and the rotating subunit in black.
This ATP synthesis reaction is called the binding change
mechanism and involves the active site of a subunit cycling
between three states.
[]
In the "open" state, ADP and phosphate
enter the active site (shown in brown in the diagram). The
protein then closes up around the molecules and binds them
loosely the "loose" state (shown in red). The enzyme then
changes shape again and forces these molecules together, with
the active site in the resulting "tight" state (shown in pink)
binding the newly produced ATP molecule with very high
affinity. Finally, the active site cycles back to the open state,
releasing ATP and binding more ADP and phosphate, ready
for the next cycle.
In some bacteria and archaea, ATP synthesis is driven by the
movement of sodium ions through the cell membrane, rather
than the movement of protons.
[63][]
Archaea such as Methanococcus also contain the A
1
A
o
synthase, a form of the
enzyme that contains additional proteins with little similarity in sequence to other bacterial and eukaryotic ATP
synthase subunits. It is possible that, in some species, the A
1
A
o
form of the enzyme is a specialized sodium-driven
ATP synthase,
[64]
but this might not be true in all cases.
[]
Reactive oxygen species
Molecular oxygen is an ideal terminal electron acceptor because it is a strong oxidizing agent. The reduction of
oxygen does involve potentially harmful intermediates.
[]
Although the transfer of four electrons and four protons
reduces oxygen to water, which is harmless, transfer of one or two electrons produces superoxide or peroxide anions,
which are dangerously reactive.
These reactive oxygen species and their reaction products, such as the hydroxyl radical, are very harmful to cells, as
they oxidize proteins and cause mutations in DNA. This cellular damage might contribute to disease and is proposed
as one cause of aging.
[65][66]
The cytochrome c oxidase complex is highly efficient at reducing oxygen to water, and it releases very few partly
reduced intermediates; however small amounts of superoxide anion and peroxide are produced by the electron
transport chain.
[]
Particularly important is the reduction of coenzyme Q in complex III, as a highly reactive
ubisemiquinone free radical is formed as an intermediate in the Q cycle. This unstable species can lead to electron
"leakage" when electrons transfer directly to oxygen, forming superoxide.
[67]
As the production of reactive oxygen
species by these proton-pumping complexes is greatest at high membrane potentials, it has been proposed that
mitochondria regulate their activity to maintain the membrane potential within a narrow range that balances ATP
production against oxidant generation.
[68]
For instance, oxidants can activate uncoupling proteins that reduce
membrane potential.
[69]
To counteract these reactive oxygen species, cells contain numerous antioxidant systems, including antioxidant
vitamins such as vitamin C and vitamin E, and antioxidant enzymes such as superoxide dismutase, catalase, and
peroxidases,
[]
which detoxify the reactive species, limiting damage to the cell.
Oxidative phosphorylation
243
Inhibitors
There are several well-known drugs and toxins that inhibit oxidative phosphorylation. Although any one of these
toxins inhibits only one enzyme in the electron transport chain, inhibition of any step in this process will halt the rest
of the process. For example, if oligomycin inhibits ATP synthase, protons cannot pass back into the mitochondrion.
[]
As a result, the proton pumps are unable to operate, as the gradient becomes too strong for them to overcome.
NADH is then no longer oxidized and the citric acid cycle ceases to operate because the concentration of NAD
+
falls
below the concentration that these enzymes can use.
Compounds Use Effect on oxidative phosphorylation
Cyanide
Carbonmonoxide
Azide
Poisons
Inhibit the electron transport chain by binding more strongly than oxygen to the FeCu center in cytochrome c
oxidase, preventing the reduction of oxygen.
[70]
Oligomycin Antibiotic
Inhibits ATP synthase by blocking the flow of protons through the F
o
subunit.
[]
CCCP
2,4-Dinitrophenol
Poisons
Ionophores that disrupt the proton gradient by carrying protons across a membrane. This ionophore uncouples
proton pumping from ATP synthesis because it carries protons across the inner mitochondrial membrane.
[71]
Rotenone Pesticide
Prevents the transfer of electrons from complex I to ubiquinone by blocking the ubiquinone-binding site.
[72]
Malonate and
oxaloacetate
Competitive inhibitors of succinate dehydrogenase (complex II).
[73]
Not all inhibitors of oxidative phosphorylation are toxins. In brown adipose tissue, regulated proton channels called
uncoupling proteins can uncouple respiration from ATP synthesis.
[74]
This rapid respiration produces heat, and is
particularly important as a way of maintaining body temperature for hibernating animals, although these proteins
may also have a more general function in cells' responses to stress.
[75]
History
The field of oxidative phosphorylation began with the report in 1906 by Arthur Harden of a vital role for phosphate
in cellular fermentation, but initially only sugar phosphates were known to be involved.
[76]
However, in the early
1940s, the link between the oxidation of sugars and the generation of ATP was firmly established by Herman
Kalckar,
[77]
confirming the central role of ATP in energy transfer that had been proposed by Fritz Albert Lipmann in
1941.
[78]
Later, in 1949, Morris Friedkin and Albert L. Lehninger proved that the coenzyme NADH linked metabolic
pathways such as the citric acid cycle and the synthesis of ATP.
[79]
For another twenty years, the mechanism by which ATP is generated remained mysterious, with scientists searching
for an elusive "high-energy intermediate" that would link oxidation and phosphorylation reactions.
[80]
This puzzle
was solved by Peter D. Mitchell with the publication of the chemiosmotic theory in 1961.
[81]
At first, this proposal
was highly controversial, but it was slowly accepted and Mitchell was awarded a Nobel prize in 1978.
[82][83]
Subsequent research concentrated on purifying and characterizing the enzymes involved, with major contributions
being made by David E. Green on the complexes of the electron-transport chain, as well as Efraim Racker on the
ATP synthase.
[84]
A critical step towards solving the mechanism of the ATP synthase was provided by Paul D.
Boyer, by his development in 1973 of the "binding change" mechanism, followed by his radical proposal of
rotational catalysis in 1982.
[][85]
More recent work has included structural studies on the enzymes involved in
oxidative phosphorylation by John E. Walker, with Walker and Boyer being awarded a Nobel Prize in 1997.
[86]
Oxidative phosphorylation
244
References
[11] [11] Medical CHEMISTRY Compendium. By Anders Overgaard Pedersen and Henning Nielsen. Aarhus University. 2008
[12] Efremov R.G., Baradaran R., & Sazanov L.A., (2010) The arcdhitecture of respiratory complex I, Nature 465, 441-445
Further reading
Introductory
Nelson DL; Cox MM (2004). Lehninger Principles of Biochemistry (4th ed.). W. H. Freeman.
ISBN0-7167-4339-6.
Schneider ED; Sagan D (2006). Into the Cool: Energy Flow, Thermodynamics and Life (1st ed.). University of
Chicago Press. ISBN0-226-73937-6.
Lane N (2006). Power, Sex, Suicide: Mitochondria and the Meaning of Life (1st ed.). Oxford University Press,
USA. ISBN0-19-920564-7.
Advanced
Nicholls DG; Ferguson SJ (2002). Bioenergetics 3 (1st ed.). Academic Press. ISBN0-12-518121-3.
Haynie D (2001). Biological Thermodynamics (1st ed.). Cambridge University Press. ISBN0-521-79549-4.
Rajan SS (2003). Introduction to Bioenergetics (1st ed.). Anmol. ISBN81-261-1364-2.
Wikstrom M (Ed) (2005). Biophysical and Structural Aspects of Bioenergetics (1st ed.). Royal Society of
Chemistry. ISBN0-85404-346-2.
External links
General resources
Animated diagrams illustrating oxidative phosphorylation (http:/ / www. wiley. com/ legacy/ college/ boyer/
0470003790/ animations/ electron_transport/ electron_transport. htm) Wiley and Co Concepts in Biochemistry
ATP synthase - the rotary engine in the cell (http:/ / www. res. titech. ac. jp/ ~seibutu/ ) Brief introduction,
including videos of microscope images of the enzyme rotating, at Tokyo Institute of Technology
On-line biophysics lectures (http:/ / www. life. uiuc. edu/ crofts/ bioph354/ ) Antony Crofts, University of Illinois
at Urbana-Champaign
Structural resources
Animations of the ATP synthase (http:/ / nature. berkeley. edu/ ~hongwang/ Project/ ATP_synthase/ ) Hongyun
Wang and George Oster, University of California, Berkeley
PDB molecule of the month:
ATP synthase (http:/ / www. rcsb. org/ pdb/ static. do?p=education_discussion/ molecule_of_the_month/
pdb72_1. html)
Cytochrome c (http:/ / www. rcsb. org/ pdb/ static. do?p=education_discussion/ molecule_of_the_month/
pdb36_1. html)
Cytochrome c oxidase (http:/ / www. rcsb. org/ pdb/ static. do?p=education_discussion/
molecule_of_the_month/ pdb5_1. html)
Interactive molecular models at Universidade Fernando Pessoa:
NADH dehydrogenase (http:/ / www2. ufp. pt/ ~pedros/ anim/ 2frame-ien. htm)
succinate dehydrogenase (http:/ / www2. ufp. pt/ ~pedros/ anim/ 2frame-iien. htm)
Coenzyme Q - cytochrome c reductase (http:/ / www2. ufp. pt/ ~pedros/ anim/ 2frame-iiien. htm)
cytochrome c oxidase (http:/ / www2. ufp. pt/ ~pedros/ anim/ 2frame-iven. htm)
245
Photosynthesis
Photosynthesis
Schematic of photosynthesis in plants. The
carbohydrates produced are stored in or used by
the plant.
Overall equation for the type of photosynthesis
that occurs in plants
Composite image showing the global distribution
of photosynthesis, including both oceanic
phytoplankton and terrestrial vegetation. Dark
blue and green indicate regions of high
photosynthetic activity in ocean and land
respectively.
Photosynthesis is a process used by plants and other autotrophic
organisms to convert light energy, normally from the sun, into
chemical energy that can be used to fuel the organisms' activities.
Carbohydrates, such as sugars, are synthesized from carbon dioxide
and water (hence the name photosynthesis, from the Greek -
[photo-], "light," and - [synthesis], "putting together") during
the process. Oxygen is also released, mostly as a waste product. Most
plants, most algae, and cyanobacteria perform the process of
photosynthesis, and are called photoautotrophs. Photosynthesis
maintains atmospheric oxygen levels and supplies most of the energy
necessary for all life on Earth,
[]
except for chemotrophs, which gain
energy through oxidative chemical reactions.
Although photosynthesis is performed differently by different species,
the process always begins when energy from light is absorbed by
proteins called reaction centres that contain green chlorophyll
pigments. In plants, these proteins are held inside organelles called
chloroplasts, which are most abundant in leaf cells, while in bacteria
they are embedded in the plasma membrane. In these light-dependent
reactions, some energy is used to strip electrons from suitable
substances such as water. This produces oxygen gas and hydrogen
ions, which are transferred to a compound called nicotinamide adenine
dinucleotide phosphate (NADP
+
), reducing it to NADPH. More light
energy is transferred to chemical energy in the generation of adenosine
triphosphate (ATP), the "energy currency" of cells.
In plants, algae and cyanobacteria, sugars are produced by a sequence
of light-independent reactions called the Calvin cycle, but some
bacteria use different mechanisms, such as the reverse Krebs cycle. In
the Calvin cycle, atmospheric carbon dioxide is incorporated into
already existing organic carbon compounds, such as ribulose
bisphosphate (RuBP).
[]
Using the ATP and NADPH produced by the
light-dependent reactions, the resulting compounds are then reduced
into triose phosphate. Of every six triose phosphate molecules
produced, one is removed to form further carbohydrates and five are
"recycled" back into the cycle to regenerate the original carbon dioxide
acceptor, RuBP.
Photosynthesis
246
The first photosynthetic organisms probably evolved early in the evolutionary history of life and most likely used
reducing agents such as hydrogen or hydrogen sulfide as sources of electrons, rather than water.
[]
Cyanobacteria
appeared later, and the excess oxygen they produced contributed to the oxygen catastrophe,
[]
which rendered the
evolution of complex life possible. Today, the average rate of energy capture by photosynthesis globally is
approximately 130terawatts,
[][][]
which is about six times larger than the current power consumption of human
civilization.
[]
Photosynthetic organisms also convert around 100115 thousand million metric tons (i.e.,
100115petagrams) of carbon into biomass per year.
[][]
Overview
Photosynthesis changes sunlight into chemical
energy, splits water to liberate O
2
, and fixes CO
2
into sugar.
Photosynthetic organisms are photoautotrophs, which means that they
are able to synthesize food directly from carbon dioxide and water
using energy from light. However, not all organisms that use light as a
source of energy carry out photosynthesis, since photoheterotrophs use
organic compounds, rather than carbon dioxide, as a source of carbon.
[]
In plants, algae and cyanobacteria, photosynthesis releases oxygen.
This is called oxygenic photosynthesis. Although there are some
differences between oxygenic photosynthesis in plants, algae, and
cyanobacteria, the overall process is quite similar in these organisms.
However, there are some types of bacteria that carry out anoxygenic
photosynthesis, which consumes carbon dioxide but does not release
oxygen.
Carbon dioxide is converted into sugars in a process called carbon
fixation. Carbon fixation is an endothermic redox reaction, so
photosynthesis needs to supply both a source of energy to drive this
process, and the electrons needed to convert carbon dioxide into a
carbohydrate. This addition of the electrons is a reduction reaction. In
general outline and in effect, photosynthesis is the opposite of cellular respiration, in which glucose and other
compounds are oxidized to produce carbon dioxide and water, and to release exothermic chemical energy to drive
the organism's metabolism. However, the two processes take place through a different sequence of chemical
reactions and in different cellular compartments.
The general equation for photosynthesis is therefore:
2n CO
2
+ 2n DH
2
+ photons 2(CH
2
O)
n
+ 2n DO
Carbon dioxide + electron donor + light energy carbohydrate + oxidized electron donor
In oxygenic photosynthesis water is the electron donor and, since its hydrolysis releases oxygen, the equation for this
process is:
2n CO
2
+ 4n H
2
O + photons 2(CH
2
O)
n
+ 2n O
2
+ 2n H
2
O
carbon dioxide + water + light energy carbohydrate + oxygen + water
Often 2n water molecules are cancelled on both sides, yielding:
2n CO
2
+ 2n H
2
O + photons 2(CH
2
O)
n
+ 2n O
2
carbon dioxide + water + light energy carbohydrate + oxygen
Other processes substitute other compounds (such as arsenite) for water in the electron-supply role; for example
some microbes use sunlight to oxidize arsenite to arsenate:
[1]
The equation for this reaction is:
CO
2
+ (AsO
3
3
) + photons (AsO
4
3
) + CO
[]
Photosynthesis
247
carbon dioxide + arsenite + light energy arsenate + carbon monoxide (used to build other compounds in
subsequent reactions)
Photosynthesis occurs in two stages. In the first stage, light-dependent reactions or light reactions capture the energy
of light and use it to make the energy-storage molecules ATP and NADPH. During the second stage, the
light-independent reactions use these products to capture and reduce carbon dioxide.
Most organisms that utilize photosynthesis to produce oxygen use visible light to do so, although at least three use
shortwave infrared or, more specifically, far-red radiation.
[2]
Photosynthetic membranes and organelles
Chloroplast ultrastructure: 1. outer membrane 2. intermembrane space3.
inner membrane (1+2+3: envelope) 4. stroma (aqueous fluid) 5. thylakoid
lumen (inside of thylakoid) 6. thylakoid membrane 7. granum (stack of
thylakoids) 8. thylakoid (lamella) 9. starch 10. ribosome 11. plastidial DNA
12. plastoglobule (drop of lipids)
In photosynthetic bacteria, the proteins that gather
light for photosynthesis are embedded within cell
membranes, which is the simplest configuration
these proteins are arranged.
[]
However, this
membrane may be tightly folded into cylindrical
sheets called thylakoids,
[]
or bunched up into
round vesicles called intracytoplasmic
membranes.
[]
These structures can fill most of the
interior of a cell, giving the membrane a very
large surface area and therefore increasing the
amount of light that the bacteria can absorb.
[]
In plants and algae, photosynthesis takes place in
organelles called chloroplasts. A typical plant cell
contains about 10 to 100 chloroplasts. The
chloroplast is enclosed by a membrane. This membrane is composed of a phospholipid inner membrane, a
phospholipid outer membrane, and an intermembrane space between them. Within the membrane is an aqueous fluid
called the stroma. The stroma contains stacks (grana) of thylakoids, which are the site of photosynthesis. The
thylakoids are flattened disks, bounded by a membrane with a lumen or thylakoid space within it. The site of
photosynthesis is the thylakoid membrane, which contains integral and peripheral membrane protein complexes,
including the pigments that absorb light energy, which form the photosystems.
Plants absorb light primarily using the pigment chlorophyll, which is the reason that most plants have a green color.
Besides chlorophyll, plants also use pigments such as carotenes and xanthophylls.
[]
Algae also use chlorophyll, but
various other pigments are present as phycocyanin, carotenes, and xanthophylls in green algae, phycoerythrin in red
algae (rhodophytes) and fucoxanthin in brown algae and diatoms resulting in a wide variety of colors.
These pigments are embedded in plants and algae in special antenna-proteins. In such proteins all the pigments are
ordered to work well together. Such a protein is also called a light-harvesting complex.
Although all cells in the green parts of a plant have chloroplasts, most of the energy is captured in the leaves, except
in certain species adapted to conditions of strong sunlight and aridity, such as many Euphorbia and Cactus species,
whose main photosynthetic organs are their stems. The cells in the interior tissues of a leaf, called the mesophyll, can
contain between 450,000 and 800,000 chloroplasts for every square millimeter of leaf. The surface of the leaf is
uniformly coated with a water-resistant waxy cuticle that protects the leaf from excessive evaporation of water and
decreases the absorption of ultraviolet or blue light to reduce heating. The transparent epidermis layer allows light to
pass through to the palisade mesophyll cells where most of the photosynthesis takes place.
Photosynthesis
248
Light reactions
Light-dependent reactions of photosynthesis at the thylakoid membrane
In the light reactions, one molecule of
the pigment chlorophyll absorbs one
photon and loses one electron. This
electron is passed to a modified form
of chlorophyll called pheophytin,
which passes the electron to a quinone
molecule, allowing the start of a flow
of electrons down an electron transport
chain that leads to the ultimate
reduction of NADP to NADPH. In
addition, this creates a proton gradient
across the chloroplast membrane; its
dissipation is used by ATP synthase
for the concomitant synthesis of ATP.
The chlorophyll molecule regains the lost electron from a water molecule through a process called photolysis, which
releases a dioxygen (O
2
) molecule. The overall equation for the light-dependent reactions under the conditions of
non-cyclic electron flow in green plants is:
[]
2 H
2
O + 2 NADP
+
+ 3 ADP + 3 P
i
+ light 2 NADPH + 2 H
+
+ 3 ATP + O
2
Not all wavelengths of light can support photosynthesis. The photosynthetic action spectrum depends on the type of
accessory pigments present. For example, in green plants, the action spectrum resembles the absorption spectrum for
chlorophylls and carotenoids with peaks for violet-blue and red light. In red algae, the action spectrum overlaps with
the absorption spectrum of phycobilins for red blue-green light, which allows these algae to grow in deeper waters
that filter out the longer wavelengths used by green plants. The non-absorbed part of the light spectrum is what gives
photosynthetic organisms their color (e.g., green plants, red algae, purple bacteria) and is the least effective for
photosynthesis in the respective organisms.
Z scheme
The "Z scheme"
In plants, light-dependent reactions occur in the thylakoid membranes of the chloroplasts and use light energy to
synthesize ATP and NADPH. The light-dependent reaction has two forms: cyclic and non-cyclic. In the non-cyclic
reaction, the photons are captured in the light-harvesting antenna complexes of photosystem II by chlorophyll and
other accessory pigments (see diagram at right). When a chlorophyll molecule at the core of the photosystem II
reaction center obtains sufficient excitation energy from the adjacent antenna pigments, an electron is transferred to
Photosynthesis
249
the primary electron-acceptor molecule, pheophytin, through a process called photoinduced charge separation. These
electrons are shuttled through an electron transport chain, the so-called Z-scheme shown in the diagram, that initially
functions to generate a chemiosmotic potential across the membrane. An ATP synthase enzyme uses the
chemiosmotic potential to make ATP during photophosphorylation, whereas NADPH is a product of the terminal
redox reaction in the Z-scheme. The electron enters a chlorophyll molecule in Photosystem I. The electron is excited
due to the light absorbed by the photosystem. A second electron carrier accepts the electron, which again is passed
down lowering energies of electron acceptors. The energy created by the electron acceptors is used to move
hydrogen ions across the thylakoid membrane into the lumen. The electron is used to reduce the co-enzyme NADP,
which has functions in the light-independent reaction. The cyclic reaction is similar to that of the non-cyclic, but
differs in the form that it generates only ATP, and no reduced NADP (NADPH) is created. The cyclic reaction takes
place only at photosystem I. Once the electron is displaced from the photosystem, the electron is passed down the
electron acceptor molecules and returns to photosystem I, from where it was emitted, hence the name cyclic reaction.
Water photolysis
The NADPH is the main reducing agent in chloroplasts, providing a source of energetic electrons to other reactions.
Its production leaves chlorophyll with a deficit of electrons (oxidized), which must be obtained from some other
reducing agent. The excited electrons lost from chlorophyll in photosystem I are replaced from the electron transport
chain by plastocyanin. However, since photosystem II includes the first steps of the Z-scheme, an external source of
electrons is required to reduce its oxidized chlorophyll a molecules. The source of electrons in green-plant and
cyanobacterial photosynthesis is water. Two water molecules are oxidized by four successive charge-separation
reactions by photosystem II to yield a molecule of diatomic oxygen and four hydrogen ions; the electron yielded in
each step is transferred to a redox-active tyrosine residue that then reduces the photoxidized paired-chlorophyll a
species called P680 that serves as the primary (light-driven) electron donor in the photosystem II reaction center. The
oxidation of water is catalyzed in photosystem II by a redox-active structure that contains four manganese ions and a
calcium ion; this oxygen-evolving complex binds two water molecules and stores the four oxidizing equivalents that
are required to drive the water-oxidizing reaction. Photosystem II is the only known biological enzyme that carries
out this oxidation of water. The hydrogen ions contribute to the transmembrane chemiosmotic potential that leads to
ATP synthesis. Oxygen is a waste product of light-dependent reactions, but the majority of organisms on Earth use
oxygen for cellular respiration, including photosynthetic organisms.
[][]
Photosynthesis
250
Light-independent reactions
Calvin cycle
In the light-independent (or "dark") reactions, the enzyme RuBisCO captures CO
2
from the atmosphere and in a
process that requires the newly formed NADPH, called the Calvin-Benson Cycle, releases three-carbon sugars,
which are later combined to form sucrose and starch. The overall equation for the light-independent reactions in
green plants is:
[]:128
3 CO
2
+ 9 ATP + 6 NADPH + 6 H
+
C
3
H
6
O
3
-phosphate + 9 ADP + 8 P
i
+ 6 NADP
+
+ 3 H
2
O
Overview of the Calvin cycle and carbon fixation
To be more specific, carbon fixation
produces an intermediate product,
which is then converted to the final
carbohydrate products. The carbon
skeletons produced by photosynthesis
are then variously used to form other
organic compounds, such as the
building material cellulose, as
precursors for lipid and amino acid
biosynthesis, or as a fuel in cellular
respiration. The latter occurs not only
in plants but also in animals when the
energy from plants gets passed through
a food chain.
The fixation or reduction of carbon
dioxide is a process in which carbon
dioxide combines with a five-carbon
sugar, ribulose 1,5-bisphosphate
(RuBP), to yield two molecules of a
three-carbon compound, glycerate
3-phosphate (GP), also known as 3-phosphoglycerate (PGA). GP, in the presence of ATP and NADPH from the
light-dependent stages, is reduced to glyceraldehyde 3-phosphate (G3P). This product is also referred to as
3-phosphoglyceraldehyde (PGAL) or even as triose phosphate. Triose is a 3-carbon sugar (see carbohydrates). Most
(5 out of 6 molecules) of the G3P produced is used to regenerate RuBP so the process can continue (see
Calvin-Benson cycle). The 1 out of 6 molecules of the triose phosphates not "recycled" often condense to form
hexose phosphates, which ultimately yield sucrose, starch and cellulose. The sugars produced during carbon
metabolism yield carbon skeletons that can be used for other metabolic reactions like the production of amino acids
and lipids.
Photosynthesis
251
Carbon concentrating mechanisms
On land
Overview of C4 carbon fixation
In hot and dry conditions, plants close their stomata
to prevent the loss of water. Under these conditions,
CO
2
will decrease, and oxygen gas, produced by
the light reactions of photosynthesis, will decrease
in the stem, not leaves, causing an increase of
photorespiration by the oxygenase activity of
ribulose-1,5-bisphosphate carboxylase/oxygenase
and decrease in carbon fixation. Some plants have
evolved mechanisms to increase the CO
2
concentration in the leaves under these conditions.
C
4
plants chemically fix carbon dioxide in the cells
of the mesophyll by adding it to the three-carbon
molecule phosphoenolpyruvate (PEP), a reaction
catalyzed by an enzyme called PEP carboxylase,
creating the four-carbon organic acid oxaloacetic
acid. Oxaloacetic acid or malate synthesized by this
process is then translocated to specialized bundle
sheath cells where the enzyme RuBisCO and other
Calvin cycle enzymes are located, and where CO
2
released by decarboxylation of the four-carbon
acids is then fixed by RuBisCO activity to the
three-carbon sugar 3-phosphoglyceric acids. The
physical separation of RuBisCO from the
oxygen-generating light reactions reduces
photorespiration and increases CO
2
fixation and, thus, photosynthetic capacity of the leaf.
[]
C
4
plants can produce
more sugar than C
3
plants in conditions of high light and temperature. Many important crop plants are C
4
plants,
including maize, sorghum, sugarcane, and millet. Plants that do not use PEP-carboxylase in carbon fixation are
called C
3
plants because the primary carboxylation reaction, catalyzed by RuBisCO, produces the three-carbon sugar
3-phosphoglyceric acids directly in the Calvin-Benson cycle. Over 90% of plants use C
3
carbon fixation, compared
to 3% that use C
4
carbon fixation.
[]
Xerophytes, such as cacti and most succulents, also use PEP carboxylase to capture carbon dioxide in a process
called Crassulacean acid metabolism (CAM). In contrast to C
4
metabolism, which physically separates the CO
2
fixation to PEP from the Calvin cycle, CAM temporally separates these two processes. CAM plants have a different
leaf anatomy from C
3
plants, and fix the CO
2
at night, when their stomata are open. CAM plants store the CO
2
mostly in the form of malic acid via carboxylation of phosphoenolpyruvate to oxaloacetate, which is then reduced to
malate. Decarboxylation of malate during the day releases CO
2
inside the leaves, thus allowing carbon fixation to
3-phosphoglycerate by RuBisCO. Sixteen thousand species of plants use CAM.
[]
Photosynthesis
252
In water
Cyanobacteria possess carboxysomes, which increase the concentration of CO
2
around RuBisCO to increase the rate
of photosynthesis. An enzyme, carbonic anhydrase, located within the carboxysome releases CO
2
from the dissolved
hydrocarbonate ions (HCO
3

). Before the CO
2
diffuses out it is quickly sponged up by RuBisCO, which is
concentrated within the carboxysomes. HCO
3

ions are made from CO
2
outside the cell by another carbonic
anhydrase and are actively pumped into the cell by a membrane protein. They cannot cross the membrane as they are
charged, and within the cytosol they turn back into CO
2
very slowly without the help of carbonic anhydrase. This
causes the HCO
3

ions to accumulate within the cell from where they diffuse into the carboxysomes.
[3]
Pyrenoids in
algae and hornworts also act to concentrate CO
2
around rubisco.
[4]
Order and kinetics
The overall process of photosynthesis takes place in four stages:
[]
Stage Description Time scale
1 Energy transfer in antenna chlorophyll (thylakoid membranes) femtosecond to picosecond
2 Transfer of electrons in photochemical reactions (thylakoid membranes) picosecond to nanosecond
3 Electron transport chain and ATP synthesis (thylakoid membranes) microsecond to millisecond
4 Carbon fixation and export of stable products millisecond to second
Efficiency
Measuring the photosynthetic efficiency of wheat
in the field using an LCpro-SD
Plants usually convert light into chemical energy with a photosynthetic
efficiency of 36%.
[5]
Actual plants' photosynthetic efficiency varies
with the frequency of the light being converted, light intensity,
temperature and proportion of carbon dioxide in the atmosphere, and
can vary from 0.1% to 8%.
[6]
By comparison, solar panels convert light
into electric energy at an efficiency of approximately 620% for
mass-produced panels, and above 40% in laboratory devices.
Photosynthesis measurement systems are not designed to directly
measure the amount of light absorbed by the leaf. Nevertheless, the
light response curves that systems like the LCpro-SD produce, do
allow comparisons in photosynthetic efficiency between plants.
Photosynthesis
253
Evolution
Plant cells with visible chloroplasts (from a moss, Plagiomnium affine)
Early photosynthetic systems, such as those
from green and purple sulfur and green and
purple nonsulfur bacteria, are thought to
have been anoxygenic, using various
molecules as electron donors. Green and
purple sulfur bacteria are thought to have
used hydrogen and sulfur as an electron
donor. Green nonsulfur bacteria used
various amino and other organic acids.
Purple nonsulfur bacteria used a variety of
nonspecific organic molecules. The use of
these molecules is consistent with the
geological evidence that the atmosphere was
highly reduced at that time.
[citation needed]
Fossils of what are thought to be
filamentous photosynthetic organisms have
been dated at 3.4 billion years old.
[7][8]
The main source of oxygen in the atmosphere is oxygenic photosynthesis, and its first appearance is sometimes
referred to as the oxygen catastrophe. Geological evidence suggests that oxygenic photosynthesis, such as that in
cyanobacteria, became important during the Paleoproterozoic era around 2 billion years ago. Modern photosynthesis
in plants and most photosynthetic prokaryotes is oxygenic. Oxygenic photosynthesis uses water as an electron donor,
which is oxidized to molecular oxygen (O
2
) in the photosynthetic reaction center.
Symbiosis and the origin of chloroplasts
Several groups of animals have formed symbiotic relationships with photosynthetic algae. These are most common
in corals, sponges and sea anemones. It is presumed that this is due to the particularly simple body plans and large
surface areas of these animals compared to their volumes.
[9]
In addition, a few marine mollusks Elysia viridis and
Elysia chlorotica also maintain a symbiotic relationship with chloroplasts they capture from the algae in their diet
and then store in their bodies. This allows the mollusks to survive solely by photosynthesis for several months at a
time.
[][]
Some of the genes from the plant cell nucleus have even been transferred to the slugs, so that the
chloroplasts can be supplied with proteins that they need to survive.
[]
An even closer form of symbiosis may explain the origin of chloroplasts. Chloroplasts have many similarities with
photosynthetic bacteria, including a circular chromosome, prokaryotic-type ribosomes, and similar proteins in the
photosynthetic reaction center.
[][]
The endosymbiotic theory suggests that photosynthetic bacteria were acquired (by
endocytosis) by early eukaryotic cells to form the first plant cells. Therefore, chloroplasts may be photosynthetic
bacteria that adapted to life inside plant cells. Like mitochondria, chloroplasts still possess their own DNA, separate
from the nuclear DNA of their plant host cells and the genes in this chloroplast DNA resemble those in
cyanobacteria.
[]
DNA in chloroplasts codes for redox proteins such as photosynthetic reaction centers. The CoRR
Hypothesis proposes that this Co-location is required for Redox Regulation.
Photosynthesis
254
Cyanobacteria and the evolution of photosynthesis
The biochemical capacity to use water as the source for electrons in photosynthesis evolved once, in a common
ancestor of extant cyanobacteria. The geological record indicates that this transforming event took place early in
Earth's history, at least 24502320 million years ago (Ma), and, it is speculated, much earlier.
[10]
Available evidence
from geobiological studies of Archean (>2500 Ma) sedimentary rocks indicates that life existed 3500 Ma, but the
question of when oxygenic photosynthesis evolved is still unanswered. A clear paleontological window on
cyanobacterial evolution opened about 2000 Ma, revealing an already-diverse biota of blue-greens. Cyanobacteria
remained principal primary producers throughout the Proterozoic Eon (2500543 Ma), in part because the redox
structure of the oceans favored photoautotrophs capable of nitrogen fixation.
[citation needed]
Green algae joined
blue-greens as major primary producers on continental shelves near the end of the Proterozoic, but only with the
Mesozoic (25165 Ma) radiations of dinoflagellates, coccolithophorids, and diatoms did primary production in
marine shelf waters take modern form. Cyanobacteria remain critical to marine ecosystems as primary producers in
oceanic gyres, as agents of biological nitrogen fixation, and, in modified form, as the plastids of marine algae.
[]
A 2010 study by researchers at Tel Aviv University discovered that the Oriental hornet (Vespa orientalis) converts
sunlight into electric power using a pigment called xanthopterin. This is the first scientific evidence of a member of
the animal kingdom engaging in photosynthesis.
[11]
Discovery
Although some of the steps in photosynthesis are still not completely understood, the overall photosynthetic equation
has been known since the 19th century.
Jan van Helmont began the research of the process in the mid-17th century when he carefully measured the mass of
the soil used by a plant and the mass of the plant as it grew. After noticing that the soil mass changed very little, he
hypothesized that the mass of the growing plant must come from the water, the only substance he added to the potted
plant. His hypothesis was partially accurate much of the gained mass also comes from carbon dioxide as well as
water. However, this was a signaling point to the idea that the bulk of a plant's biomass comes from the inputs of
photosynthesis, not the soil itself.
Joseph Priestley, a chemist and minister, discovered that, when he isolated a volume of air under an inverted jar, and
burned a candle in it, the candle would burn out very quickly, much before it ran out of wax. He further discovered
that a mouse could similarly "injure" air. He then showed that the air that had been "injured" by the candle and the
mouse could be restored by a plant.
In 1778, Jan Ingenhousz, court physician to the Austrian Empress, repeated Priestley's experiments. He discovered
that it was the influence of sunlight on the plant that could cause it to revive a mouse in a matter of hours.
In 1796, Jean Senebier, a Swiss pastor, botanist, and naturalist, demonstrated that green plants consume carbon
dioxide and release oxygen under the influence of light. Soon afterward, Nicolas-Thodore de Saussure showed that
the increase in mass of the plant as it grows could not be due only to uptake of CO
2
but also to the incorporation of
water. Thus, the basic reaction by which photosynthesis is used to produce food (such as glucose) was outlined.
Cornelis Van Niel made key discoveries explaining the chemistry of photosynthesis. By studying purple sulfur
bacteria and green bacteria he was the first scientist to demonstrate that photosynthesis is a light-dependent redox
reaction, in which hydrogen reduces carbon dioxide.
Robert Emerson discovered two light reactions by testing plant productivity using different wavelengths of light.
With the red alone, the light reactions were suppressed. When blue and red were combined, the output was much
more substantial. Thus, there were two photosystems, one absorbing up to 600nm wavelengths, the other up to
700nm. The former is known as PSII, the latter is PSI. PSI contains only chlorophyll a, PSII contains primarily
chlorophyll a with most of the available chlorophyll b, among other pigment. These include phycobilins, which are
the red and blue pigments of red and blue algae respectively, and fucoxanthol for brown algae and diatoms. The
Photosynthesis
255
process is most productive when absorption of quanta are equal in both the PSII and PSI, assuring that input energy
from the antenna complex is divided between the PSI and PSII system, which in turn powers the photochemistry.
[]
Melvin Calvin works in his photosynthesis
laboratory.
Robert Hill thought that a complex of reactions consisting of an
intermediate to cytochrome b
6
(now a plastoquinone), another is from
cytochrome f to a step in the carbohydrate-generating mechanisms. These
are linked by plastoquinone, which does require energy to reduce
cytochrome f for it is a sufficient reductant. Further experiments to prove
that the oxygen developed during the photosynthesis of green plants
came from water, were performed by Hill in 1937 and 1939. He showed
that isolated chloroplasts give off oxygen in the presence of unnatural
reducing agents like iron oxalate, ferricyanide or benzoquinone after
exposure to light. The Hill reaction is as follows:
2 H
2
O + 2 A + (light, chloroplasts) 2 AH
2
+ O
2
where A is the electron acceptor. Therefore, in light, the electron acceptor
is reduced and oxygen is evolved.
Samuel Ruben and Martin Kamen used radioactive isotopes to determine
that the oxygen liberated in photosynthesis came from the water.
Melvin Calvin and Andrew Benson, along with James Bassham, elucidated the path of carbon assimilation (the
photosynthetic carbon reduction cycle) in plants. The carbon reduction cycle is known as the Calvin cycle, which
ignores the contribution of Bassham and Benson. Many scientists refer to the cycle as the Calvin-Benson Cycle,
Benson-Calvin, and some even call it the Calvin-Benson-Bassham (or CBB) Cycle.
Nobel Prize-winning scientist Rudolph A. Marcus was able to discover the function and significance of the electron
transport chain.
Otto Heinrich Warburg and Dean Burk discovered the I-quantum photosynthesis reaction that splits the CO
2
,
activated by the respiration.
[12]
Louis N.M. Duysens and Jan Amesz discovered that chlorophyll a will absorb one light, oxidize cytochrome f,
chlorophyll a (and other pigments) will absorb another light, but will reduce this same oxidized cytochrome, stating
the two light reactions are in series.
Factors
The leaf is the primary site of photosynthesis in
plants.
There are three main factors affecting photosynthesis and several
corollary factors. The three main are:
Light irradiance and wavelength
Carbon dioxide concentration
Temperature.
Light intensity (irradiance), wavelength and
temperature
In the early 20th century, Frederick Blackman and Gabrielle Matthaei
investigated the effects of light intensity (irradiance) and temperature
on the rate of carbon assimilation.
Photosynthesis
256
At constant temperature, the rate of carbon assimilation varies with irradiance, initially increasing as the
irradiance increases. However, at higher irradiance, this relationship no longer holds and the rate of carbon
assimilation reaches a plateau.
At constant irradiance, the rate of carbon assimilation increases as the temperature is increased over a limited
range. This effect is seen only at high irradiance levels. At low irradiance, increasing the temperature has little
influence on the rate of carbon assimilation.
These two experiments illustrate vital points: First, from research it is known that, in general, photochemical
reactions are not affected by temperature. However, these experiments clearly show that temperature affects the rate
of carbon assimilation, so there must be two sets of reactions in the full process of carbon assimilation. These are, of
course, the light-dependent 'photochemical' stage and the light-independent, temperature-dependent stage. Second,
Blackman's experiments illustrate the concept of limiting factors. Another limiting factor is the wavelength of light.
Cyanobacteria, which reside several meters underwater, cannot receive the correct wavelengths required to cause
photoinduced charge separation in conventional photosynthetic pigments. To combat this problem, a series of
proteins with different pigments surround the reaction center. This unit is called a phycobilisome.
Carbon dioxide levels and photorespiration
Photorespiration
As carbon dioxide concentrations rise,
the rate at which sugars are made by
the light-independent reactions
increases until limited by other factors.
RuBisCO, the enzyme that captures
carbon dioxide in the light-independent
reactions, has a binding affinity for
both carbon dioxide and oxygen. When
the concentration of carbon dioxide is
high, RuBisCO will fix carbon dioxide.
However, if the carbon dioxide
concentration is low, RuBisCO will
bind oxygen instead of carbon dioxide. This process, called photorespiration, uses energy, but does not produce
sugars.
RuBisCO oxygenase activity is disadvantageous to plants for several reasons:
1. One product of oxygenase activity is phosphoglycolate (2 carbon) instead of 3-phosphoglycerate (3 carbon).
Phosphoglycolate cannot be metabolized by the Calvin-Benson cycle and represents carbon lost from the cycle. A
high oxygenase activity, therefore, drains the sugars that are required to recycle ribulose 5-bisphosphate and for
the continuation of the Calvin-Benson cycle.
2. 2. Phosphoglycolate is quickly metabolized to glycolate that is toxic to a plant at a high concentration; it inhibits
photosynthesis.
3. Salvaging glycolate is an energetically expensive process that uses the glycolate pathway, and only 75% of the
carbon is returned to the Calvin-Benson cycle as 3-phosphoglycerate. The reactions also produce ammonia (NH
3
),
which is able to diffuse out of the plant, leading to a loss of nitrogen.
A highly simplified summary is:
2 glycolate + ATP 3-phosphoglycerate + carbon dioxide + ADP + NH
3
The salvaging pathway for the products of RuBisCO oxygenase activity is more commonly known as
photorespiration, since it is characterized by light-dependent oxygen consumption and the release of carbon dioxide.
Photosynthesis
257
References
[1] Anaerobic Photosynthesis, Chemical & Engineering News, 86, 33, August 18, 2008, p. 36
[5] Chapter 1 Biological energy production">
[6] Govindjee, What is photosynthesis? (http:/ / www. life.uiuc. edu/ govindjee/ whatisit. htm)
[7] Photosynthesis got a really early start (http:/ / www. newscientist. com/ article/ mg18424671. 600-photosynthesis-got-a-really-early-start.
html), New Scientist, 2 October 2004
[8] Revealing the dawn of photosynthesis (http:/ / www. newscientist. com/ article/ mg19125654. 200-revealing-the-dawn-of-photosynthesis.
html), New Scientist, 19 August 2006
[12] Otto Warburg Biography (http:/ / nobelprize. org/ nobel_prizes/ medicine/ laureates/ 1931/ warburg. html). Nobelprize.org (1970-08-01).
Retrieved on 2011-11-03.
Further reading
Books
Asimov, Isaac (1968). Photosynthesis. New York, London: Basic Books, Inc. ISBN0-465-05703-9.
Bidlack JE; Stern KR, Jansky S (2003). Introductory plant biology. New York: McGraw-Hill.
ISBN0-07-290941-2.
Blankenship RE (2008). Molecular Mechanisms of Photosynthesis (2nd ed.). John Wiley & Sons Inc.
ISBN0-470-71451-4.
Govindjee (1975). Bioenergetics of photosynthesis. Boston: Academic Press. ISBN0-12-294350-3.
Govindjee Beatty JT,Gest H, Allen JF (2006). Discoveries in Photosynthesis. Advances in Photosynthesis and
Respiration 20. Berlin: Springer. ISBN1-4020-3323-0.
Gregory RL (1971). Biochemistry of photosynthesis. New York: Wiley-Interscience. ISBN0-471-32675-5.
Rabinowitch E, Govindjee (1969). Photosynthesis. London: J. Wiley. ISBN0-471-70424-5.
Reece, J, Campbell, N (2005). Biology. San Francisco: Pearson, Benjamin Cummings. ISBN0-8053-7146-X.
Papers
Gupta RS, Mukhtar T, Singh B (June 1999). "Evolutionary relationships among photosynthetic prokaryotes
(Heliobacterium chlorum, Chloroflexus aurantiacus, cyanobacteria, Chlorobium tepidum and proteobacteria):
implications regarding the origin of photosynthesis". Mol. Microbiol. 32 (5): 893906. PMID 10361294 (http:/ /
www. ncbi. nlm. nih. gov/ pubmed/ 10361294). "implications regarding the origin of photosynthesis"
Blankenship RE (1992). "Origin and early evolution of photosynthesis". Photosyn. Res. 33: 91111. PMID
11538390 (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 11538390).
Rutherford AW, Faller P (January 2003). "Photosystem II: evolutionary perspectives" (http:/ / www. ncbi. nlm.
nih. gov/ pmc/ articles/ PMC1693113). Philos. Trans. R. Soc. Lond., B, Biol. Sci. 358 (1429): 24553. doi:
10.1098/rstb.2002.1186 (http:/ / dx. doi. org/ 10. 1098/ rstb. 2002. 1186). PMC 1693113 (http:/ / www. ncbi. nlm.
nih. gov/ pmc/ articles/ PMC1693113). PMID 12594932 (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 12594932).
Photosynthesis
258
External links
A collection of photosynthesis pages for all levels from a renowned expert (Govindjee) (http:/ / www. life. uiuc.
edu/ govindjee/ linksPSed. htm)
In depth, advanced treatment of photosynthesis, also from Govindjee (http:/ / www. life. uiuc. edu/ govindjee/
paper/ gov. html)
Science Aid: Photosynthesis (http:/ / scienceaid. co. uk/ biology/ biochemistry/ photosynthesis. html) Article
appropriate for high school science
Metabolism, Cellular Respiration and Photosynthesis The Virtual Library of Biochemistry and Cell Biology
(http:/ / www. biochemweb. org/ metabolism. shtml)
Overall examination of Photosynthesis at an intermediate level (http:/ / www. chemsoc. org/ networks/ learnnet/
cfb/ Photosynthesis. htm)
Overall Energetics of Photosynthesis (http:/ / www. life. uiuc. edu/ govindjee/ photosynBook. html)
Photosynthesis Discovery Milestones (http:/ / www. juliantrubin. com/ bigten/ photosynthesisexperiments. html)
experiments and background
The source of oxygen produced by photosynthesis (http:/ / bcs. whfreeman. com/ thelifewire/ content/ chp08/
0802001. html) Interactive animation, a textbook tutorial
Jessica Marshall (2011-03-29). "First practical artificial leaf makes debut" (http:/ / news. discovery. com/ earth/
artificial-leaf-technology-solar-110329. html). Discovery News.
Photosynthesis Light Dependent & Light Independent Stages (http:/ / www. biology-innovation. co. uk/ pages/
plant-biology-ecology/ photosynthesis/ )
Khan Academy, video introduction (http:/ / www. khanacademy. org/ video/ photosynthesis?playlist=Biology)
259
Lipid metabolism
Fatty acid synthesis
Fatty acid synthesis is the creation of fatty acids from acetyl-CoA and malonyl-CoA precursors through action of
enzymes called fatty acid synthases. It is an important part of the lipogenesis process, which - together with
glycolysis - stands behind creating fats from blood sugar in living organisms.
Straight-Chain Fatty Acids
Straight-chain fatty acids occur in two types; saturated and unsaturated.
Saturated Straight-Chain Fatty Acids
Synthesis of saturated fatty acids via Fatty Acid
Synthase II in E. coli
Much like -oxidation, straight-chain fatty acid synthesis occurs via
the six recurring reactions shown below, until the 16-carbon palmitic
acid is produced.
[1]
The diagrams presented show how fatty acids are synthesized in
microorganisms and list the enzymes found in Escherichia coli.
[1]
These reactions are performed by fatty acid synthase II (FASII), which
in general contain multiple enzymes that act as one complex. FASII is
present in prokaryotes, plants, fungi, and parasites, as well as in
mitochondria.
[2]
In animals, as well as yeast and some fungi, these same reactions occur
on fatty acid synthase I (FASI), a large dimeric protein that has all of
the enzymatic activities required to create a fatty acid. FASI is less
efficient than FASII; however, it allows for the formation of more molecules, including medium-chain fatty acids
via early chain termination.
[2]
Once a 16:0 carbon fatty acid has been formed, it can undergo a number of modifications, in particular by fatty acid
synthase III (FASIII), which uses 2 carbon molecules to elongate preformed fatty acids.
[2]
Step Enzyme Reaction Description
(a) Acetyl CoA:ACP transacylase Activates acetyl CoA for reaction with malonyl-ACP
(b) Malonyl CoA:ACP
transacylase
Activates malonyl CoA for reaction with acetyl-ACP
Fatty acid synthesis
260
(c) 3-ketoacyl-ACP synthetase Reacts priming acetyl-ACP with chain-extending
malonyl-ACP.
(d) 3-ketoacyl-ACP reductase Reduces the carbon 3 ketone to a hydroxyl group
(e) 3-Hydroxyacyl ACP dehydrase Removes water
(f) Enoyl-ACP reductase Reduces the C3-C4 double bond.
Abbreviations: ACP - Acyl carrier protein, CoA - Coenzyme A, NADP - Nicotinamide adenine dinucleotide
phosphate.
Regulation
Acetyl-CoA is formed into malonyl-CoA by acetyl-CoA carboxylase, at which point malonyl-CoA is destined to
feed into the fatty acid synthesis pathway. Acetyl-CoA carboxylase is the point of regulation in saturated
straight-chain fatty acid synthesis, and is subject to both phosphorylation and allosteric regulation. Regulation by
phosphorylation occurs mostly in mammals, while allosteric regulation occurs in most organisms. Allosteric control
occurs as feedback inhibition by palmitoyl-CoA and activation by citrate. When there are high levels of
palmitoyl-CoA, the final product of saturated fatty acid synthesis, it allosterically inactivates acetyl-CoA carboxylase
to prevent a build-up of fatty acids in cells. Citrate acts to activate acetyl-CoA carboxylase under high levels,
because high levels indicate that there is enough acetyl-CoA to feed into the Krebs cycle and produce energy.
[3]
De Novo Synthesis in Humans
In humans, fatty acids are formed predominantly in the liver and lactating mammary glands, and, to a lesser extent,
the adipose tissue. Most acetyl-CoA is formed from pyruvate by pyruvate dehydrogenase in the mitochondria.
Acetyl-CoA produced in the mitochondria is condensed with oxaloacetate by citrate synthase to form citrate, which
is then transported into the cytosol and broken down to yield acetyl-CoA and oxaloacetate by ATP citrate lyase.
Oxaloacetate in the cytosol is reduced to malate by cytoplasmic malate dehydrogenase, and malate is transported
back into the mitochondria to participate in the Citric acid cycle.
[4]
Desaturation
Desaturation of fatty acids involves a process that requires molecular oxygen (O
2
), NADH, and cytochrome b
5
. The
reaction, which occurs in the endoplasmic reticulum, results in the oxidation of both the fatty acid and NADH. The
most common desaturation reactions involve the placement of a double bond between carbons 9 and 10 (as in the
conversion of palmitic acid to palmitoleic acid and the conversion of stearic acid to oleic acid, facilitated by the
action of
9
-desaturase). Other positions that can be desaturated in humans include carbon 4, 5, and 6, via
4
-,
5
-,
and
6
-desaturases, respectively.
Fatty acid synthesis
261
Unsaturated fatty acids are essential components to prokaryotic and eukaryotic cell membranes. These fatty acids
function primarily in maintaining membrane fluidity.
[5]
They have also been associated with serving as signaling
molecules in other processes such as cell differentiation and DNA replication.
[5]
There are two pathways organisms
use for desaturation: Aerobic and Anaerobic.
Anaerobic Desaturation
Many bacteria use the anaerobic pathway for synthesizing unsaturated fatty acids. This pathway does not utilize
oxygen and is dependent on enzymes to insert the double bond before elongation utilizing the normal fatty acid
synthesis machinery. In Escherichia coli, this pathway is well understood.
Synthesis of unsaturated fatty acids via anaerobic
desaturation
FabA is a -hydroxydecanoyl-ACP dehydrase - it is specific for the
10-carbon saturated fatty acid synthesis intermediate
(-hydroxydecanoyl-ACP).
FabA catalyzes the dehydration of -hydroxydecanoyl-ACP,
causing the release of water and insertion of the double bond
between C7 and C8 counting from the methyl end. This creates the
trans-2-decenoyl intermediate.
Either the trans-2-decenoyl intermediate can be shunted to the
normal saturated fatty acid synthesis pathway by FabB, where the
double bond will be hydrolyzed and the final product will be a
saturated fatty acid, or FabA will catalyze the isomerization into the
cis-3-decenoyl intermediate.
FabB is a -ketoacyl-ACP synthase that elongates and channels
intermediates into the mainstream fatty acid synthesis pathway.
When FabB reacts with the cis-decenoyl intermediate, the final product after elongation will be an unsaturated
fatty acid.
[6]
The two main unsaturated fatty acids made are Palmitoleoyl-ACP (16:17) and cis-vaccenoyl-ACP (18:17).
[7]
Most bacteria that undergo anaerobic desaturation contain homologues of FabA and FabB.
[8]
Clostridia are the main
exception; they have a novel enzyme, yet to be identified, that catalyzes the formation of the cis double bond.
[7]
Regulation
This pathway undergoes transcriptional regulation by FadR and FabR. FadR is the more extensively studied protein
and has been attributed bifunctional characteristics. It acts as an activator of fabA and fabB transcription and as a
repressor for the -oxidation regulon. In contrast, FabR acts as a repressor for the transcription of fabA and fabB.
[6]
Fatty acid synthesis
262
Aerobic Desaturation
Synthesis of unsaturated fatty acids via aerobic
desaturation
Aerobic desaturation is the most widespread pathway for the synthesis
of unsaturated fatty acids. It is utilized in all eukaryotes and some
prokaryotes. This pathway utilizes desaturases to synthesize
unsaturated fatty acids from full-length saturated fatty acid
substrates.
[9]
All desaturases require oxygen and ultimately consume
NADH even though desaturation is an oxidative process. Desaturases
are specific for the double bond they induce in the substrate. In
Bacillus subtilis, the desaturase,
5
-Des, is specific for inducing a
cis-double bond at the
5
position.
[5][9]
Saccharomyces cerevisiae
contains one desaturase, Ole1p, which induces the cis-double bond at

9
.
[5]
Regulation
In B. subtilis, this pathway is regulated by a two-component system:
DesK and DesR. DesK is a membrane-associated kinase and DesR is a
transcriptional regulator of the des gene.
[5][9]
The regulation responds
to temperature; when there is a drop in temperature, this gene is upregulated. Unsaturated fatty acids increase the
fluidity of the membrane and stabilize it under lower temperatures. DesK is the sensor protein that, when there is a
decrease in temperature, will autophosphorylate. DesK-P will transfer its phosphoryl group to DesR. Two DesR-P
proteins will dimerize and bind to the DNA promoters of the des gene and recruit RNA polymerase to begin
transcription.
[5][9]
Pseudomonas aeruginosa
In general, both anaerobic and aerobic unsaturated fatty acid synthesis will not occur within the same system,
however Pseudomonas aeruginosa and Vibrio ABE-1 are exceptions.
[10][11][12]
While, P. aeruginosa undergoes
primarily anaerobic desaturation, it also undergoes two aerobic pathways. One pathway utilizes a
9
-desaturase
(DesA) that catalyzes a double bond formation in membrane lipids. Another pathway uses two proteins, DesC and
DesB, together to act as a
9
-desaturase, which inserts a double bond into a saturated fatty acid-CoA molecule. This
second pathway is regulated by repressor protein DesT. DesT is also a repressor of fabAB expression for anaerobic
desaturation when in presence of exogenous unsaturated fatty acids. This functions to coordinate the expression of
the two pathways within the organism.
[11][13]
Fatty acid synthesis
263
Branched-chain fatty acids
Branched-chain fatty acids are usually saturated and are found in two distinct families: the iso-series and
anteiso-series. It has been found that Actinomycetales contain unique branch-chain fatty acid synthesis mechanisms,
including that which forms tuberculosteric acid.
Branch-Chain Fatty Acid Synthesizing System
Valine primer
Leucine primer
Fatty acid synthesis
264
Isoleucine primer
Synthetic pathways of the branched-chain fatty acid synthesizing system given differing primers
The branched-chain fatty acid synthesizing system uses -keto acids as primers. This system is distinct from the
branched-chain fatty acid synthetase that utilizes short-chain acyl-CoA esters as primers.
[14]
-Keto acid primers are
derived from the transamination and decarboxylation of valine, leucine, and isoleucine to form
2-methylpropanyl-CoA, 3-methylbutyryl-CoA, and 2-Methylbutyryl-CoA, respectively.
[15]
2-Methylpropanyl-CoA
primers derived from valine are elongated to produce even-numbered iso-series fatty acids such as
14-methyl-pentadecanoic (isopalmitic) acid, and 3-methylbutyryl-CoA primers from leucine may be used to form
odd-numbered iso-series fatty acids such as 13-methyl-tetradecanoic acid. 2-Methylbutyryl-CoA primers from
isoleucine are elongated to form anteiso-series fatty acids containing an odd number of carbon atoms such as
12-Methyl tetradecanoic acid.
[16]
Decarboxylation of the primer precursors occurs through the branched-chain
-keto acid decarboxylase (BCKA) enzyme. Elongation of the fatty acid follows the same biosynthetic pathway in
Escherichia coli used to produce straight-chain fatty acids where malonyl-CoA is used as a chain extender.
[17]
The
major end products are 12-17 carbon branched-chain fatty acids and their composition tends to be uniform and
characteristic for many bacterial species.
[16]
BCKA decarboxylase and relative activities of -keto acid substrates
The BCKA decarboxylase enzyme is composed of two subunits in a tetrameric structure (A
2
B
2
) and is essential for
the synthesis of branched-chain fatty acids. It is responsible for the decarboxylation of -keto acids formed by the
transamination of valine, leucine, and isoleucine and produces the primers used for branched-chain fatty acid
synthesis. The activity of this enzyme is much higher with branched-chain -keto acid substrates than with
straight-chain substrates, and in Bacillus species its specificity is highest for the isoleucine-derived
-keto--methylvaleric acid, followed by -ketoisocaproate and -ketoisovalerate.
[16][17]
The enzymes high affinity
toward branched-chain -keto acids allows it to function as the primer donating system for branched-chain fatty acid
synthetase.
[17]
Fatty acid synthesis
265
Substrate BCKA activity CO2 Produced (nmol/min mg) Km (M) Vmax (nmol/min mg)
L
--keto--methyl-valerate 100% 19.7 <1 17.8
-Ketoisovalerate 63% 12.4 <1 13.3
-Ketoisocaproate 38% 7.4 <1 5.6
Pyruvate 25% 4.9 51.1 15.2
Factors affecting chain length and pattern distribution
-Keto acid primers are used to produce branched-chain fatty acids that, in general, are between 12 and 17 carbons
in length. The proportions of these branched-chain fatty acids tend to be uniform and consistent among a particular
bacterial species but may be altered due to changes in malonyl-CoA concentration, temperature, or heat-stable
factors (HSF) present.
[16]
All of these factors may affect chain length, and HSFs have been demonstrated to alter the
specificity of BCKA decarboxylase for a particular -keto acid substrate, thus shifting the ratio of branched-chain
fatty acids produced.
[16]
An increase in malonyl-CoA concentration has been shown to result in a larger proportion
of C17 fatty acids produced, up until the optimal concentration (20M) of malonyl-CoA is reached. Decreased
temperatures also tend to shift the fatty-acid distribution slightly toward C17 fatty-acids in Bacillus species.
[14][16]
Branch-Chain Fatty Acid Synthase
This system functions similarly to the branch-chain fatty acid synthesizing system, however it uses short-chain
carboxylic acids as primers instead of alpha-keto acids. In general, this method is used by bacteria that do not have
the ability to perform the branch-chain fatty acid system using alpha-keto primers. Typical short-chain primers
include isovalerate, isobutyrate, and 2-methyl butyrate. In general, the acids needed for these primers are taken up
from the environment; this is often seen in ruminal bacteria.
[18]
The overall reaction is:
Isobutyryl-CoA + 6 malonyl-CoA +12 NADPH + 12H
+
Isopalmitic acid + 6 CO
2
12 NADP + 5 H
2
O + 7
CoA
[14]
The difference between (straight-chain) fatty acid synthase and branch-chain fatty acid synthase is substrate
specificity of the enzyme that catalyzes the reaction of acyl-CoA to acyl-ACP.
[14]
Omega-alicyclic fatty acids
Omega-alicyclic fatty acids typically contain an omega-terminal propyl
or butyryl cyclic group and are some of the major membrane fatty
acids found in several species of bacteria. The fatty acid synthetase
used to produce omega-alicyclic fatty acids is also used to produce
membrane branched-chain fatty acids. In bacteria with membranes
composed mainly of omega-alicyclic fatty acids, the supply of cyclic
carboxylic acid-CoA esters is much greater than that of branched-chain primers.
[14]
The synthesis of cyclic primers
is not well understood but it has been suggested that mechanism involves the conversion of sugars to shikimic acid
which is then converted to cyclohexylcarboxylic acid-CoA esters that serve as primers for omega-alicyclic fatty acid
synthesis
[18]
Fatty acid synthesis
266
Tuberculostearic Acid Synthesis
Mechanism of the synthesis of Tuberculostearic
acid
Tuberculostearic acid (
D
-10-Methylstearic acid) is a saturated fatty
acid that is known to be produced by Mycobacterium spp. and two
species of Streptomyces. It is formed from the precursor oleic acid (a
monosaturated fatty acid).
[19]
After oleic acid is esterified to a
phospholipid, S-adenosyl-methionine donates a methyl group to the
double bond of oleic acid.
[20]
This methylation reaction forms the
intermediate 10-methylene-octadecanoyal. Successive reduction of the
residue, with NADPH as a cofactor, results in 10-methylstearic acid
[15]
References
[1] [1] Dijkstra, Albert J., R. J. Hamilton, and Wolf Hamm. "Fatty Acid Biosynthesis."
Trans Fatty Acids. Oxford: Blackwell Pub., 2008. 12. Print.
[2] "Fatty Acids: Straight-chain Saturated, Structure, Occurrence and Biosynthesis."
Lipid Library - Lipid Chemistry, Biology, Technology and Analysis. Web. 30 Apr.
2011. <http://lipidlibrary.aocs.org/lipids/fa_sat/index.htm>.
[3] Diwan, Joyce J. "Fatty Acid Synthesis." Rensselaer Polytechnic Institute (RPI) :: Architecture, Business, Engineering, IT, Humanities,
Science. Web. 30 Apr. 2011. <http://rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/fasynthesis.htm>.
[5] [5] Aguilar, Pablo S, and Diegode Mendoza. "Control of fatty acid desaturation: a mechanism conserved from bacteria to humans." Molecular
microbiology 62.6 (2006):1507-14.
[6] [6] Feng, Youjun, and John ECronan. "Complex binding of the FabR repressor of bacterial unsaturated fatty acid biosynthesis to its cognate
promoters." Molecular microbiology 80.1 (2011):195-218.
[7] [7] Zhu, Lei, et al. "Functions of the Clostridium acetobutylicium FabF and FabZ proteins in unsaturated fatty acid biosynthesis." BMC
microbiology 9(2009):119.
[8] [8] Wang, Haihong, and John ECronan. "Functional replacement of the FabA and FabB proteins of Escherichia coli fatty acid synthesis by
Enterococcus faecalis FabZ and FabF homologues." Journal of biological chemistry 279.33 (2004):34489-95.
[9] [9] Mansilla, Mara C, and Diegode Mendoza. "The Bacillus subtilis desaturase: a model to understand phospholipid modification and
temperature sensing." Archives of microbiology 183.4 (2005):229-35.
[10] [10] Wada, M, N. Fukunaga, and S. Sasaki. "Mechanism of biosynthesis of unsaturated fatty acids in Pseudomonas sp. strain E-3, a
psychrotrophic bacterium." Journal of bacteriology 171.8 (1989):4267-71.
[11] [11] Subramanian, Chitra, Charles ORock, and Yong-MeiZhang. "DesT coordinates the expression of anaerobic and aerobic pathways for
unsaturated fatty acid biosynthesis in Pseudomonas aeruginosa." Journal of bacteriology 192.1 (2010):280-5.
[12] [12] Morita, N, et al. "Both the anaerobic pathway and aerobic desaturation are involved in the synthesis of unsaturated fatty acids in Vibrio sp.
strain ABE-1." FEBS letters 297.1-2 (1992):9-12.
[13] [13] Zhu, Kun, et al. "Two aerobic pathways for the formation of unsaturated fatty acids in Pseudomonas aeruginosa." Molecular microbiology
60.2 (2006):260-73.
[14] [14] Kaneda, Toshi. "Iso- and Anteiso-Fatty Acids in Bacteria: Biosynthesis, Function, and Taxonomic Significance." Microbiological Reviews
55.2 (1991): 288-302
[15] "Branched-chain Fatty Acids, Phytanic Acid, Tuberculostearic Acid Iso/anteiso- Fatty Acids." Lipid Library - Lipid Chemistry, Biology,
Technology and Analysis. Web. 01 May 2011. http:/ / lipidlibrary. aocs. org/ lipids/ fa_branc/ index. htm.
[16] [16] Naik, Devaray N., and Toshi Kaneda. "Biosynthesis of Branched Long-chain Fatty Acids by Species of Bacillus: Relative Activity of Three
-keto Acid Substrates and Factors Affecting Chain Length." Can. J. Microbiol. 20 (1974): 1701-708.
[17] [17] Oku, Hirosuke, and Toshi Kaneda. "Biosynthesis of Branched-chain Fatty Acids in Bacillis Subtilis." The Journal of Biological Chemistry
263.34 (1988): 18386-8396.
[18] Christie, William W. "Fatty Acids: Natural Alicyclic Structures, Occurrence, and Biochemistry." The AOCS Lipid Library. 5 Apr. 2011.
Web. 24 Apr. 2011. <http://lipidlibrary.aocs.org/lipids/fa_cycl/file.pdf>.
[19] [19] Ratledge, Colin, and John Stanford. The Biology of the Mycobacteria. London: Academic, 1982. Print.
[20] [20] Kubica, George P., and Lawrence G. Wayne. The Mycobacteria: a Sourcebook. New York: Dekker, 1984. Print.
Fatty acid synthesis
267
External links
Overview (http:/ / www. rpi. edu/ dept/ bcbp/ molbiochem/ MBWeb/ mb2/ part1/ fasynthesis. htm) at Rensselaer
Polytechnic Institute
Overview (http:/ / web. indstate. edu/ thcme/ mwking/ lipid-synthesis. html#synthesis) at Indiana State
UniversityWikipedia:Link rot
Lipogenesis
Lipogenesis is the process by which acetyl-CoA is converted to fats. The former is an intermediate stage in
metabolism of simple sugars, such as glucose, a source of energy of living organisms. Through lipogenesis, the
energy can be efficiently stored in the form of fats. Lipogenesis encompasses the processes of fatty acid synthesis
and subsequent triglyceride synthesis (when fatty acids are esterified with glycerol to form fats).
[1]
The products are
secreted from the liver in the form of very-low-density lipoproteins (VLDL).
Fatty acid synthesis
Fatty acids synthesis starts with acetyl-CoA and builds up by the addition of two carbon units. The synthesis occurs
in the cytoplasm in contrast to the degradation (oxidation), which occurs in the mitochondria. Many of the enzymes
for the fatty acid synthesis are organized into a multienzyme complex called fatty acid synthetase.
[2]
The major sites
of fatty acid synthesis are adipose tissue and the liver.
[3]
Control and regulation
Insulin is an indicator of the blood sugar level of the body, as its concentration increases proportionally with blood
sugar levels. Thus, a large insulin level is associated with the fed state. As one might expect, it increases the rate of
storage pathways, such as lipogenesis. Insulin stimulates lipogenesis in two main ways: The enzymes pyruvate
dehydrogenase (PDH), which forms acetyl-CoA, and acetyl-CoA carboxylase (ACC), which forms malonyl-CoA
from acetyl-CoA, are obvious control points. These are activated by insulin. So a high insulin level leads to an
overall increase in the levels of malonyl-CoA, which is the substrate required for fatty acids synthesis.
PDH dephosphorylation
Pyruvate dehydrogenase is dephosphorylated in response to increased insulin signalling. The dephosphorylated form
is more active.
As insulin binds to cellular surface transmembrane receptors that intracellularly activate the adenylate cyclase
enzyme that catalyze cAMP (cyclic AMP) production from ATP. The increased intracellular cAMP, acts as a second
messenger, in response to the insulin binding. cAMP activates protein kinase enzyme that in turn activates
phosphorylase enzyme that phosphorylates and in doing so activates a number of different intracellular enzymes
such as the pyruvate dehydrogenase that dehydrates pyruvate to form AcCoa. So, an extracellular hormone, insulin,
can in multistep activation (cascade) activate an enzyme in the cellular matrix.
This mechanism leads to the increased rate of catalysis of this enzyme, so increases the levels of acetyl-CoA.
Increased levels of acetyl-CoA will increase the flux through not only the fat synthesis pathway but also the citric
acid cycle.
Note: The above is incorrect; insulin does not activate adenylate cyclase. Epinephrine via the beta adrenergic
receptor and glucagon via its receptor activate adenylate cyclase, increasing cAMP levels and PKA activity. Insulin
activates cAMP phosphodiesterase, which breaks down cAMP. This leads to a decrease in cAMP levels and
therefore a decrease in PKA activity. With regard to Pyruvate dehydrogenase, this enzyme is inhibited when
Lipogenesis
268
phosphorylated. Insulin stimulates the activity of pyruvate dehydrogenase phosphatase. This enzyme removes the
phosphate from pyruvate dehydrogenase, allowing for conversion of pyruvate to acetyl-CoA.
Acetyl-CoA carboxylase
Insulin affects ACC in a similar way to PDH. It leads to its dephosphorylation which activates the enzyme. Glucagon
has an antagonistic effect and increases phosphorylation, deactivation, thereby inhibiting ACC and slowing fat
synthesis.
Affecting ACC affects the rate of acetyl-CoA conversion to malonyl-CoA. Increased malonyl-CoA level pushes the
equilibrium over to increase production of fatty acids through biosynthesis. Long chain fatty acids are negative
allosteric regulators of ACC and so when the cell has sufficient long chain fatty acids, they will eventually inhibit
ACC activity and stop fatty acid synthesis.
AMP and ATP concentrations of the cell act as a measure of the ATP needs of a cell and as ATP levels get low it
activates the ATP synthetase which in turn phosphorylates ACC. When ATP is depleted, there is a rise in 5'AMP.
This rise activates AMP-activated protein kinase, which phosphorylates ACC, thereby inhibits fat synthesis. This is a
useful way to ensure that glucose is not diverted down a storage pathway in times when energy levels are low.
ACC is also activated by citrate. This means that, when there is abundant acetyl-CoA in the cell cytoplasm for fat
synthesis, it proceeds at an appropriate rate.
Note: Research now shows that glucose metabolism (exact metabolite to be determined), aside from insulin's
influence on lipogenic enzyme genes, can induce the gene products for liver's pyruvate kinase, acetyl-CoA
carboxylase, and fatty acid synthase. These genes are induced by the transcription factors ChREBP/Mlx via high
blood glucose levels
[4]
and presently unknown signaling events. Insulin induction is due to SREBP-1c, which is also
involved in cholesterol metabolism.
Fatty acid esterification
Experiments were conducted to study in vivo the over-all fatty acid specificity of the mechanisms involved in
chylomicron cholesterol ester and triglyceride formation during fat absorption in the rat. Mixtures containing similar
amounts of two, three, or four C14-labeled fatty acids (palmitic, stearic, oleic, and linoleic acids), but with varying
ratios of unlabeled fatty acids, were given by gastric intubation to rats with cannulated thoracic ducts. The chyle or
chylomicron lipid so obtained was chromatographed on silicic acid columns to separate cholesterol esters and
glycerides (the latter being 98.2% triglycerides). After assaying each lipid class for total radioactivity, gas-liquid
chromatography was employed to measure the total mass and the distribution of mass and of radioactivity in the
individual fatty acid components of each lipid fraction. The specific radioactivity of each fatty acid in each fraction
could then be calculated. The data provided quantitative information on the relative specificity of incorporation of
each fatty acid into each chylomicron lipid class and on the relative extent to which each fatty acid in each lipid
fraction was diluted with endogenous fatty acid. With the exception of a slight discrimination against stearic acid, the
processes of fatty acid absorption and chylomicron triglyceride formation displayed no specificity for one fatty acid
relative to another. In contrast, chylomicron cholesterol ester formation showed marked specificity for oleic acid,
relative to the other three fatty acids. This specificity was not significantly altered by varying the composition of the
test meal, by including cholesterol in the test meal, or by feeding the animal a high-cholesterol diet for several weeks
preceding the study. Considerable dilution of the dietary fatty acids with endogenous fatty acids was observed. In
one experiment, 43% of the chylomicron triglyceride fatty acids was of endogenous origin. Relatively more (54%) of
the cholesterol ester fatty acids was of endogenous origin.
Lipogenesis
269
In Industry
About 100,000 metric tons of the natural fatty acids are consumed in the preparation of various fatty acid esters. The
simple esters with lower chain alcohols (methyl-, ethyl-, n-propyl-, isopropyl- and butyl esters) are used as
emollients in cosmetics and other personal care products and as lubricants. Esters of fatty acids with more complex
alcohols, such as sorbitol, ethylene glycol, diethylene glycol, and polyethylene glycol are consumed in foods,
personal care, paper, water treatment, metal working fluids, rolling oils, and synthetic lubricants.
References
[4] [4] Work from Howard Towle, Catherine Postic, and K. Uyeda.
Acetyl-CoA carboxylase
Acetyl-CoA carboxylase
Identifiers
EC number
6.4.1.2
[1]
CAS number
9023-93-2
[2]
Databases
IntEnz
IntEnz view
[3]
BRENDA
BRENDA entry
[4]
ExPASy
NiceZyme view
[5]
KEGG
KEGG entry
[6]
MetaCyc
metabolic pathway
[7]
PRIAM
profile
[8]
PDB structures
RCSB PDB
[9]
PDBe
[10]
PDBsum
[11]
Gene Ontology
AmiGO
[12]
/ EGO
[13]
Search
PMC
articles
[14]
PubMed
articles
[15]
NCBI
proteins
[16]
Acetyl-CoA carboxylase
270
Acetyl-CoA
carboxylase alpha
Identifiers
Symbol ACACA
Alt. symbols ACAC, ACC1, ACCA
Entrez
31
[17]
HUGO
84
[18]
OMIM
601557
[19]
RefSeq
NM_198839
[20]
UniProt
Q13085
[21]
Other data
EC number
6.4.1.2
[22]
Locus
Chr. 17 q21
[23]
Acetyl-CoA
carboxylase beta
Identifiers
Symbol ACACB
Alt. symbols ACC2, ACCB
Entrez
32
[24]
HUGO
85
[25]
OMIM
200350
[26]
RefSeq
NM_001093
[27]
UniProt
O00763
[28]
Other data
EC number
6.4.1.2
[22]
Locus
Chr. 12 q24.1
[29]
Acetyl-CoA carboxylase (ACC) is a biotin-dependent enzyme that catalyzes the irreversible carboxylation of
acetyl-CoA to produce malonyl-CoA through its two catalytic activities, biotin carboxylase (BC) and
carboxyltransferase (CT). ACC is a multi-subunit enzyme in most prokaryotes and in the chloroplasts of most plants
and algae, whereas it is a large, multi-domain enzyme in the endoplasmic reticulum of most eukaryotes. The most
important function of ACC is to provide the malonyl-CoA substrate for the biosynthesis of fatty acids.
[]
The activity
of ACC can be controlled at the transcriptional level as well as by small molecule modulators and covalent
modification. The human genome contains the genes for two different ACCs
[]
ACACA
[]
and ACACB.
[]
Acetyl-CoA carboxylase
271
Structure
Prokaryotes and plants have multi-subunit ACCs composed of several polypeptides encoded by distinct genes. Biotin
carboxylase (BC) activity, biotin carboxyl carrier protein (BCCP), and carboxyl transferase (CT) activity are each
contained on a different subunit. The stoichiometry of these subunits in the ACC holoenzyme differs amongst
organisms.
[]
Humans and most eukaryotes have evolved an ACC with CT and BC catalytic domains and biotin
carboxyl carrier domains on a single polypeptide. ACC functional regions, starting from the N-terminus to
C-terminus are the biotin carboxylase (BC), biotin binding (BB), carboxyltransferase (CT), and ATP-binding (AB).
AB lies within BC. Biotin is covalently attached through an amide bond to the long side chain of a lysine reside in
BB. As BB is between BC and CT regions, biotin can be easily translocate to both of the active sites where it is
required.
In mammals where two isoforms of ACC are expressed, the main structural difference between these isoforms is the
extended ACC2 N-terminus containing a mitochondria targeting sequence.
[]
Crystallographic structures of E. coli acetyl-CoA carboxylase
Biotin carboxylase subunit of E. coli acetyl-CoA
carboxylase
Acetyl-CoA carboxylase
272
Biotin carboxyl carrier protein subunit of E. coli acetyl-CoA
carboxylase
Carboxyl transferase subunit of E. coli acetyl-CoA
carboxylase
Acetyl-CoA carboxylase
273
Mechanism
The overall reaction of ACAC(A,B) proceeds by a two-step mechanism.
[]
The first reaction is carried out by BC and
involves the ATP-dependent carboxylation of biotin with bicarbonate serving as the source of CO
2
. The carboxyl
group is transferred from biotin to acetyl CoA to form malonyl CoA in the second reaction, which is catalyzed by
CT.
The reaction mechanism of ACAC(A,B).The color scheme is as follows: enzyme, coenzymes, substrate names, metal ions, phosphate, and carbonate
In the context of the active site, the reaction proceeds with extensive interaction of the residues Glu296 and
positively charged Arg338 and Arg292 with the substrates.
[]
Two Mg2+ are coordinated by the phosphate groups on
the ATP, and are required for ATP binding to the enzyme. Bicarbonate is deprotonated by Glu296, although in
solution, this proton transfer is unlikely as the pKa of bicarbonate is 10.3. The enzyme apparently manipulates pKas
to facilitate the deprotonation of bicarbonate. The pKa of bicarbonate is decreased by its interaction with positively
charged side chains of Arg338 and Arg292. Furthermore, Glu296 interacts with the side chain of Glu211, an
interaction that has been shown to cause an increase in the apparent pKa. Following deprotonation of bicarbonate,
the oxygen of the bicarbonate acts as a nucleophile and attacks the gamma phosphate on ATP. The
carboxyphosphate intermediate quickly decomposes to CO
2
and PO
4
3-
. The PO
4
3-
deprotonates biotin, creating an
enolate, stabilized by Arg338, that subsequently attacks CO₂ resulting in the production of
carboxybiotin.
[]
The carboxybiotin translocates to the carboxytransferase (CT) active site, where the carboxyl group
is transferred to acetyl-CoA. In contrast to the BC domain, little is known about the reaction mechanism of CT. A
proposed mechanism is the release of carbon dioxide from biotin, which subsequently abstracts a proton from the
methyl group from acetyl CoA carboxylase. The resulting enolate attacks CO
2
to form malonyl CoA. In a competing
mechanism, proton abstraction is concerted with the attack of acetyl CoA.
Function
The function of ACC is to regulate the metabolism of fatty acids. When the enzyme is active, the product,
malonyl-CoA is produced which is a building block for new fatty acids and can inhibit the transfer of the fatty acyl
group from acyl CoA to carnitine with carnitine acyltransferase, which inhibits the beta-oxidation of fatty acids in
the mitochondria.
In mammals, two main isoforms of ACC are expressed, ACC1 and ACC2, which differ in both tissue distribution
and function. ACC1 is found in the cytoplasm of all cells but is enriched in lipogenic tissue, such as adipose tissue
and lactating mammary glands, where fatty acid synthesis is important.
[]
In oxidative tissues, such as the skeletal
muscle and the heart, the ratio of ACC2 expressed is higher. ACC1 and ACC2 are both highly expressed in the liver
where both fatty acid oxidation and synthesis is important.
[]
The differences in tissue distribution indicate that ACC1
Acetyl-CoA carboxylase
274
maintains regulation of fatty acid synthesis whereas ACC2 mainly regulates fatty acid oxidation.
Regulation
Control of Acetyl CoA Carboxylase. The AMP regulated kinase triggers the
phosphorylation of the enzyme (thus inactivating it) and the phosphatase enzyme removes
the phosphate group.
The regulation of mammalian ACC is
complex, in order to control two
distinct pools of malonyl CoA that
direct either the inhibition of beta
oxidation or the activation of lipid
biosynthesis
[]
.
Mammalian ACC1 and ACC2 are
regulated transcriptionally by multiple
promoters which mediate ACC abundance in response to the cells nutritional status. Activation of gene expression
through different promoters results in alternative splicing; however, the physiological significance of specific ACC
isozymes remains unclear.
[]
The sensitivity to nutritional status results from the control of these promoters by
transcription factors such as SREBP1c, controlled by insulin at the transcriptional level, and ChREBP, which
increases in expression with high carbohydrates diets.
[][]
Through a feedforward loop, citrate allosterically activates ACC.
[]
Citrate may increase ACC polymerization to
increases enzymatic activity; however, it is unclear if polymerization is citrate's main mechanism of increasing ACC
activity or if polymerization is an artifact of in vitro experiments. Other allosteric activators include glutamate and
other dicarboxylic acids.
[]
Long and short chain fatty acyl CoAs are negative feedback inhibitors of ACC.
[]
Phosphorylation can result when the hormones glucagon or epinephrine bind to cell surface receptors, but the main
cause of phosphorylation is due to a rise in AMP levels when the energy status of the cell is low, leading to the
activation of the AMP-activated protein kinase (AMPK). AMPK is the main kinase regulator of ACC, able to
phosphorylate a number of serine residues on both isoforms of ACC.
[]
On ACC1, AMPK phosphorylates Ser79,
Ser1200, and Ser1215. On ACC2, AMPK phosphorylates Ser218.
[]
Protein kinase A also has the ability to
phosphorylate ACC, with a much greater ability to phosphorylate ACC2 than ACC1. However, the physiological
significance of protein kinase A in the regulation of ACC is currently unknown. Researchers hypothesize there are
other ACC kinases important to its regulation as there are many other possible phosphorylation sites on ACC.
[]
When insulin binds to its receptors on the cellular membrane, it activates a phosphatase to dephosphorylate the
enzyme; thereby removing the inhibitory effect.
This protein may use the morpheein model of allosteric regulation.
[]
Clinical implications
At the juncture of lipid synthesis and oxidation pathways, ACC presents many clinical possibilities for the
production of novel antibiotics and the development of new therapies for diabetes, obesity, and other manifestations
of metabolic syndrome.
[]
Researchers aim to take advantage of structural differences between bacterial and human
ACCs to create antibiotics specific to the bacterial ACC, in efforts to minimize side effects to patients. Promising
results for the usefulness of an ACC inhibitor include the finding that ACC2 -/- mice (mice with no expression of
ACC2) have continuous fatty acid oxidation, reduced body fat mass, and reduced body weight despite an increase in
food consumption. ACC2 -/- mice are also protected from diabetes.
[]
It should be noted that mutant mice lacking
ACC1 are embryonically lethal. However, it is unknown whether drugs targeting ACCs in humans must be specific
for ACC2.
[]
Acetyl-CoA carboxylase
275
References
[1] http:/ / www. chem. qmul.ac. uk/ iubmb/ enzyme/ EC6/ 4/ 1/ 2. html
[2] http:/ / toolserver.org/ ~magnus/ cas.php?language=en& cas=9023-93-2& title=
[3] http:/ / www. ebi. ac. uk/ intenz/ query?cmd=SearchEC& ec=6. 4. 1. 2
[4] http:/ / www. brenda-enzymes. org/ php/ result_flat.php4?ecno=6. 4. 1. 2
[5] http:/ / www. expasy. org/ enzyme/ 6. 4.1.2
[6] http:/ / www. genome. ad. jp/ dbget-bin/ www_bget?enzyme+ 6. 4. 1. 2
[7] http:/ / biocyc. org/ META/ substring-search?type=NIL& object=6. 4. 1. 2
[8] http:/ / priam. prabi. fr/ cgi-bin/ PRIAM_profiles_CurrentRelease. pl?EC=6. 4. 1. 2
[9] http:/ / www. rcsb.org/ pdb/ search/ smartSubquery. do?smartSearchSubtype=EnzymeClassificationQuery& Enzyme_Classification=6. 4. 1.
2
[10] http:/ / www.ebi. ac.uk/ pdbe-srv/ PDBeXplore/ enzyme/ ?ec=6. 4. 1. 2
[11] http:/ / www.ebi. ac.uk/ thornton-srv/ databases/ cgi-bin/ enzymes/ GetPage. pl?ec_number=6. 4. 1. 2
[12] http:/ / amigo. geneontology.org/ cgi-bin/ amigo/ go.cgi?query=GO:0003989& view=details
[13] http:/ / www.ebi. ac.uk/ ego/ DisplayGoTerm?id=GO:0003989& format=normal
[14] http:/ / www.ncbi. nlm.nih. gov/ entrez/ query. fcgi?db=pubmed& term=6. 4. 1. 2%5BEC/
RN%20Number%5D%20AND%20pubmed%20pmc%20local%5Bsb%5D
[15] http:/ / www.ncbi. nlm.nih. gov/ entrez/ query. fcgi?db=pubmed& term=6. 4. 1. 2%5BEC/ RN%20Number%5D
[16] http:/ / www.ncbi. nlm.nih. gov/ protein?term=6.4.1.2%5BEC/ RN%20Number%5D
[17] http:/ / www.ncbi. nlm.nih. gov/ entrez/ query. fcgi?db=gene& amp;cmd=retrieve& amp;dopt=default& amp;list_uids=31& rn=1
[18] http:/ / www.genenames.org/ data/ hgnc_data. php?hgnc_id=84
[19] http:/ / www.omim. org/ 601557
[20] http:/ / genome. ucsc.edu/ cgi-bin/ hgTracks?Submit=Submit& position=NM_198839& rn=1
[21] http:/ / www.uniprot. org/ uniprot/ Q13085
[22] http:/ / www.genome. jp/ dbget-bin/ www_bget?enzyme+ 6. 4. 1. 2
[23] http:/ / omim. org/ search?index=geneMap& search=17q21
[24] http:/ / www.ncbi. nlm.nih. gov/ entrez/ query. fcgi?db=gene& amp;cmd=retrieve& amp;dopt=default& amp;list_uids=32& rn=1
[25] http:/ / www.genenames.org/ data/ hgnc_data. php?hgnc_id=85
[26] http:/ / www.omim. org/ 200350
[27] http:/ / genome. ucsc.edu/ cgi-bin/ hgTracks?Submit=Submit& position=NM_001093& rn=1
[28] http:/ / www.uniprot. org/ uniprot/ O00763
[29] http:/ / omim. org/ search?index=geneMap& search=12q24. 1
Further reading
Voet, Donald; Voet, Judith G. (2004). Biochemistry (3rd ed.). Wiley. ISBN0-471-19350-X.
edited by (2000). In Buchanan, Bob B.; Gruissem, Wilhelm; Jones, Russell L. Biochemistry and molecular
biology of plants. American Society of Plant Physiologists. ISBN0-943088-37-2.
Levert K, Waldrop G, Stephens J (2002). "A biotin analog inhibits acetyl-CoA carboxylase activity and
adipogenesis". J. Biol. Chem. 277 (19): 1634750. doi: 10.1074/jbc.C200113200 (http:/ / dx. doi. org/ 10. 1074/
jbc. C200113200). PMID 11907024 (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 11907024).
Fatty acid degradation
276
Fatty acid degradation
Fatty acid degradation is the process in which fatty acids are broken down into their metabolites, in the end
generating acetyl-CoA, the entry molecule for the citric acid cycle, the main energy supply of animals. It includes
three major steps:
Lipolysis of and release from adipose tissue
Activation and transport into mitochondria
-oxidation
Lipolysis and release
Initially in the process of degradation, fatty acids are stored in fat cells (adipocytes). The breakdown of this fat is
known as lipolysis. The products of lipolysis, free fatty acids, are released into the bloodstream and circulate
throughout the body.
Activation and transport into mitochondria
Fatty acids must be activated before they can be carried into the mitochondria, where fatty acid oxidation occurs.
This process occurs in two steps catalyzed by the enzyme fatty acyl-CoA synthetase.
Formation of an activated thioester bond
The enzyme first catalyzes nucleophilic attack on the -phosphate of ATP to form pyrophosphate and an acyl chain
linked to AMP. The next step is formation of an activated thioester bond between the fatty acyl chain and Coenzyme
A.
The balanced equation for the above is:
RCOO
-
+ CoA + ATP + H
2
O RCO-CoA + AMP + PP
i
+ 2H
+
This two-step reaction is freely reversible and its equilibrium lies near 1. To drive the reaction forward, the reaction
is coupled to a strongly exergonic hydrolysis reaction: the enzyme inorganic pyrophosphatase cleaves the
pyrophosphate liberated from ATP to two phosphate ions. Thus the net reaction becomes:
RCOO
-
+ CoA + ATP + H
2
O RCO-CoA + AMP + 2P
i
+ 2H
+
Fatty acid degradation
277
Transport into the mitochondrial matrix
The inner mitochondrial membrane is impermeable to fatty acids and a specialized carnitine carrier system
operates to transport activated fatty acids from cytosol to mitochondria.
Once activated, the acyl CoA is transported into the mitochondrial matrix. This occurs via a series of similar steps:
1. Acyl CoA is conjugated to carnitine by carnitine acyltransferase (palmitoyltransferase) I located on the outer
mitochondrial membrane
2. Acyl carnitine is shuttled inside by a translocase
3. Acyl carnitine (such as Palmitoylcarnitine) is converted to acyl CoA by carnitine acyltransferase
(palmitoyltransferase) II located on the inner mitochondrial membrane. The liberated carnitine returns to the
cytosol.
It is important to note that carnitine acyltransferase I undergoes allosteric inhibition as a result of malonyl-CoA, an
intermediate in fatty acid biosynthesis, in order to prevent futile cycling between beta-oxidation and fatty acid
synthesis.
-oxidation
Once inside the mitochondria, the -oxidation of fatty acids occurs via five recurring steps:
1. 1. Activation by ATP
2. Oxidation by FAD,
3. Hydration,
4. Oxidation by NAD
+
,
5. Thiolysis,
6. The final product is acetyl-CoA, the entry molecule for the citric acid cycle.
Beta oxidation
278
Beta oxidation
Schematic demonstrating mitochondrial fatty acid beta-oxidation and effects of
long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency, LCHAD deficiency
Beta-oxidation is the process by
which fatty acid molecules are broken
down in the mitochondria to generate
acetyl-coA, which enters the citric acid
cycle, and NADH and FADH2, which
are used by the electron transport
chain.
Fatty Acid Catabolism involve three
stages. The first stage of fatty acid
catabolism is Beta-Oxidation. The
second stage is acetyl CoA oxidation
to carbon dioxide. The third stage is
electron transfer from electron carries
to the electron transfer chain.
Priming the fatty acid to for oxidation:
Carnitine Shuttle
1. Acyl CoA is transferred to the hydroxyl group of carnitine by carnitine acyltransferase I (palmitoyltransferase)
located on the outer mitochondrial membrane
2. Acylcarnitine is shuttled inside by a carnitine-acylcarnitine translocase
3. Acylcarnitine is converted back to acyl CoA by carnitine acyltransferase II (palmitoyltransferase) located on the
inner mitochondrial membrane. The liberated carnitine returns to the cytosol.
Once the fatty acid is inside the mitochondrial matrix, Beta Oxidation can begin. It has 4 steps. Step 1 of
Beta-Oxidation: Long chain fatty acid is dehydrogenated to create a trans double bond between C2 and C3. This is
catalyzed by the fatty acyl CoA dehydrogenase to produce trans-delta 2-enoyl CoA. It uses FAD as an electron
acceptor and it is reduced to FADH2.
Step 2 of Beta-Oxidation: Trans-delta2-enoyl CoA is hydrated at the double bond to produce L-B-hydroxyacyl CoA.
This is catalyzed by enoyl CoA hydratase.
Step 3 of Beta- Oxidation: L-B-hydroxyacyl CoA is dehydrogenated again to create B-ketoacyl CoA by
B-hydroxyacyl CoA dehydrogenase. This enzyme uses NAD as an electron acceptor.
Step 4 of Beta-Oxidation: Thiolysis occurs between C2 and C3 (alpha and beta carbons) of B-ketoacyl CoA.
Thiolase enzyme catalyzes the reaction when a new molecule of coenzyme A breaks the bond by nucleophilic attach
on C2. This releases the first two carbon units, as acetyl CoA, and an fatty acyl CoA without the two first carbons.
The process continues until all of the carbons in the fatty acid are turned into acetyl CoA.
Fatty acids are oxidized by most of the tissues in the body. However, some tissues such as the adrenal medulla do not
use fatty acids for their energy requirements and instead use carbohydrates.
Activation and transport
Free fatty acids cannot penetrate the plasma membrane due to their negative charge. Once in the cytosol, activation
of the fatty acid is catalyzed by long fatty acyl CoA synthetase. A fatty acid reacts with ATP to give a fatty acyl
adenylate, plus inorganic pyrophosphate, which then reacts with free coenzyme A to give a fatty acyl-CoA ester plus
AMP. If the fatty acyl-CoA has a long chain (10 or more carbons) then it is reacted with carnitine to form
acylcarnitine, which is transported across the inner mitochondrial membrane by a Carnitine-acylcarnitine
Beta oxidation
279
translocase. If the fatty acyl-CoA contains a short chain (less than 10 carbons) it can simply diffuse through the inner
mitochondrial membrane.
Even-numbered saturated fatty acids
Once inside the mitochondria, each cycle of -oxidation, liberating a two carbon unit (acetyl-CoA), occurs in a
sequence of four reactions:
Description Diagram Enzyme End product
Dehydrogenation by FAD: The first step is the
oxidation of the fatty acid by
Acyl-CoA-Dehydrogenase. The enzyme catalyzes
the formation of a double bond between the C-2
and C-3.
acyl CoA
dehydrogenase
trans-
2
-enoyl-CoA
Hydration: The next step is the hydration of the
bond between C-2 and C-3. The reaction is
stereospecific, forming only the L isomer.
enoyl CoA
hydratase
L--hydroxyacyl CoA
Oxidation by NAD
+
: The third step is the oxidation
of L--hydroxyacyl CoA by NAD
+
. This converts
the hydroxyl group into a keto group.
L--hydroxyacyl
CoA
dehydrogenase
-ketoacyl CoA
Thiolysis: The final step is the cleavage of
-ketoacyl CoA by the thiol group of another
molecule of Coenzyme A. The thiol is inserted
between C-2 and C-3.
-ketothiolase An acetyl-CoA molecule,
and an acyl-CoA molecule
that is two carbons shorter
This process continues until the entire chain is cleaved into acetyl CoA units. The final cycle produces two separate
acetyl CoAs, instead of one acyl CoA and one acetyl CoA. For every cycle, the Acyl CoA unit is shortened by two
carbon atoms. Concomitantly, one molecule of FADH
2
, NADH and acetyl CoA are formed.
Odd-numbered saturated fatty acids
In general, fatty acids with an odd number of carbons are found in the lipids of plants and some marine organisms.
Many ruminant animals form a large amount of 3-carbon propionate during the fermentation of carbohydrates in the
rumen.
[1]
Chains with an odd-number of carbons are oxidized in the same manner as even-numbered chains, but the final
products are propionyl-CoA and acetyl-CoA.
Propionyl-CoA is first carboxylated using a bicarbonate ion into D-stereoisomer of methylmalonyl-CoA, in a
reaction that involves a biotin co-factor, ATP, and the enzyme propionyl-CoA carboxylase. The bicarbonate ion's
carbon is added to the middle carbon of propionyl-CoA, forming a D-methylmalonyl-CoA. However, the D
conformation is enzymatically converted into the L conformation by methylmalonyl-CoA epimerase, then it
undergoes intramolecular rearrangement, which is catalyzed by methylmalonyl-CoA mutase (requiring B
12
as a
coenzyme) to form succinyl-CoA. The succinyl-CoA formed can then enter the citric acid cycle.
However, whereas acetyl-CoA enters the citric acid cycle by condensing with an existing molecule of oxaloacetate,
succinyl-CoA enters the cycle as a principal in its own right. Thus the succinate just adds to the population of
circulating molecules in the cycle and undergoes no net metabolization while in it. When this infusion of citric acid
cycle intermediates exceeds cataplerotic demand (such as for aspartate or glutamate synthesis), some of them can be
extracted to the gluconeogenesis pathway, in the liver and kidneys, through phosphoenolpyruvate carboxykinase,
Beta oxidation
280
and converted to free glucose.
[2]
Unsaturated fatty acids
-Oxidation of unsaturated fatty acids poses a problem since the location of a cis bond can prevent the formation of a
trans-
2
bond. These situations are handled by an additional two enzymes, Enoyl CoA isomerase or 2,4 Dienoyl
CoA reductase.
Whatever the conformation of the hydrocarbon chain, -oxidation occurs normally until the acyl CoA (because of
the presence of a double bond) is not an appropriate substrate for acyl CoA dehydrogenase, or enoyl CoA hydratase:
If the acyl CoA contains a cis-
3
bond, then cis-
3
-Enoyl CoA isomerase will convert the bond to a trans-
2
bond, which is a regular substrate.
If the acyl CoA contains a cis-
4
double bond, then its dehydrogenation yields a 2,4-dienoyl intermediate, which
is not a substrate for enoyl CoA hydratase. However, the enzyme 2,4 Dienoyl CoA reductase reduces the
intermediate, using NADPH, into trans-
3
-enoyl CoA. As in the above case, this compound is converted into a
suitable intermediate by 3,2-Enoyl CoA isomerase.
To summarize:
Odd-numbered double bonds are handled by the isomerase.
Even-numbered double bonds by the reductase (which creates an odd-numbered double bond)
Oxidation in peroxisomes
Fatty acid oxidation also occurs in peroxisomes, when the fatty acid chains are too long to be handled by the
mitochondria. However, the oxidation ceases at octanoyl-CoA. It is believed that very long chain (greater than C-22)
fatty acids undergo initial oxidation in peroxisomes which is followed by mitochondrial oxidation.
One significant difference is that oxidation in peroxisomes is not coupled to ATP synthesis. Instead, the
high-potential electrons are transferred to O
2
, which yields H
2
O
2
. The enzyme catalase, found exclusively in
peroxisomes, converts the hydrogen peroxide into water and oxygen.
Peroxisomal -oxidation also requires enzymes specific to the peroxisome and to very long fatty acids. There are
three key differences between the enzymes used for mitochondrial and peroxisomal -oxidation:
1. -oxidation in the peroxisome requires the use of a peroxisomal carnitine acyltransferase (instead of carnitine
acyltransferase I and II used by the mitochondria) for transport of the activated acyl group into the peroxisome.
2. The first oxidation step in the peroxisome is catalyzed by the enzyme acyl-CoA oxidase.
3. The -ketothiolase used in peroxisomal -oxidation has an altered substrate specificity, different from the
mitochondrial -ketothiolase.
Peroxisomal oxidation is induced by high-fat diet and administration of hypolipidemic drugs like clofibrate.
Energy yield
The ATP yield for every oxidation cycle is 14 ATP (according to the P/O ratio), broken down as follows:
Beta oxidation
281
Source ATP Total
1 FADH
2
x 1.5 ATP
= 1.5 ATP (some sources say 2 ATP)
[citation needed]
1 NADH x 2.5 ATP = 2.5 ATP (some sources say 3 ATP)
1 acetyl CoA x 10 ATP = 10 ATP (some sources say 12 ATP)
TOTAL = 14 ATP
For an even-numbered saturated fat (C
2n
), n - 1 oxidations are necessary, and the final process yields an additional
acetyl CoA. In addition, two equivalents of ATP are lost during the activation of the fatty acid. Therefore, the total
ATP yield can be stated as:
(n - 1) * 14 + 10 - 2 = total ATP
For instance, the ATP yield of palmitate (C
16
, n = 8) is:
(8 - 1) * 14 + 10 - 2 = 106 ATP
Represented in table form:
Source ATP Total
7 FADH
2
x 1.5 ATP = 10.5 ATP
7 NADH x 2.5 ATP = 17.5 ATP
8 acetyl CoA x 10 ATP = 80 ATP
Activation = -2 ATP
NET = 106 ATP
For sources that use the larger ATP production numbers described above, the total would be 129 ATP
={(8-1)*17+12-2} equivalents per palmitate.
Beta-oxidation of unsaturated fatty acids changes the ATP yield due to the requirement of two possible additional
enzymes.
Clinical
There are at least 25 enzymes and specific transport proteins in the -oxidation pathway.
[3]
Of these 18 have been
associated with human disease.
External links
The chemical logic behind fatty acid metabolism
[4]
at ufp.pt
JEREMY M. BERG,JOHN L. TYMOCZKO and LUBERT STRYER Biochemistry, 2002
[5]
Animations
[6]
at brookscole.com
Beta oxidation
282
References
[1] Nelson, D. L. & Cox, M. M. (2005). Lehninger Principles of Biochemistry, 4th Edition. New York: W. H. Freeman and Company, pp.
648-649. ISBN 0-7167-4339-6.
[3] [3] Tein I (2013) Disorders of fatty acid oxidation. Handb Clin Neurol 113:1675-88. doi: 10.1016/B978-0-444-59565-2.00035-6.
[4] http:/ / www2.ufp.pt/ ~pedros/ bq/ fatty.htm
[5] http:/ / www. ncbi. nlm.nih. gov/ entrez/ query.fcgi?cmd=Search& db=books& doptcmdl=GenBookHL& term=oxidation+ yield+ AND+
stryer%5Bbook%5D+ AND+ 216592%5Buid%5D& rid=stryer. section. 3050#3060
[6] http:/ / www. brookscole.com/ chemistry_d/ templates/ student_resources/ shared_resources/ animations/ carnitine/ carnitine1. html
283
Nitrogen metabolism
Nitrogen fixation
Nitrogen fixation is a process by which nitrogen (N
2
) in the atmosphere is converted into ammonia (NH
3
).
[]
Atmospheric nitrogen or molecular nitrogen (N
2
) is relatively inert: it does not easily react with other chemicals to
form new compounds. Fixation processes free up the nitrogen atoms from their diatomic form (N
2
) to be used in
other ways.
Nitrogen fixation, natural and synthetic, is essential for all forms of life because nitrogen is required to biosynthesize
basic building blocks of plants, animals and other life forms, e.g., nucleotides for DNA and RNA and amino acids
for proteins. Therefore nitrogen fixation is essential for agriculture and the manufacture of fertilizer. It is also an
important process in the manufacture of explosives (e.g. gunpowder, dynamite, TNT, etc.). Nitrogen fixation occurs
naturally in the air by means of lightning.
[1][2]
Nitrogen fixation also refers to other biological conversions of nitrogen, such as its conversion to nitrogen dioxide.
Microorganisms that can fix nitrogen are prokaryotes (both bacteria and archaea, distributed throughout their
respective kingdoms) called diazotrophs. Some higher plants, and some animals (termites), have formed associations
(symbioses) with diazotrophs. Biological nitrogen fixation was discovered by the German agronomist Hermann
Hellriegel and Dutch microbiologist Martinus Beijerinck.
Biological nitrogen fixation
Schematic representation of the nitrogen cycle. Abiotic nitrogen fixation has been
omitted.
Biological nitrogen fixation (BNF)
occurs when atmospheric nitrogen is
converted to ammonia by an enzyme
called nitrogenase.
[]
The reaction for
BNF is:
N
2
+ 8 H
+
+ 8 e

2 NH
3
+ H
2
The process is coupled to the hydrolysis
of 16 equivalents of ATP and is
accompanied by the co-formation of
one molecule of H
2
. In free-living
diazotrophs, the nitrogenase-generated
ammonium is assimilated into
glutamate through the glutamine
synthetase/glutamate synthase pathway.
Enzymes responsible for nitrogenase
action are very susceptible to
destruction by oxygen. Many bacteria
cease production of the enzyme in the presence of oxygen.
[]
Many nitrogen-fixing organisms exist only in anaerobic
conditions, respiring to draw down oxygen levels, or binding the oxygen with a protein such as leghemoglobin.
[]
Nitrogen fixation
284
Microorganisms that fix nitrogen (diazotrophs)
Cyanobacteria, e.g. the highly significant Trichodesmium
Green sulfur bacteria
Azotobacteraceae
Rhizobia
Frankia
Nitrogen fixation by Frankia
Frankia, Gram-positive soil bacteria, induce the formation of nitrogen-fixing nodules in actinorhizal plants.
Nitrogen fixation by cyanobacteria
Cyanobacteria inhabit nearly all illuminated environments on Earth and play key roles in the carbon and nitrogen
cycle of the biosphere. In general, cyanobacteria are able to utilize a variety of inorganic and organic sources of
combined nitrogen, like nitrate, nitrite, ammonium, urea, or some amino acids. Several cyanobacterial strains are
also capable of diazotrophic growth, an ability that may have been present in their last common ancestor in the
Archaean.
[3]
Nitrogen fixation by cyanobacteria in coral reefs can fix twice the amount of nitrogen than on
landaround 1.8kg of nitrogen is fixed per hectare per day. The colonial marine cyanobacterium Trichodesmium is
thought to fix nitrogen on such a scale that it accounts for almost half of the nitrogen-fixation in marine systems on a
global scale.
[4]
Root nodule symbioses
Legume family
Plants that contribute to nitrogen fixation include the legume family Fabaceae with taxa such as kudzu, clovers,
soybeans, alfalfa, lupines, peanuts, and rooibos. They contain symbiotic bacteria called Rhizobia within nodules in
their root systems, producing nitrogen compounds that help the plant to grow and compete with other plants. When
the plant dies, the fixed nitrogen is released, making it available to other plants and this helps to fertilize the soil.
[][5]
The great majority of legumes have this association, but a few genera (e.g., Styphnolobium) do not. In many
traditional and organic farming practices, fields are rotated through various types of crops, which usually includes
one consisting mainly or entirely of clover or buckwheat (non-legume family Polygonaceae), which are often
referred to as "green manure".
Inga alley farming relies on the leguminous genus Inga, a small tropical, tough-leaved, nitrogen-fixing tree.
[6]
Non-leguminous
A whole alder tree root nodule.
Although by far the majority of plants able to form nitrogen-fixing root
nodules are in the legume family Fabaceae, there are a few exceptions:
Parasponia, a tropical Celtidaceae also able to interact with rhizobia
and form nitrogen-fixing nodules
[7]
Actinorhizal plants such as alder and bayberry, can also form
nitrogen-fixing nodules, thanks to a symbiotic association with
Frankia bacteria. These plants belong to 25 genera
[8]
distributed
among 8 plant families. The ability to fix nitrogen is far from
universally present in these families. For instance, of 122 genera in
the Rosaceae, only 4 genera are capable of fixing nitrogen. All these
Nitrogen fixation
285
A sectioned alder tree root nodule.
families belong to the orders Cucurbitales, Fagales, and Rosales,
which together with the Fabales form a clade of eurosids. In this
clade, Fabales were the first lineage to branch off; thus, the ability
to fix nitrogen may be plesiomorphic and subsequently lost in most
descendants of the original nitrogen-fixing plant; however, it may
be that the basic genetic and physiological requirements were
present in an incipient state in the last common ancestors of all these
plants, but only evolved to full function in some of them:
Family: Genera
Betulaceae: Alnus
(alders)
Cannabaceae: Trema
Casuarinaceae:
Allocasuarina
Casuarina
Ceuthostoma
Gymnostoma
...... Coriariaceae: Coriaria
Datiscaceae: Datisca
Elaeagnaceae:
Elaeagnus
(silverberries)
Hippophae
(sea-buckthorns)
Shepherdia
(buffaloberries)
...... Myricaceae:
Comptonia
(sweetfern)
Morella
Myrica
(bayberries)
...... Rhamnaceae:
Ceanothus
Colletia
Discaria
Kentrothamnus
Retanilla
Talguenea
Trevoa
...... Rosaceae:
Cercocarpus (mountain
mahoganies)
Chamaebatia
(mountain miseries)
Dryas
Purshia/Cowania
(bitterbrushes/cliffroses)
There are also several nitrogen-fixing symbiotic associations that involve cyanobacteria (such as Nostoc):
Some lichens such as Lobaria and Peltigera
Mosquito fern (Azolla species)
Cycads
Gunnera
Industrial nitrogen fixation
The possibility that atmospheric nitrogen reacts with certain chemicals was first observed by Desfosses in 1828. He
observed that mixtures of alkali metal oxides and carbon react at high temperatures with nitrogen. With the use of
barium carbonate as starting material the first commercially used process became available in the 1860s developed
by Margueritte and Sourdeval. The resulting barium cyanide could be reacted with steam yielding ammonia. In 1898
Adolph Frank and Nikodem Caro decoupled the process and first produced calcium carbide and in a subsequent step
reacted it with nitrogen to calcium cyanamide. The Ostwald process for the production of nitric acid was discovered
in 1902. Frank-Caro process and Ostwald process dominated the industrial fixation of nitrogen until the discovery of
the Haber process in 1909.
[9][10]
Haber process
Artificial fertilizer production is now the largest source of human-produced fixed nitrogen in the Earth's ecosystem.
Ammonia is a required precursor to fertilizers, explosives, and other products. The most common method is the
Haber process. The Haber process requires high pressures (around 200 atm) and high temperatures (at least 400C),
routine conditions for industrial catalysis. This highly efficient process uses natural gas as a hydrogen source and air
as a nitrogen source.
[11]
Much research has been conducted on the discovery of catalysts for nitrogen fixation, often with the goal of reducing
the energy required for this conversion. However, such research has thus far failed to even approach the efficiency
and ease of the Haber process. Many compounds react with atmospheric nitrogen to give dinitrogen complexes. The
Nitrogen fixation
286
first dinitrogen complex to be reported was based on ruthenium,[Ru(NH
3
)
5
(N
2
)]
2+
.
[12]
Ambient nitrogen reduction
Catalytic chemical nitrogen fixation at temperatures considerably lower than the Haber process is an ongoing
scientific endeavor. Nitrogen was converted to ammonia and hydrazine by Alexander E. Shilov in 1970.
[13][14]
Few compounds will cleave the N
2
molecule. Under an atmosphere of nitrogen, lithium metal converts to lithium
nitride. Treatment of the resulting nitride gives ammonia. Another example of homolytic cleavage of dinitrogen
under mild conditions was published in 1995. Two equivalents of a molybdenum complex reacted with one
equivalent of dinitrogen, creating a triple bonded MoN complex.
[15]
Since then, this triple bonded complex has been
used to make nitriles.
[16]
Trimethylsilyl chloride, lithium, and nitrogen molecule react to give tris(trimethylsilyl)amine, under catalysis by
nichrome wire or chromium trichloride in tetrahydrofuran.
3 Me
3
SiCl + 3 Li + 1/2 N2 (Me
3
Si)
3
N + 3 LiCl
Tris(trimethylsilyl)amine can then be used for reaction with ,,-triketones to give tricyclic pyrroles.
[17]
Catalytic systems for converting nitrogen to ammonia have been developed since the 1980s.
[18]
In 2003 another was
reported based on molybdenum compound, a proton source, and a strong reducing agent.
[19][20][21][22]
However, this
catalytic reduction fixates only a few nitrogen molecules.
In 2011 Arashiba et al. reported yet another system with a catalyst again based on molybdenum but with a
diphosphorus pincer ligand.
[23]
Nitrogen fixation
287
References
[2] http:/ / www. biology. ed.ac. uk/ archive/ jdeacon/ microbes/ nitrogen. htm
[3] "The evolution of nitrogen fixation in cyanobacteria" N. Latysheva, V. L. Junker, W. J. Palmer, G. A. Codd and D. Barker; Bioinformatics;
2012: 28(5) pp 603606; (Article)
[6] Elkan, Daniel. "Slash-and-burn farming has become a major threat to the world's rainforest". The Guardian, 21 April 2004.
[11] http:/ / www.epa. gov/ watertrain/ nitroabstr.html US Enivronmental Protection Agency: Human Alteration of the Global Nitrogen Cycle:
Causes and Consequences by Peter M. Vitousek, Chair, John Aber, Robert W. Howarth, Gene E. Likens, Pamela A. Matson, David W.
Schindler, William H. Schlesinger, and G. David Tilman
[13] "Catalytic reduction of molecular nitrogen in solutions" A. E. Shilov Russian Chemical Bulletin Volume 52, Number 12, 25552562,
[14] "Reduction of dinitrogen" Richard R. Schrock PNAS 14 November 2006 vol. 103 no. 46 17087
[15] "Dinitrogen Cleavage by a Three-Coordinate Molybdenum(III) Complex" Catalina E. Laplaza and Christopher C. Cummins Science 12 May
1995: 861863.10.1126/science.268.5212.861
[16] "A Cycle for Organic Nitrile Synthesis via Dinitrogen Cleavage" John J. Curley, Emma L. Sceats, and Christopher C. Cummins J. Am.
Chem. Soc., 2006, 128 (43), pp. 1403614037
[18] C. J. Pickett, "The Chatt Cycle and the Mechanism of Enzymic Reduction of Molecular Nitrogen", J. Biol. Inorg. Chem. 1996 1, 601606.
[19] Synthesis and Reactions of Molybdenum Triamidoamine Complexes Containing Hexaisopropylterphenyl Substituents Dmitry V. Yandulov,
Richard R. Schrock, Arnold L. Rheingold, Christopher Ceccarelli, and William M. Davis Inorg. Chem.; 2003; 42(3) pp 796813; (Article)
[20] "Catalytic Reduction of Dinitrogen to Ammonia at a Single Molybdenum Center" Dmitry V. Yandulov and Richard R. Schrock Science 4
July 2003: Vol. 301. no. 5629, pp. 7678
[21] The catalyst is based on molybdenum(V) chloride and tris(2-aminoethyl)amine substituted with three very bulky hexa-isopropylterphenyl
(HIPT) groups. Nitrogen adds end-on to the molybdenum atom, and the bulky HIPT substituents prevent the formation of the stable and
nonreactive Mo-N=N-Mo dimer, and the nitrogen is reduced in an isolated pocket. The proton donor is a pyridinium cation, which is
accompanied by a tetraborate counter ion. The reducing agent is decamethylchromocene. All ammonia formed is collected as the HCl salt by
trapping the distillate with a HCl solution
[22] [22] Note also that, although the dinitrogen complex is shown in brackets, this species can be isolated and characterized. Here the brackets do not
indicate that the intermediate is not observed.
[23] Kazuya Arashiba, Yoshihiro Miyake Yoshiaki Nishibayashi "A molybdenum complex bearing PNP-type pincer ligands leads to the catalytic
reduction of dinitrogen into ammonia" Nature Chemistry Volume: 3, Pages: 120125 Year published:(2011)
External links
"A Brief History of the Discovery of Nitrogen-fixing Organisms", Ann M. Hirsch (2009) (http:/ / www. mcdb.
ucla. edu/ Research/ Hirsch/ imagesb/ HistoryDiscoveryN2fixingOrganisms. pdf)
Marine Nitrogen Fixation laboratory at the University of Southern California (http:/ / dornsife. usc. edu/ labs/
capone)
Amino acid synthesis
288
Amino acid synthesis
Amino acid synthesis is the set of biochemical processes (metabolic pathways) by which the various amino acids
are produced from other compounds. The substrates for these processes are various compounds in the organism's diet
or growth media. Not all organisms are able to synthesise all amino acids. For example, humans are able to
synthesise only 12 of the 20 standard amino acids.
A fundamental problem for biological systems is to obtain nitrogen in an easily usable form. This problem is solved
by certain microorganisms capable of reducing the inert NN molecule (nitrogen gas) to two molecules of ammonia
in one of the most remarkable reactions in biochemistry. Ammonia is the source of nitrogen for all the amino acids.
The carbon backbones come from the glycolytic pathway, the pentose phosphate pathway, or the citric acid cycle.
In amino acid production, one encounters an important problem in biosynthesis, namely stereochemical control.
Because all amino acids except glycine are chiral, biosynthetic pathways must generate the correct isomer with high
fidelity. In each of the 19 pathways for the generation of chiral amino acids, the stereochemistry at the -carbon
atom is established by a transamination reaction that involves pyridoxal phosphate. Almost all the transaminases that
catalyze these reactions descend from a common ancestor, illustrating once again that effective solutions to
biochemical problems are retained throughout evolution.
Biosynthetic pathways are often highly regulated such that building-blocks are synthesized only when supplies are
low. Very often, a high concentration of the final product of a pathway inhibits the activity of enzymes that function
early in the pathway. Often present are allosteric enzymes capable of sensing and responding to concentrations of
regulatory species. These enzymes are similar in functional properties to aspartate transcarbamoylase and its
regulators. Feedback and allosteric mechanisms ensure that all twenty amino acids are maintained in sufficient
amounts for protein synthesis and other processes.
Amino acid synthesis
Amino acids are synthesized from -ketoacids, and later transaminated from another aminoacid, usually Glutamate.
The enzyme involved in this reaction is an aminotransferase.
-ketoacid + glutamate amino acid + -ketoglutarate
Glutamate itself is formed by amination of -ketoglutarate:
-ketoglutarate + NH+
4 glutamate
Nitrogen fixation: Microorganisms use ATP and a powerful reductant to
reduce atmospheric nitrogen to ammonia
Microorganisms use ATP and reduced ferredoxin, a powerful reductant, to reduce N2 to NH3. An iron-molybdenum
cluster in nitrogenase deftly catalyzes the fixation of N2, a very inert molecule. Higher organisms consume the fixed
nitrogen to synthesize amino acids, nucleotides, and other nitrogen-containing biomolecules. The major points of
entry of NH4+ into metabolism are glutamine or glutamate.
Amino acids are made from intermediates of the citric acid cycle and other
major pathways
Of the basic set of 20 amino acids (not counting selenocysteine), there are 8 that human beings cannot synthesize. In
addition, the amino acids arginine, cysteine, glycine, glutamine, histidine, proline, serine, and tyrosine are considered
conditionally essential, meaning they are not normally required in the diet, but must be supplied exogenously to
specific populations that do not synthesize it in adequate amounts.
[1][2]
For example, enough arginine is synthesized
Amino acid synthesis
289
by the urea cycle to meet the needs of an adult but perhaps not those of a growing child. Amino acids that must be
obtained from the diet are called essential amino acids. Nonessential amino acids are produced in the body. The
pathways for the synthesis of nonessential amino acids are quite simple. Glutamate dehydrogenase catalyzes the
reductive amination of -ketoglutarate to glutamate. A transamination reaction takes place in the synthesis of most
amino acids. At this step, the chirality of the amino acid is established. Alanine and aspartate are synthesized by the
transamination of pyruvate and oxaloacetate, respectively. Glutamine is synthesized from NH4+ and glutamate, and
asparagine is synthesized similarly. Proline and arginine are derived from glutamate. Serine, formed from
3-phosphoglycerate, is the precursor of glycine and cysteine. Tyrosine is synthesized by the hydroxylation of
phenylalanine, an essential amino acid. The pathways for the biosynthesis of essential amino acids are much more
complex than those for the nonessential ones. Activated Tetrahydrofolate, a carrier of one-carbon units, plays an
important role in the metabolism of amino acids and nucleotides. This coenzyme carries one-carbon units at three
oxidation states, which are interconvertible: most reducedmethyl; intermediatemethylene; and most
oxidizedformyl, formimino, and methenyl. The major donor of activated methyl groups is S-adenosylmethionine,
which is synthesized by the transfer of an adenosyl group from ATP to the sulfur atom of methionine.
S-Adenosylhomocysteine is formed when the activated methyl group is transferred to an acceptor. It is hydrolyzed to
adenosine and homocysteine, the latter of which is then methylated to methionine to complete the activated methyl
cycle.
Cortisol inhibits protein synthesis.
[3]
Amino acid biosynthesis is regulated by feedback inhibition
Most of the pathways of amino acid biosynthesis are regulated by feedback inhibition, in which the committed step
is allosterically inhibited by the final product. Branched pathways require extensive interaction among the branches
that includes both negative and positive regulation. The regulation of glutamine synthetase from E. coli is a striking
demonstration of cumulative feedback inhibition and of control by a cascade of reversible covalent modifications.
Additional regulation based on amino acid families
-ketoglutarate Family
A: The -ketoglutarate family of amino acid synthesis (synthesis of glutamate, glutamine, proline and arginine)
begins with -ketoglutarate, an intermediate in the Citric Acid Cycle. The concentration of -ketoglutarate is
dependent on the activity and metabolism within the cell along with the regulation of enzymatic activity. In E. coli
citrate synthase, the enzyme involved in the condensation reaction initiating the Citric Acid Cycle is strongly
inhibited by -ketoglutarate feedback inhibition and can be inhibited by DPNH as well high concentrations of
ATP.
[4]
This is one of the initial regulations of the -ketoglutarate family of amino acid synthesis.
B: The regulation of the synthesis of glutamate from -ketoglutarate is subject to regulatory control of the Citric
Acid Cycle as well as mass action dependent on the concentrations of reactants involved due to the reversible nature
of the transamination and glutamate dehydrogenase reactions.
[4]
C: The conversion of glutamate to glutamine is regulated by glutamine synthetase (GS) and is a highly significant
step in nitrogen metabolism.
[4]
This enzyme is regulated by at least four different mechanisms: 1. Repression and
depression due to nitrogen levels; 2. Activation and inactivation due to enzymatic forms (taut and relaxed); 3.
Cumulative feedback inhibition through end product metabolites; and 4. Alterations of the enzyme due to
adenylation and deadenylation.
[4]
In rich nitrogenous media or growth conditions containing high quantities of
ammonia there is a low level of GS, whereas in limiting quantities of ammonia the specific activity of the enzyme is
20-fold higher.
[4]
The confirmation of the enzyme plays a role in regulation depending on if GS is in the taut or
relaxed form. The taut form of GS is fully active but, the removal of manganese converts the enzyme to the relaxed
state. The specific conformational state occurs based on the binding of specific divalent cations and is also related to
Amino acid synthesis
290
adenylation.
[4]
The feedback inhibition of GS is due to a cumulative feedback due to several metabolites including
L-tryptophan, L-histidine, AMP, CTP, glucosamine-6-phosphate and carbamyl phosphate, alanine, and glycine.
[4]
An excess of any one product does not individually inhibit the enzyme but a combination or accumulation of all the
end products have a strong inhibitory effect on the synthesis of glutamine.
[4]
Glutamine synthase actiivty is also
inhibited via adenylation. The adenylation actividty is catalyzed by the bifunctional adenylyltransferasl/adenylyl
removal (AT/AR) enzyme. Glutamine and a regulatory protein called PII act together to stimulate adenylation.
[5]
D: The regulation of proline biosynthesis can be dependent on the initial controlling step through negative feedback
inhibition.
[]
In E. coli, proline allosterically inhibits Glutamate 5-kinase which catalyzes the reaction from
L-glutamate to an unstable intermediate L--Glutamyl phosphate.
[]
E: Arginine synthesis also utilizes negative feedback as well as repression through a repressor encoded by the gene
argR. The gene product of argR, ArgR an aporepressor, and arginine as a corepressor affect the operon of arginine
biosynthesis. The degree of repression is determined by the concentrations of the repressor protein and corepressor
level.
[6]
Erythrose 4-phosphate and Phosphoenolpyruvate Family
Regulation of the Aromatic Amino Acids
Phenylalanine, tyrosine, and tryptophan are known as the aromatic amino acids. The synthesis of all three share a
common beginning to their pathways; the formation of chorismate from phosphoenolpyruvate (PEP) and erythrose
4- phosphate (E4P). The first step, condensation of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) from
PEP/E4P, uses three isoenzymes AroF, AroG, and AroH. Each one of these has its synthesis regulated from tyrosine,
phenylalanine, and tryptophan, respectively. These isoenzymes all have the ability to help regulate synthesis of
DAHP by the method of feedback inhibition. This acts in the cell by monitoring the concentrations of each of the
three aromatic amino acids. When there is too much of any one of them, that one will allosterically control the
DAHP synthetase by turning it off. With the first step of the common pathway shut off, synthesis of the three
amino acids can not proceed. The rest of the enzymes in the common pathway (conversion of DAHP to chorismate)
appear to be synthesized constitutively, except for shikimate kinase which can be inhibited by shikimate through
linear mixed-type inhibition. If too much shikimate has been produced then it can bind to shikimate kinase to stop
further production.
Besides the regulations described above, each amino acids terminal pathway can be regulated. These terminal
pathways progress from chorismate to the final end product, either tyrosine, phenylalanine, or tryptophan. Each one
of these pathways is regulated in a similar fashion to the common pathway; with feedback inhibition on the first
committed step of the pathway.
Tyrosine and phenylalanine share the same initial step in their terminal pathways, chorismate converted to
prephenate which is converted to an amino acid-specific intermediate. This process is mediated by a phenylalanine
(PheA) or tyrosine (TyrA) specific chorismate mutase-prephenate dehydrogenase. The reason for the amino
acid-specific enzymes is because PheA uses a simple dehydrogenase to convert prephenate to phenylpyruvate, while
TyrA uses a NAD-dependent dehydrogenase to make 4-hydroxylphenylpyruvate. Both PheA and TyrA are feedback
inhibited by their respective amino acids. Tyrosine can also be inhibited at the transcriptional level by the TyrR
repressor. TyrR binds to the TyrR boxes on the operon near the promoter of the gene that it wants to repress.
In the terminal-tryptophan synthesis pathway, the initial step converts chorismate to anthranilate using anthranilate
synthase. This enzyme requires either ammonia or glutamine as the amino group donor. Anthranilate synthase is
regulated by the gene products of trpE and trpD. trpE encodes the first subunit, which binds to chorismate and moves
the amino group from the donor to chorismate. trpD encodes the second subunit, which is simply used to bind
glutamine and use it as the amino group donor so that the amine group can transfer to the chorismate. Anthranilate
synthase is also regulated by feedback inhibition. The finished product of tryptophan, once produced in great enough
quantities, is able to act as the co-repressor to the TrpR repressor which represses expression of the trp operon.
Amino acid synthesis
291
Oxaloacetate/Aspartate Family
The oxaloacetate/aspartate family of amino acids is composed of lysine, asparagine, methionine, threonine, and
isoleucine. Aspartate can be converted into lysine, asparagine, methionine and threonine. Threonine also gives rise to
isoleucine. All of these amino acids contain different mechanisms for their regulation, some being more complex
than others. All the enzymes in this biosynthetic pathway are subject to regulation via feedback inhibition and/or
repression at the genetic level. As is typical in highly branched metabolic pathways, there is additional regulation at
each branch point of the pathway. This type of regulatory scheme allows control over the total flux of the aspartate
pathway in addition to the total flux of individual amino acids. The aspartate pathway uses L-aspartic acid as the
precursor for the biosynthesis of one fourth of the building block amino acids. Without this pathway, protein
synthesis would not be possible.
Aspartate
The enzyme aspartokinase, which catalyzes the phosphorylation of aspartate and initiates its conversion into other
amino acids, can be broken up into 3 isozymes, AK-I, II and III. AK-I is feed-back inhibited by threonine, while
AK-II and III are inhibited by lysine. As a sidenote, AK-III catalyzes the phosphorylation of aspartic acid that is the
commitment step in this biosynthetic pathway. The higher the concentration of threonine or lysine, the more
aspartate kinase becomes downregulated.
Lysine
Lysine is synthesized from aspartate via the diaminopimelate (DAP) pathway. The initial two stages of the DAP
pathway are catalyzed by aspartokinase and aspartate semialdehyde dehydrogenase and play a key role in the
biosynthesis of lysine, threonine and methionine. There are two bifuctional aspartokinase/homoserine
dehydrogenases, ThrA and MetL, in addition to a monofunctional aspartokinase, LysC. Transcription of
aspartokinase genes is regulated by concentrations of the subsequently produced amino acids, lysine, threonine and
methionine. The higher these amino acids concentrations, the less the gene is transcribed. ThrA and LysC are also
feed-back inhibited by threonine and lysine. Finally, DAP decarboxylase LysA mediates the last step of the lysine
synthesis and is common for all studied bacterial species. The formation of aspartate kinase (AK), which catalyzes
the phosphorylation of aspartate and initiates its conversion into other amino acids, is also inhibited by both lysine
and threonine, which prevents the formation of the amino acids derived from aspartate. Additionally, high lysine
concentrations inhibit the activity of dihydrodipicolinate synthase (DHPS). So, in addition to inhibiting the first
enzyme of the aspartate families biosynthetic pathway, lysine also inhibits the activity of the first enzyme after the
branch point, i.e. the enzyme that is specific for lysines own synthesis.
Asparagine
There are two different asparagine synthetases found in bacterial species. These two synthetases, which are both
referred to as the AsnC protein, are coded for by two genes: AsnA and AsnB. AsnC is autogenously regulated, which
is where the product of a structural gene regulates the expression of the operon in which the genes reside. The
stimulating effect of AsnC on AsnA transcription is downregulated by asparagine. However, the autoregulation of
AsnC is not affected by asparagine.
Methionine
Methionine synthesis is under tight regulation. The repressor protein MetJ, in cooperation with the corepressor
protein S-adenosyl-methionine, mediates the repression of methionines biosynthetic pathway. Recently, a new
regulator focus, MetR has been identified. The MetR protein is required for MetE and MetH gene expression and
functions as a transactivator of transcription for these genes. MetR transcriptional activity is regulated by
homocystein, which is the metabolic precursor of methionine. It is also known that vitamin B12 can repress MetE
gene expression, which is mediated by the MetH holoenzyme.
Threonine
Amino acid synthesis
292
The biosynthesis of threonine is regulated via allosteric regulation of its precursor, homoserine, by structurally
altering the enzyme homoserine dehydrogenase. This reaction occurs at a key branch point in the pathway, with the
substrate homoserine serving as the precursor for the biosynthesis of lysine, methionine, threonin and isoleucine.
High levels of threonine result in low levels of homoserine synthesis. The synthesis of aspartate kinase (AK), which
catalyzes the phosphorylation of aspartate and initiates its conversion into other amino acids, is feed-back inhibited
by lysine, isoleucine, and threonine, which prevents the synthesis of the amino acids derived from aspartate. So, in
addition to inhibiting the first enzyme of the aspartate families biosynthetic pathway, threonine also inhibits the
activity of the first enzyme after the branch point, i.e. the enzyme that is specific for threonines own synthesis.
Isoleucine
The enzymes threonine deaminase, dihydroxy acid dehydrase and transaminase are controlled by end-product
regulation. I.e. the presence of isoleucine will downregulate the formation of all three enzymes, resulting in the
downregulation of threonine biosynthesis. High concentrations of isoleucine also result in the downregulation of
aspartates conversion into the aspartyl-phosphate intermediate, hence halting further biosynthesis of lysine,
methionine, threonine, and isoleucine.
The Ribose 5-phosphate family
Regulation of Histidine in Escherichia coli
The synthesis of histidine in "E. coli" is a complex pathway involving 10 reactions and 10 enzymes. Synthesis begins
with 5-phosphoribosyl-pyrophosphate (PRPP) and finishes with histidine and occurs through the reactions of the
following enzymes:
[]
HisG-> HisE/HisI-> HisA-> HisH-> HisF-> HisB-> HisC-> HisB-> HisD (HisE/I and HisB are both bifunctional
enzymes)
All of the enzymes are coded for on the his operon. This operon has a distinct block of the leader sequence, called
block 1:
Met-Thr-Arg-Val-Gln-Phe-Lys-His-His-His-His-His-His-His-Pro-Asp
This leader sequence is very important for the regulation of histidine in "E. coli". The his operon operates under a
system of coordinated regulation where all the gene products will be repressed or depressed equally. The main factor
in the repression or derepression of histidine synthesis is the concentration of histidine charged tRNAs. The
regulation of histidine is actually quite simple considering the complexity of its biosynthesis pathway and, it closely
resembles regulation of tryptophan. In this system the full leader sequence has 4 blocks of complementary strands
that can form hairpin loops structures.
[]
Block one, shown above, is the key to regulation. When histidine charged
tRNA levels are low in the cell the ribosome will stall at the string of His residues in block 1. This stalling of the
ribosome will allow complementary strands 2 and 3 to form a hairpin loop. The loop formed by strands 2 and 3
forms an anti-terminator and translation of the his genes will continue and histidine will be produced. However when
histidine charged tRNA levels are high the ribosome will not stall at block 1, this will not allow strands 2 and 3 to
form a hairpin. Instead strands 3 and 4 will form a hairpin loop further downstream of the ribosome. The hairpin
loop formed by strands 3 and 4 is a terminating loop, when the ribosome comes into contact with the loop, it will be
knocked off the transcript. When the ribosome is removed the his genes will not be translated and histidine will not
be produced by the cell.
[7]
Amino acid synthesis
293
The 3-phosphoglycerate family
Regulation of Serine in Escherichia coli
Serine is the first amino acid in this family to be produced; it is then modified to produce both glycine and cysteine
(and many other biologically important molecules). Serine is formed from 3-phosphoglycerate in the following
pathway:
3-phosphoglycerate-> phosphohydroxyl-pyruvate-> phosphoserine-> serine
The conversion from 3-phosphoglycerate to phosphohydroxyl-pyruvate is achieved by the enzyme phosphoglycerate
dehydrogenase. This enzyme is the key regulatory step in this pathway. Phosphoglycerate dehydrogenase is
regulated by the concentration of serine in the cell. At high concentrations this enzyme will be inactive and serine
will not be produced. At low concentrations of serine the enzyme will be fully active and serine will be produced by
the bacterium.
[8]
Since serine is the first amino acid produced in this family both glycine and cysteine will be
regulated by the available concentration of serine in the cell.
[9]
Regulation of Glycine in Escherichia coli
Glycine is synthesized from serine using the enzyme serine hydromethyltransferase (SHMT), which is coded by the
gene glyA. The enzyme effectively removes a hydroxyl group from serine and replaces it with a methyl group to
yield glycine. This reaction is the only way E. coli can produce glycine. The regulation of glyA is very complex and
is known to incorporate serine, glycine, methionine, purines, thymine, and folates however, the full mechanism has
yet to be elucidated.
[]
The methionine gene product MetR and the methionine intermediate homocysteine are known
to positively regulate glyA. Homocysteine is a coactivator of glyA and must act in concert with MetR.
[][10]
On the
other hand, PurR, a protein which plays a role in purine synthesis and S-adeno-sylmethionine are known to down
regulate glyA. PurR binds directly to the control region of glyA and effectively turns the gene off so that glycine will
not be produced by the bacterium.
Regulation of Cysteine in Escherichia coli
Cysteine is a very important molecule for a bacteriums survival. This amino acid harbors a sulfur atom and can
actively participate in disulfide bond formation. The genes required for the synthesis of cysteine are coded for on the
cys regulon. The integration of sulfur into the molecule is positively regulated by CysB. CysB is the main focus of
cysteine regulation. Effective inducers of this regulon are N-acetyl-serine (NAS) and very small amounts of reduced
sulfur. CysB functions by binding to DNA half sites on the cys regulon. These half sites differ in quantity and
arrangement depending on the promoter of interest. There is however one half site that is conserved. It lies just
upstream of the -35 site of the promoter. There are also multiple accessory sites depending on the promoter. In the
absence of the inducer, NAS, CysB will bind the DNA and cover many of the accessory half sites. Without the
accessory half sites the regulon cannot be transcribed and cysteine will not be produced. It is believed that the
presence of NAS causes CysB to undergo a conformational change. This conformational change allows CysB to bind
properly to all the half sites and causes the recruitment of the RNA polymerase. The RNA polymerase will then
transcribe the cys regulon and cysteine will be produced.
Further regulation is required for this pathway, however. CysB can actually down regulate its own transcription by
binding to its own DNA sequence and blocking the RNA polymerase. In this case NAS will act to disallow the
binding of CysB to its own DNA sequence. OAS is a precursor of NAS, cysteine itself can inhibit CysE which
functions to create OAS. Without the necessary OAS, NAS will not be produced and cysteine will not be produced.
There are two other negative regulators of cysteine. These are the molecules sulfide and thiosulfate, they act to bind
to CysB and they compete with NAS for the binding of CysB.
[11]
Amino acid synthesis
294
The Pyruvate Family
Pyruvate is the end result of glycolysis and can feed into both the TCA cycle and fermentation processes.
[12]
Reactions beginning with either one or two molecules of pyruvate cause the synthesis of alanine, valine, and leucine.
Feedback inhibition of final products is the main method of inhibition, and, in E. coli, the ilvEDA operon also plays a
part in this regulation.
Alanine Regulation
Alanine is produced by the transamination of one molecule of pyruvate using two alternate steps: 1) conversion of
glutamate to -ketoglutarate using a glutamate-alanine transaminase, and 2) conversion of valine to
-ketoisovalerate via Transaminase C.
Not much is known about the regulation of alanine synthesis. The only definite method is the bacteriums ability to
repress Transaminase C activity by either valine or leucine (see ilvEDA operon). Other than that, alanine
biosynthesis does not seem to be regulated.
[13]
Valine Regulation
Valine is produced by a four-enzyme pathway. It begins with the reaction of two pyruvate molecules catalyzed by
Acetohydroxy acid synthase yielding -acetolactate. Step two is the NADPH+ + H+ - dependent reduction of
-acetolactate and migration of the methane groups to produce , -dihydroxyisovalerate. This is catalyzed by
Acetohydroxy isomeroreductase. The third reaction is the dehydration reaction of , -dihydroxyisovalerate
catalyzed by Dihydroxy acid dehydrase resulting in -ketoisovalerate. Finally, a transamination catalyzed either by
an alanine-valine transaminase or a glutamate-valine transaminase results in valine.
Valine performs feedback inhibition to inhibit the Acetohydroxy acid synthase used to combine the first two
pyruvate molecules.
[14]
Leucine Regulation
The leucine synthesis pathway diverges from the valine pathway beginning with -ketoisovalerate.
-Isopropylmalate synthase reacts with this substrate and Acetyl CoA to produce -isopropylmalate. An isomerase
then isomerizes -isopropylmalate to -isopropylmalate. The third step is the NAD+-dependent oxidation of
-isopropylmalate via the action of a dehydrogenase to yield -ketoisocaproate. Finally is the transamination via the
action of a glutamate-leucine transaminase to result in leucine.
Leucine, like valine, regulates the first step of its pathway by inhibiting the action of the -Isopropylmalate
synthase.
[14]
Because leucine is synthesized by a diversion from the valine synthetic pathway, the feedback
inhibition of valine on its pathway also can inhibit the synthesis of leucine.
ilvEDA operon in E. coli
The genes that encode both the Dihydroxy acid dehydrase used in the creation of -ketoisovalerate and
Transaminase E, as well as other enzymes are encoded on the ilvEDA operon. This operon is bound and inactivated
by valine, leucine, and isoleucine. (Isoleucine is not a direct derivative of pyruvate, but is produced by the use of
many of the same enzymes used to produce valine and, indirectly, leucine.) When one of these amino acids is
limited, the gene furthest from the amino-acid binding site of this operon can be transcribed. When a second of these
amino acids is limited, the next-closest gene to the binding site can be transcribed, and so forth.
[14]
Amino acids are precursors of many biomolecules
Amino acids are precursors of a variety of biomolecules. Glutathione (-Glu-Cys-Gly) serves as a sulfhydryl buffer
and detoxifying agent. Glutathione peroxidase, a selenoenzyme, catalyzes the reduction of hydrogen peroxide and
organic peroxides by glutathione. Nitric oxide, a short-lived messenger, is formed from arginine. Porphyrins are
synthesized from glycine and succinyl CoA, which condense to give -aminolevulinate. Two molecules of this
intermediate become linked to form porphobilinogen. Four molecules of porphobilinogen combine to form a linear
Amino acid synthesis
295
tetrapyrrole, which cyclizes to uroporphyrinogen III. Oxidation and side-chain modifications lead to the synthesis of
protoporphyrin IX, which acquires an iron atom to form heme.
[15]
References
[3] Manchester, K.L., Sites of Hormonal Regulation of Protein Metabolism. p. 229, Mammalian Protein [Munro, H.N., Ed.]. Academic Press,
New York. On p273.
[4] Shapiro, Bennett M. and E. R. Stadtman (1970). The Regulation of Glutamine Synthesis in Microorganisms. Annu. Rev. Microbiol.
24:501-524
[5] [5] White, David. (2007). The Physiology and Biochemistry of Prokaryotes, Third Edition. Oxford University Press, New York
[6] Maas, Wener K. (1991). The Regulation of Arginine Biosynthesis: Its Contribution to Understanding the Control of Gene Expression.
Genetics 128: 489-494
[12] [12] Lehninger, Albert L.; Nelson, David L.; Cox, Michael M. (2008). Principles of Biochemistry (5th ed.). New York, NY: W.H. Freeman and
Company. p. 528. ISBN 978-0-7167-7108-1
[13] Umbarger, H. E. (1978). Amino acid biosynthesis and its regulation. Annual Reviews Biochemistry, 47, 533-606. Retrieved from http:/ /
www.annualreviews.org/ doi/ pdf/ 10.1146/ annurev.bi. 47. 070178. 002533
[14] [14] ibid
[15] Biochemistry. Berg, Jeremy M.; Tymoczko, John L.; and Stryer, Lubert. New York: W. H. Freeman and Co. ; c2002
External links
NCBI Bookshelf Free Textbook Access (http:/ / www. ncbi. nlm. nih. gov/ books/ bv. fcgi?rid=stryer. TOC&
depth=2)
Nucleotide
Structural elements of common nucleic acid constituents. The compounds marked
nucleoside monophosphate, nucleoside diphosphate and nucleoside triphosphate are all
nucleotides.
Nucleotides are biological molecules
that form the building blocks of
nucleic acids (DNA and RNA) and
serve to carry packets of energy within
the cell (ATP). In the form of the
nucleoside triphosphates (ATP, GTP,
CTP and UTP), nucleotides play
central roles in metabolism.
[1]
In
addition, nucleotides participate in cell
signaling (cGMP and cAMP), and are
incorporated into important cofactors
of enzymatic reactions (e.g. coenzyme A, FAD, FMN, NAD, and NADP
+
).
Nucleotide
296
Structure
Ribose structure indicating numbering of carbon
atoms
A nucleotide is composed of a nucleobase (nitrogenous base), a
five-carbon sugar (either ribose or 2-deoxyribose), and one or more
phosphate groups.
[2]
Without the phosphate group, the nucleobase and
sugar compose a nucleoside. A nucleotide can thus also be called a
nucleoside monophosphate. The phosphate groups form bonds with
either the 2, 3, or 5-carbon of the sugar, with the 5-carbon site most
common. Cyclic nucleotides form when the phosphate group is bound
to two of the sugar's hydroxyl groups.
[1]
Nucleotides contain either a
purine or a pyrimidine base. Ribonucleotides are nucleotides in which
the sugar is ribose. Deoxyribonucleotides are nucleotides in which the
sugar is deoxyribose.
Nucleic acids are polymeric macromolecules made from nucleotide
monomers. In DNA, the purine bases are adenine and guanine, while
the pyrimidines are thymine and cytosine. RNA uses uracil in place of thymine. Adenine always pairs with thymine
by 2 hydrogen bonds, while guanine pairs with cytosine through 3 hydrogen bonds, each due to their unique
structures.
Synthesis
Nucleotides can be synthesized by a variety of means both in vitro and in vivo.
In vivo, nucleotides can be synthesized de novo or recycled through salvage pathways.
[3]
The components used in de
novo nucleotide synthesis are derived from biosynthetic precursors of carbohydrate and amino acid metabolism, and
from ammonia and carbon dioxide. The liver is the major organ of de novo synthesis of all four nucleotides. De novo
synthesis of pyrimidines and purines follows two different pathways. Pyrimidines are synthesized first from aspartate
and carbamoyl-phosphate in the cytoplasm to the common precursor ring structure orotic acid, onto which a
phosphorylated ribosyl unit is covalently linked. Purines, however, are first synthesized from the sugar template onto
which the ring synthesis occurs. For reference, the syntheses of the purine and pyrimidine nucleotides are carried out
by several enzymes in the cytoplasm of the cell, not within a specific organelle. Nucleotides undergo breakdown
such that useful parts can be reused in synthesis reactions to create new nucleotides.
In vitro, protecting groups may be used during laboratory production of nucleotides. A purified nucleoside is
protected to create a phosphoramidite, which can then be used to obtain analogues not found in nature and/or to
synthesize an oligonucleotide.
Nucleotide
297
Pyrimidine ribonucleotide synthesis
The synthesis of Uridine monophosphateUMP.The color scheme is as follows: enzymes,
coenzymes, substrate names, inorganic molecules
The synthesis of the pyrimidines CTP
and UTP occurs in the cytoplasm and
starts with the formation of carbamoyl
phosphate from glutamine and CO
2
.
Next, aspartate undergoes a
condensation reaction with
carbamoyl-phosphate to form orotic
acid. In a subsequent cyclization
reaction, the enzyme Aspartate
carbamoyltransferase forms
N-carbamoyl-aspartate which is
converted into dihydroorotic acid by
dihydroorotase. The latter is converted
to orotate by dihydroorotate oxidase.
The net reaction is:
(S)-Dihydroorotate + O
2
= Orotate +
H
2
O
2
Orotate is covalently linked with a
phosphorylated ribosyl unit. The
covalent linkage between the ribose
and pyrimidine occurs at position C
1
[4]
of the ribose unit, which contains a
pyrophosphate, and N
1
of the pyrimidine ring. Orotate phosphoribosyltransferase (aka "PRPP transferase") catalyzes
the net reaction yielding orotidine monophosphate (OMP):
Orotate + 5-Phospho--D-ribose 1-diphosphate (aka. "PRPP") = Orotidine 5'-phosphate + Pyrophosphate
Orotidine-5-phosphate is decarboxylated by orotidine-5'-phosphate decarboxylase to form uridine monophosphate
(UMP). PRPP transferase catalyzes both the ribosylation and decarboxylation reactions, forming UMP from orotic
acid in the presence of PRPP. It is from UMP that other pyrimidine nucleotides are derived. UMP is phosphorylated
by two kinases to uridine triphosphate (UTP) via two sequential reactions with ATP. First the diphosphate form UDP
is produced, which in turn is phosphorylated to UTP. Both steps are fueled by ATP hydrolysis:
ATP + UMP = ADP + UDP
UDP + ATP = UTP + ADP
CTP is subsequently formed by amination of UTP by the catalytic activity of CTP synthetase. Glutamine is the NH
3
donor and the reaction is fueled by ATP hydrolysis, too:
UTP + Glutamine + ATP + H
2
O = CTP + ADP + P
i
Cytidine monophosphate (CMP) is derived from cytidine triphosphate (CTP) with subsequent loss of two
phosphates.
[5]

[6]
Nucleotide
298
Purine ribonucleotide synthesis
The atoms which are used to build the purine nucleotides come from a variety of sources:
The synthesis of IMP. The color scheme is as follows: enzymes, coenzymes, substrate
names, metal ions, inorganic molecules
The biosynthetic origins of purine ring atoms
N
1
arises from the amine group of Asp
C
2
and C
8
originate from formate
N
3
and N
9
are contributed by the amide group of
Gln
C
4
, C
5
and N
7
are derived from Gly
C
6
comes from HCO
3
-
(CO
2
)
The de novo synthesis of purine nucleotides by which these precursors are incorporated into the purine ring proceeds
by a 10-step pathway to the branch-point intermediate IMP, the nucleotide of the base hypoxanthine. AMP and GMP
are subsequently synthesized from this intermediate via separate, two-step pathways. Thus, purine moieties are
initially formed as part of the ribonucleotides rather than as free bases.
Six enzymes take part in IMP synthesis. Three of them are multifunctional:
GART (reactions 2, 3, and 5)
PAICS (reactions 6, and 7)
ATIC (reactions 9, and 10)
The pathway starts with the formation of PRPP. PRPS1 is the enzyme that activates R5P, which is formed primarily
by the pentose phosphate pathway, to PRPP by reacting it with ATP. The reaction is unusual in that a
pyrophosphoryl group is directly transferred from ATP to C
1
of R5P and that the product has the configuration
about C1. This reaction is also shared with the pathways for the synthesis of Trp, His, and the pyrimidine
nucleotides. Being on a major metabolic crossroad and requiring much energy, this reaction is highly regulated.
Nucleotide
299
In the first reaction unique to purine nucleotide biosynthesis, PPAT catalyzes the displacement of PRPP's
pyrophosphate group (PP
i
) by an amide nitrogen donated from either glutamine (N), glycine (N&C), aspartate (N),
folic acid (C
1
), or CO
2
. This is the committed step in purine synthesis. The reaction occurs with the inversion of
configuration about ribose C
1
, thereby forming -5-phosphorybosylamine (5-PRA) and establishing the anomeric
form of the future nucleotide.
Next, a glycine is incorporated fueled by ATP hydrolysis and the carboxyl group forms an amine bond to the NH
2
previously introduced. A one-carbon unit from folic acid coenzyme N
10
-formyl-THF is then added to the amino
group of the substituted glycine followed by the closure of the imidazole ring. Next, a second NH
2
group is
transferred from a glutamine to the first carbon of the glycine unit. A carboxylation of the second carbon of the
glycin unit is concomittantly added. This new carbon is modified by the additional of a third NH
2
unit, this time
transferred from an aspartate residue. Finally, a second one-carbon unit from formyl-THF is added to the nitrogen
group and the ring covalently closed to form the common purine precursor inosine monophosphate (IMP).
Inosine monophosphate is converted to adenosine monophosphate in two steps. First, GTP hydrolysis fuels the
addition of aspartate to IMP by adenylosuccinate synthase, substituting the carbonyl oxygen for a nitrogen and
forming the intermediate adenylosuccinate. Fumarate is then cleaved off forming adenosine monophosphate. This
step is catalyzed by adenylosuccinate lyase.
Inosine monophosphate is converted to guanosine monophosphate by the oxidation of IMP forming xanthylate,
followed by the insertion of an amino group at C
2
. NAD
+
is the electron acceptor in the oxidation reaction. The
amide group transfer from glutamine is fueled by ATP hydrolysis.
Pyrimidine and purine degradation
In humans, pyrimidine rings (C, T, U) can be degraded completely to CO
2
and NH
3
(urea excretion). That having
been said, purine rings (G, A) cannot. Instead they are degraded to the metabolically inert uric acid which is then
excreted from the body. Uric acid is formed when GMP is split into the base guanine and ribose. Guanine is
deaminated to xanthine which in turn is oxidized to uric acid. This last reaction is irreversible. Similarly, uric acid
can be formed when AMP is deaminated to IMP from which the ribose unit is removed to form hypoxanthine.
Hypoxanthine is oxidized to xanthine and finally to uric acid. Instead of uric acid secretion, guanine and IMP can be
used for recycling purposes and nucleic acid synthesis in the presence of PRPP and aspartate (NH
3
donor).
Length unit
Nucleotide (abbreviated "nt") is a common unit of length for single-stranded nucleic acids, similar to how base pair
is a unit of length for double-stranded nucleic acids.
Abbreviation codes for degenerate bases
The IUPAC has designated the symbols for nucleotides.
[]
Apart from the five (A, G, C, T/U) bases, often degenerate
bases are used especially for designing PCR primers. These nucleotide codes are listed here. Some primer sequences
may also include the character "I", which codes for the non-standard nucleotide Inosine. Inosine occurs in tRNAs,
and will pair with Adenine, Cytosine, or Thymine. This character does not appear in the following table however,
because it does not represent a degeneracy. While Inosine can serve a similar function as the degeneracy "H", it is an
actual nucleotide, rather than a representation of a mix of nucleotides that covers each possible pairing needed.
Nucleotide
300
Symbol
[]
Description Bases represented
A adenosine A 1
C cytidine C
G guanosine G
T thymidine T
U uridine U
W weak A T 2
S strong C G
M amino A C
K keto G T
R purine A G
Y pyrimidine C T
B not A (B comes after A) C G T 3
D not C (D comes after C) A G T
H not G (H comes after G) A C T
V not T (V comes after T and U) A C G
N or - any base (not a gap) A C G T 4
References
[1] Alberts B, Johnson A, Lewis J, Raff M, Roberts K & Wlater P (2002). Molecular Biology of the Cell (4th ed.). Garland Science. ISBN
0-8153-3218-1. pp. 120121.
[4] See IUPAC nomenclature of organic chemistry for details on carbon residue numbering
External links
Abbreviations and Symbols for Nucleic Acids, Polynucleotides and their Constituents (http:/ / www. chem. qmul.
ac. uk/ iupac/ misc/ naabb. html) (IUPAC)
Provisional Recommendations 2004 (http:/ / www. iupac. org/ reports/ provisional/ abstract04/ BB-prs310305/
Chapter10. pdf) (IUPAC)
Chemistry explanation of nucleotide structure (http:/ / dl. clackamas. cc. or. us/ ch106-09/ nucleoti. htm)
Urea cycle
301
Urea cycle
The urea cycle (also known as the ornithine cycle) is a cycle of biochemical reactions occurring in many animals
that produces urea ((NH
2
)
2
CO) from ammonia (NH
3
). This cycle was the first metabolic cycle discovered (Hans
Krebs and Kurt Henseleit, 1932), five years before the discovery of the TCA cycle. In mammals, the urea cycle takes
place primarily in the liver, and to a lesser extent in the kidney.
Function
Organisms that cannot easily and quickly remove ammonia usually have to convert it to some other substance, like
urea or uric acid, which are much less toxic. Insufficiency of the urea cycle occurs in some genetic disorders (inborn
errors of metabolism), and in liver failure. The result of liver failure is accumulation of nitrogenous waste, mainly
ammonia, which leads to hepatic encephalopathy.
Reactions
The urea cycle consists of five reactions: two mitochondrial and three cytosolic. The cycle converts two amino
groups, one from NH
4
+
and one from Asp, and a carbon atom from HCO
3

, to the relatively nontoxic excretion

product urea at the cost of four "high-energy" phosphate bonds (3 ATP hydrolyzed to 2 ADP and one AMP).
Ornithine is the carrier of these carbon and nitrogen atoms.
Reactions of the urea cycle
Step Reactants Products Catalyzed by Location
1
NH
4
+
+ HCO
3

+ 2ATP
carbamoyl phosphate + 2ADP + P
i
CPS1 mitochondria
2 carbamoyl phosphate + ornithine citrulline + P
i
OTC mitochondria
3 citrulline + aspartate + ATP argininosuccinate + AMP + PP
i
ASS cytosol
4 argininosuccinate Arg + fumarate ASL cytosol
5 Arg + H
2
O ornithine + urea ARG1 cytosol
The reactions of the urea cycle
Urea cycle
302
1 L-ornithine
2 carbamoyl phosphate
3 L-citrulline
4 argininosuccinate
5 fumarate
6 L-arginine
7 urea
L-Asp L-aspartate
CPS-1 carbamoyl phosphate synthetase I
OTC Ornithine transcarbamoylase
ASS argininosuccinate synthetase
ASL argininosuccinate lyase
ARG1 arginase 1
In the first reaction, NH
4
+
+ HCO
3

is equivalent to NH
3
+ CO
2
+ H
2
O.
Thus, the overall equation of the urea cycle is:
NH
3
+ CO
2
+ aspartate + 3 ATP + 2 H
2
O urea +
fumarate + 2 ADP + 2 P
i
+ AMP + PP
i
Since fumarate is obtained by removing NH
3
from
aspartate (by means of reactions 3 and 4), and PP
i
+ H
2
O
2 P
i
, the equation can be simplified as follows:
2 NH
3
+ CO
2
+ 3 ATP + H
2
O urea + 2 ADP + 4 P
i
+ AMP
Note that reactions related to the urea cycle also cause the production of 2 NADH, so the urea cycle releases slightly
more energy than it consumes. These NADH are produced in two ways:
One NADH molecule is reduced by the enzyme glutamate dehydrogenase in the conversion of glutamate to
ammonium and -ketoglutarate. Glutamate is the non-toxic carrier of amine groups. This provides the ammonium
ion used in the initial synthesis of carbamoyl phosphate.
The fumarate released in the cytosol is converted to malate by cytosolic fumarase. This malate is then converted
to oxaloacetate by cytosolic malate dehydrogenase, generating a reduced NADH in the cytosol. Oxaloacetate is
one of the keto acids preferred by transaminases, and so will be recycled to aspartate, maintaining the flow of
nitrogen into the urea cycle.
The two NADH produced can provide energy for the formation of 4 ATP(cytosolic NADH provides only 1.5 ATP
due to the glycerol-3-phosphate shuttle who transfers the electrons from cytosolic NADH to FADH2 and that gives
1.5 ATP), a net production of one high-energy phosphate bond for the urea cycle. However, if gluconeogenesis is
underway in the cytosol, the latter reducing equivalent is used to drive the reversal of the GAPDH step instead of
generating ATP.
The fate of oxaloacetate is either to produce aspartate via transamination or to be converted to phosphoenol pyruvate,
which is a substrate to glucose.
Urea cycle
303
Regulation
N-Acetylglutamic acid
The synthesis of carbamoyl phosphate and the urea cycle are dependent on the presence of NAcGlu, which
allosterically activates CPS1. Synthesis of NAcGlu by NAGS, is stimulated by both Arg, allosteric stimulator of
NAGS, and Glu, a product in the transamination reactions and one of NAGS's substrates, both of which are elevated
when free amino acids are elevated. So, Glu is not only a substrate for NAGS but also serves as an activator for the
urea cycle.
Substrate concentrations
The remaining enzymes of the cycle are controlled by the concentrations of their substrates. Thus, inherited
deficiencies in the cycle enzymes other than ARG1 do not result in significant decrease in urea production (the total
lack of any cycle enzyme results in death shortly after birth). Rather, the deficient enzyme's substrate builds up,
increasing the rate of the deficient reaction to normal.
The anomalous substrate buildup is not without cost, however. The substrate concentrations become elevated all the
way back up the cycle to NH
4
+
, resulting in hyperammonemia (elevated [NH
4
+
]
P
).
Although the root cause of NH
4
+
toxicity is not completely understood, a high [NH
4
+
] puts an enormous strain on the
NH
4
+
-clearing system, especially in the brain (symptoms of urea cycle enzyme deficiencies include mental
retardation and lethargy). This clearing system involves GLUD1 and GLUL, which decrease the 2-oxoglutarate
(2OG) and Glu pools. The brain is most sensitive to the depletion of these pools. Depletion of 2OG decreases the
rate of TCAC, whereas Glu is both a neurotransmitter and a precursor to GABA, another neurotransmitter.
[1](p.734)
Pathology
Anomalies of the urea cycle cause urea cycle disorders:
ornithine transcarbamoylase deficiency
Carbamoyl phosphate synthetase deficiency
Argininosuccinic aciduria
Argininemia
Hyperornithinemia, hyperammonemia, homocitrullinuria syndrome (HHH syndrome, ornithine translocase
deficiency)
Lysinuric protein intolerance
Citrullinemia
N-Acetylglutamate synthase deficiency
Most of them are associated with hyperammonemia.
Urea cycle
304
Additional images
Urea cycle. Urea cycle colored.
External links
The chemical logic behind the urea cycle
[2]
Basic Neurochemistry
[3]
- amino acid disorders
References
[1] http:/ / www. wiley. com/ college/ math/ chem/ cg/ sales/ voet. html
[2] http:/ / homepage. ufp.pt/ pedros/ bq/ urea.htm
[3] http:/ / www. ncbi. nlm.nih. gov/ books/ bv. fcgi?rid=bnchm. figgrp. 3102
305
Integration of metabolism
Hormone
Epinephrine (adrenaline), a catecholamine-type
hormone
A hormone (from Greek , "impetus") is a chemical released by a
cell, a gland, or an organ in one part of the body that affects cells in
other parts of the organism. Only a small amount of hormone is
required to alter cell metabolism. In essence, it is a chemical messenger
that transports a signal from one cell to another.
[1]
All multicellular
organisms produce hormones; plant hormones are also called
phytohormones. Hormones in animals are often transported in the
blood. Cells respond to a hormone when they express a specific
receptor for that hormone. The hormone binds to the receptor protein,
resulting in the activation of a signal transduction mechanism that
ultimately leads to cell type-specific responses.
Endocrine hormone molecules are secreted (released) directly into the bloodstream, typically into fenestrated
capillaries. Hormones with paracrine function diffuse through the interstitial spaces to nearby target tissues.
A variety of exogenous chemical compounds, both natural and synthetic, have hormone-like effects on both humans
and wildlife. Their interference with the synthesis, secretion, transport, binding, action, or elimination of natural
hormones in the body can change the homeostasis, reproduction, development, and/or behavior, just as endogenously
produced hormones do.
[]
Hormones as signals
See also Signal transduction.
Hormonal signaling involves the following:
[citation needed]
1. Biosynthesis of a particular hormone in a particular tissue
2. Storage and secretion of the hormone
3. Transport of the hormone to the target cell(s)
4. Recognition of the hormone by an associated cell membrane or intracellular receptor protein
5. Relay and amplification of the received hormonal signal via a signal transduction process: This then leads to a
cellular response. The reaction of the target cells may then be recognized by the original hormone-producing
cells, leading to a down-regulation in hormone production. This is an example of a homeostatic negative feedback
loop.
6. Degradation of the hormone.
Hormone cells are typically of a specialized cell type, residing within a particular endocrine gland, such as thyroid
gland, ovaries, and testes. Hormones exit their cell of origin via exocytosis or another means of membrane transport.
The hierarchical model is an oversimplification of the hormonal signaling process. Cellular recipients of a particular
hormonal signal may be one of several cell types that reside within a number of different tissues, as is the case for
insulin, which triggers a diverse range of systemic physiological effects. Different tissue types may also respond
differently to the same hormonal signal. Because of this, hormonal signaling is elaborate and hard to dissect.
[citation
needed]
Hormone
306
Interactions with receptors
Most hormones initiate a cellular response by initially combining with either a specific intracellular or cell
membrane associated receptor protein. A cell may have several different receptors that recognize the same hormone
and activate different signal transduction pathways, or a cell may have several different receptors that recognize
different hormones and activate the same biochemical pathway.
For many hormones, including most protein hormones, the receptor is membrane-associated and embedded in the
plasma membrane at the surface of the cell. The interaction of hormone and receptor typically triggers a cascade of
secondary effects within the cytoplasm of the cell, often involving phosphorylation or dephosphorylation of various
other cytoplasmic proteins, changes in ion channel permeability, or increased concentrations of intracellular
molecules that may act as secondary messengers (e.g., cyclic AMP). Some protein hormones also interact with
intracellular receptors located in the cytoplasm or nucleus by an intracrine mechanism.
For hormones such as steroid or thyroid hormones, their receptors are located intracellularly within the cytoplasm of
their target cell. To bind their receptors, these hormones must cross the cell membrane. They can do so because they
are lipid-soluble. The combined hormone-receptor complex then moves across the nuclear membrane into the
nucleus of the cell, where it binds to specific DNA sequences, effectively amplifying or suppressing the action of
certain genes, and affecting protein synthesis.
[]
However, it has been shown that not all steroid receptors are located
intracellularly. Some are associated with the plasma membrane.
[]
An important consideration, dictating the level at which cellular signal transduction pathways are activated in
response to a hormonal signal, is the effective concentration of hormone-receptor complexes that are formed.
Hormone-receptor complex concentrations are effectively determined by three factors:
1. 1. The number of hormone molecules available for complex formation
2. 2. The number of receptor molecules available for complex formation
3. The binding affinity between hormone and receptor.
The number of hormone molecules available for complex formation is usually the key factor in determining the level
at which signal transduction pathways are activated, the number of hormone molecules available being determined
by the concentration of circulating hormone, which is in turn influenced by the level and rate at which they are
secreted by biosynthetic cells. The number of receptors at the cell surface of the receiving cell can also be varied, as
can the affinity between the hormone and its receptor.
Physiology of hormones
Most cells are capable of producing one or more molecules, which act as signaling molecules to other cells, altering
their growth, function, or metabolism. The classical hormones produced by cells in the endocrine glands mentioned
so far in this article are cellular products, specialized to serve as regulators at the overall organism level. However,
they may also exert their effects solely within the tissue in which they are produced and originally released.
The rate of hormone biosynthesis and secretion is often regulated by a homeostatic negative feedback control
mechanism. Such a mechanism depends on factors that influence the metabolism and excretion of hormones. Thus,
higher hormone concentration alone cannot trigger the negative feedback mechanism. Negative feedback must be
triggered by overproduction of an "effect" of the hormone.
Hormone secretion can be stimulated and inhibited by:
Other hormones (stimulating- or releasing -hormones)
Plasma concentrations of ions or nutrients, as well as binding globulins
Neurons and mental activity
Environmental changes, e.g., of light or temperature
Hormone
307
One special group of hormones is the tropic hormones that stimulate the hormone production of other endocrine
glands. For example, thyroid-stimulating hormone (TSH) causes growth and increased activity of another endocrine
gland, the thyroid, which increases output of thyroid hormones.
A recently identified class of hormones is that of the "hunger hormones" - ghrelin, orexin, and PYY 3-36 - and
"satiety hormones" - e.g., cholecystokinin, leptin, nesfatin-1, obestatin.
To release active hormones quickly into the circulation, hormone biosynthetic cells may produce and store
biologically inactive hormones in the form of pre- or prohormones. These can then be quickly converted into their
active hormone form in response to a particular stimulus.
Effects of hormones
In mammals
Hormones have the following effects on the body:
stimulation or inhibition of growth
mood swings
induction or suppression of apoptosis (programmed cell death)
activation or inhibition of the immune system
regulation of metabolism
preparation of the body for mating, fighting, fleeing, and other activity
preparation of the body for a new phase of life, such as puberty, parenting, and menopause
control of the reproductive cycle
hunger cravings
sexual arousal
A hormone may also regulate the production and release of other hormones. Hormone signals control the internal
environment of the body through homeostasis.
Chemical classes of hormones
Vertebrate hormones fall into three chemical classes:
Peptide hormones consist of chains of amino acids. Examples of small peptide hormones are TRH and
vasopressin. Peptides composed of scores or hundreds of amino acids are referred to as proteins. Examples of
protein hormones include insulin and growth hormone. More complex protein hormones bear carbohydrate
side-chains and are called glycoprotein hormones. Luteinizing hormone, follicle-stimulating hormone and
thyroid-stimulating hormone are glycoprotein hormones. There is also another type of hydrophilic hormone called
nonpeptide hormones. Although they don't have peptide connections, they are assimilated as peptide hormones.
Lipid and phospholipid-derived hormones derive from lipids such as linoleic acid and arachidonic acid and
phospholipids. The main classes are the steroid hormones that derive from cholesterol and the eicosanoids.
Examples of steroid hormones are testosterone and cortisol. Sterol hormones such as calcitriol are a homologous
system. The adrenal cortex and the gonads are primary sources of steroid hormones. Examples of eicosanoids are
the widely studied prostaglandins.
Monoamines derived from aromatic amino acids like phenylalanine, tyrosine, tryptophan by the action of
aromatic amino acid decarboxylase enzymes.
Hormone
308
Pharmacology
Many hormones and their analogues are used as medication. The most commonly prescribed hormones are estrogens
and progestagens (as methods of hormonal contraception and as HRT), thyroxine (as levothyroxine, for
hypothyroidism) and steroids (for autoimmune diseases and several respiratory disorders). Insulin is used by many
diabetics. Local preparations for use in otolaryngology often contain pharmacologic equivalents of adrenaline, while
steroid and vitamin D creams are used extensively in dermatological practice.
A "pharmacologic dose" or "supraphysiological dose" of a hormone is a medical usage referring to an amount of a
hormone far greater than naturally occurs in a healthy body. The effects of pharmacologic doses of hormones may be
different from responses to naturally occurring amounts and may be therapeutically useful, though not without
potentially adverse side effects. An example is the ability of pharmacologic doses of glucocorticoids to suppress
inflammation.
Important human hormones
See: List of human hormones
References
External links
The Hormone Foundation (http:/ / www. hormone. org)
Article on hormones and their receptors (http:/ / www. biomedcentral. com/ 1471-2164/ 10/ 307/ )
HMRbase: A database of hormones and their receptors (http:/ / crdd. osdd. net/ raghava/ hmrbase/ )
Hormones (http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2011/ MB_cgi?mode=& term=Hormones) at the US National
Library of Medicine Medical Subject Headings (MeSH)
Signal transduction
309
Signal transduction
An overview of major signal transduction
pathways.
Signal transduction occurs when an extracellular signaling
[1]
molecule activates a cell surface receptor. In turn, this receptor alters
intracellular molecules creating a response.
[2]
There are two stages in
this process:
1. 1. A signaling molecule activates a specific receptor protein on the
cell membrane.
2. 2. A second messenger transmits the signal into the cell, eliciting a
physiological response.
In either step, the signal can be amplified. Thus, one signalling
molecule can cause many responses.
[]
A signal transduction functions
much like a switch.
[citation needed]
History
Occurrence of the term signal transduction in papers since 1977. These figures
were derived by an analysis of the papers contained within the MEDLINE
database.
In 1970, Martin Rodbell examined the
effects of glucagon on a rat's liver cell
membrane receptor. He noted that guanosine
triphosphate disassociated glucagon from
this receptor and stimulated the G-protein,
which strongly influenced the cell's
metabolism. Thus he deduced that the
G-protein was a transducer that accepted
glucagon molecules and affected the cell.
[]
For this he shared the 1994 Nobel Prize in
Physiology or Medicine with Alfred G.
Gilman.
The earliest MEDLINE entry for "signal
transduction" dates from 1972.
[]
Some early
articles used the terms signal transmission and sensory transduction.
[][]
In 1977, a total of 48,377 scientific papers --
including 11,211 e review papers -- were published on the subject. The term first appeared in a paper's title in
1979.
[][]
Widespread use of the term has been traced to a 1980 review article by Rodbell:
[][]
Research papers focusing
on signal transduction first appeared in large numbers in the late 1980s and early 1990s.
[3]
Signal transduction involves the binding of extracellular signalling molecules and ligands to cell-surface receptors
that trigger events inside the cell. The combination of messenger with receptor causes a change in the conformation
of the receptor, known as receptor activation. This activation is always the initial step (the cause) leading to the cell's
ultimate responses (effect) to the messenger. Despite the myriad of these ultimate responses, they are all directly due
to changes in particular cell proteins. Intracellular signaling cascades can be started through cell-substratum
interactions; examples are the integrin that binds ligands in the extracellular matrix and steroids.
[]
Most steroid
hormones have receptors within the cytoplasm and act by stimulating the binding of their receptors to the promoter
region of steroid-responsive genes.
[]
Examples of signaling molecules include the hormone melatonin,
[]
the
neurotransmitter acetylcholine
[]
and the cytokine interferon .
[]
The classifications of signalling molecules do not take into account the molecular nature of each class member;
neurotransmitters range in size from small molecules such as dopamine
[]
to neuropeptides such as endorphins.
[]
Signal transduction
310
Some molecules may fit into more than one class; for example, epinephrine is a neurotransmitter when secreted by
the central nervous system and a hormone when secreted by the adrenal medulla.
Environmental stimuli
With single-celled organisms, the variety of signal transduction processes influence its reaction to its
environment.
[citation needed]
With multicellular organisms, numerous processes are required for coordinating
individual cells to support the organism as a whole; the complexity of these processes tend to increase with the
complexity of the organism.
[citation needed]
Sensing of environments at the cellular level relies on signal
transduction;
[citation needed]
many disease processes, such as diabetes and heart disease arise from defects in these
pathways, highlighting the importance of this process in biology and medicine.
Various environmental stimuli exist that initiate signal transmission processes in multicellular organisms; examples
include photons hitting cells in the retina of the eye,
[]
and odorants binding to odorant receptors in the nasal
epithelium.
[]
Certain microbial molecules, such as viral nucleotides and protein antigens, can elicit an immune
system response against invading pathogens mediated by signal transduction processes. This may occur independent
of signal transduction stimulation by other molecules, as is the case for the toll-like receptor. It may occur with help
from stimulatory molecules located at the cell surface of other cells, as with T-cell receptor signaling. Single-celled
organisms may respond to environmental stimuli through the activation of signal transduction pathways. For
example, slime molds secrete cyclic adenosine monophosphate upon starvation, stimulating individual cells in the
immediate environment to aggregate,
[]
and yeast cells use mating factors to determine the mating types of other cells
and to participate in sexual reproduction.
[]
Receptors
Receptors can be roughly divided into two major classes: intracellular receptors and extracellular receptors.
Extracellular
Extracellular receptors are integral transmembrane proteins and make up most receptors. They span the plasma
membrane of the cell, with one part of the receptor on the outside of the cell and the other on the inside. Signal
transduction occurs as a result of a ligand binding to the outside; the molecule does not pass through the membrane.
This binding stimulates a series of events inside the cell; different types of receptor stimulate different responses and
receptors typically respond to only the binding of a specific ligand. Upon binding, the ligand induces a change in the
conformation of the inside part of the receptor.
[4]
These result in either the activation of an enzyme in the receptor or
the exposure of a binding site for other intracellular signaling proteins within the cell, eventually propagating the
signal through the cytoplasm.
In eukaryotic cells, most intracellular proteins activated by a ligand/receptor interaction possess an enzymatic
activity; examples include tyrosine kinase and phosphatases. Some of them create second messengers such as cyclic
AMP and IP
3
, the latter controlling the release of intracellular calcium stores into the cytoplasm. Other activated
proteins interact with adaptor proteins that facilitate signalling protein interactions and coordination of signalling
complexes necessary to respond to a particular stimulus. Enzymes and adaptor proteins are both responsive to
various second messenger molecules.
Many adaptor proteins and enzymes activated as part of signal transduction possess specialized protein domains that
bind to specific secondary messenger molecules. For example, calcium ions bind to the EF hand domains of
calmodulin, allowing it to bind and activate calmodulin-dependent kinase. PIP
3
and other phosphoinositides do the
same thing to the Pleckstrin homology domains of proteins such as the kinase protein AKT.
Signal transduction
311
G protein-coupled
G protein-coupled receptors (GPCRs) are a family of integral transmembrane proteins that possess seven
transmembrane domains and are linked to a heterotrimeric G protein. Many receptors are in this family, including
adrenergic receptors and chemokine receptors.
Signal transduction by a GPCR begins with an inactive G protein coupled to the receptor; it exists as a heterotrimer
consisting of G, G, and G. Once the GPCR recognizes a ligand, the conformation of the receptor changes to
activate the G protein, causing G to bind a molecule of GTP and dissociate from the other two G-protein subunits.
The dissociation exposes sites on the subunits that can interact with other molecules.
[5]
The activated G protein
subunits detach from the receptor and initiate signaling from many downstream effector proteins such as
phospholipases and ion channels, the latter permitting the release of second messenger molecules.
[6]
The total
strength of signal amplification by a GPCR is determined by the lifetimes of the ligand-receptor complex and
receptor-effector protein complex and the deactivation time of the activated receptor and effectors through intrinsic
enzymatic activity.
A study was conducted where a point mutation was inserted into the gene encoding the chemokine receptor CXCR2;
mutated cells underwent a malignant transformation due to the expression of CXCR2 in an active conformation
despite the absence of chemokine-binding. This meant that chemokine receptors can contribute to cancer
development.
[]
Tyrosine and histidine kinase
Receptor tyrosine kinases (RTKs) are transmembrane proteins with an intracellular kinase domain and an
extracellular domain that binds ligands; examples include growth factor receptors such as the insulin receptor.
[]
To
perform signal transduction, RTKs need to form dimers in the plasma membrane;
[]
the dimer is stabilized by ligands
binding to the receptor. The interaction between the cytoplasmic domains stimulates the autophosphorylation of
tyrosines within the domains of the RTKs, causing conformational changes. The receptors' kinase domains are
subsequently activated, initiating phosphorylation signaling cascades of downstream cytoplasmic molecules that
facilitate various cellular processes such as cell differentiation and metabolism.
[]
As is the case with GPCRs, proteins that bind GTP play a major role in signal transduction from the activated RTK
into the cell. In this case, the G proteins are members of the Ras, Rho, and Raf families, referred to collectively as
small G proteins. They act as molecular switches usually tethered to membranes by isoprenyl groups linked to their
carboxyl ends. Upon activation, they assign proteins to specific membrane subdomains where they participate in
signaling. Activated RTKs in turn activate small G proteins that activate guanine nucleotide exchange factors such as
SOS1. Once activated, these exchange factors can activate more small G proteins, thus amplifying the receptor's
initial signal. The mutation of certain RTK genes, as with that of GPCRs, can result in the expression of receptors
that exist in a constitutively activate state; such mutated genes may act as oncogenes.
[]
Histidine-specific protein kinases are structurally distinct from other protein kinases and are found in prokaryotes,
fungi, and plants as part of a two-component signal transduction mechanism: a phosphate group from ATP is first
added to a histidine residue within the kinase, then transferred to an aspartate residue on a receiver domain on a
different protein or the kinase itself, thus activating the aspartate residue.
[7]
Signal transduction
312
Integrin
An overview of integrin-mediated signal transduction, adapted from Hehlgens et
al. (2007).
[]
Integrins are produced by a wide variety of
cells; they play a role in cell attachment to
other cells and the extracellular matrix and
in the transduction of signals from
extracellular matrix components such as
fibronectin and collagen. Ligand binding to
the extracellular domain of integrins
changes the protein's conformation,
clustering it at the cell membrane to initiate
signal transduction. Integrins lack kinase
activity; hence integrin-mediated signal
transduction is achieved through a variety of
intracellular protein kinases and adaptor
molecules, the main coordinator being
integrin-linked kinase.
[]
As shown in the
picture to the right, cooperative
integrin-RTK signalling determines the
timing of cellular survival, apoptosis,
proliferation, and differentiation.
Important differences exist between integrin-signalling in circulating blood cells and non-circulating cells such as
epithelial cells; integrins of circulating cells are normally inactive. For example, cell membrane integrins on
circulating leukocytes are maintained in an inactive state to avoid epithelial cell attachment; they are only activated
in response to stimuli such as those received at the site of an inflammatory response. In a similar manner, integrins at
the cell membrane of circulating platelets are normally kept inactive to avoid thrombosis. Epithelial cells (which are
non-circulating) normally have active integrins at their cell membrane, helping maintain their stable adhesion to
underlying stromal cells that provide signals to maintain normal functioning.
[]
Toll gate
When activated, toll-like receptors (TLRs) take adapter molecules within the cytoplasm of cells in order to propagate
a signal. Four adaptor molecules are known to be involved in signaling, which are Myd88, TIRAP, TRIF, and
TRAM.
[][][]
These adapters activate other intracellular molecules such as IRAK1, IRAK4, TBK1, and IKKi that
amplify the signal, eventually leading to the induction or suppression of genes that cause certain responses.
Thousands of genes are activated by TLR signaling, implying that this method constitutes an important gateway for
gene modulation.
Ligand-gated ion channel
A ligand-gated ion channel, upon binding with a ligand, changes conformation to open a channel in the cell
membrane through which ions relaying signals can pass. An example of this mechanism is found in the receiving cell
of a neural synapse. The influx of ions that occurs in response to these channels opening induces action potentials,
such as those that travel along nerves, by depolarizing the membrane of post-synaptic cells, resulting in the opening
of voltage-gated ion channels.
An example of an ion allowed into the cell during a ligand-gated ion channel opening is Ca
2+
; it acts as a second
messenger initiating signal transduction cascades and altering the physiology of the responding cell. This results in
amplification of the synapse response between synaptic cells by remodelling the dendritic spines involved in the
synapse.
Signal transduction
313
Intracellular
Intracellular receptors, such as nuclear receptors and cytoplasmic receptors, are soluble proteins localized within
their respective areas. The typical ligands for nuclear receptors are lipophilic hormones like the steroid hormones
testosterone and progesterone and derivatives of vitamins A and D. To initiate signal transduction, the ligand must
pass through the plasma membrane by passive diffusion. On binding with the receptor, the ligands pass through the
nuclear membrane into the nucleus, enabling gene transcription and protein production.
Activated nuclear receptors attach to the DNA at receptor-specific hormone-responsive element (HRE) sequences,
located in the promoter region of the genes activated by the hormone-receptor complex. Due to them enabling gene
transcription, they are alternatively called inductors of gene expression. All hormones that act by regulation of gene
expression have two consequences in their mechanism of action; their effects are produced after a characteristically
long period of time and their effects persist for another long period of time, even after their concentration has been
reduced to zero, due to a relatively slow turnover of most enzymes and proteins that would either deactivate or
terminate ligand binding onto the receptor.
Signal transduction via these receptors involves little proteins, but the details of gene regulation by this method are
not well understood. Nucleic receptors have DNA-binding domains containing zinc fingers and a ligand-binding
domain; the zinc fingers stabilize DNA binding by holding its phosphate backbone. DNA sequences that match the
receptor are usually hexameric repeats of any kind; the sequences are similar but their orientation and distance
differentiate them. The ligand-binding domain is additionally responsible for dimerization of nucleic receptors prior
to binding and providing structures for transactivation used for communication with the translational apparatus.
Steroid receptors are a subclass of nuclear receptors located primarily within the cytosol; in the absence of steroids,
they cling together in an aporeceptor complex containing chaperone or heatshock proteins (HSPs). The HSPs are
necessary to activate the receptor by assisting the protein to fold in a way such that the signal sequence enabling its
passage into the nucleus is accessible. Steroid receptors, on the other hand, may be repressive on gene expression
when their transactivation domain is hidden; activity can be enhanced by phosphorylation of serine residues at their
N-terminal as a result of another signal transduction pathway, a process called crosstalk.
Retinoic acid receptors are another subset of nuclear receptors. They can be activated by an endocrine-synthesized
ligand that entered the cell by diffusion, a ligand synthesised from a precursor like retinol brought to the cell through
the bloodstream or a completely intracellularly synthesised ligand like prostaglandin. These receptors are located in
the nucleus and are not accompanied by HSPs; they repress their gene by binding to their specific DNA sequence
when no ligand binds to them and vice versa.
Certain intracellular receptors of the immune system are cytoplasmic receptors; recently identified NOD-like
receptors (NLRs) reside in the cytoplasm of some eukaryotic cells and interact with ligands using a leucine-rich
repeat (LRR) motif similar to TLRs. Some of these molecules like NOD2 interact with RIP2 kinase that activates
NF-B signaling, whereas others like NALP3 interact with inflammatory caspases and initiate processing of
particular cytokines like interleukin-1.
[8][9]
Signal transduction
314
Second messengers
First messengers are the intercellular chemical messengers (hormones, neurotransmitters, and paracrine/autocrine
agents) which reach the cell from the extracellular fluid and bind to their specific receptors. Second messengers are
the substances which enter the cytoplasm and act within the cell to trigger a response. Second messengers essentially
serve as chemical relays from the plasma membrane to the cytoplasm, thus carrying out intracellular signal
transduction.
Calcium
The release of calcium ions from the endoplasmic reticulum into the cytosol results in its binding to signaling
proteins that are then activated; it is then sequestered in the smooth endoplasmic reticulum and the mitochondria.
Two combined receptor/ion channel proteins control the transport of calcium: the InsP
3
-receptor that transports
calcium upon interaction with inositol triphosphate on its cytosolic side and the ryanodine receptor named after the
alkaloid ryanodine, similar to the InsP
3
receptor but having a feedback mechanism that releases more calcium upon
binding with it. The nature of calcium in the cytosol means that it is active for only a very short time, meaning its
free state concentration is very low and is mostly bound to organelle molecules like calreticulin when inactive.
Calcium is used in many processes including muscle contraction, neurotransmitter release from nerve endings and
cell migration. The three main pathways that lead to its activation are GPCR pathways, RTK pathways and gated ion
channels; it regulates proteins either directly or by binding to an enzyme.
Lipophilics
Lipophilic second messenger molecules are derived from lipids residing in cellular membranes; enzymes stimulated
by activated receptors activate the lipids by modifying them. Examples include diacylglycerol and ceramide, the
former required for the activation of protein kinase C.
Nitric oxide
Nitric oxide (NO) acts as a second messenger because it is a free radical that can diffuse through the plasma
membrane and affect nearby cells. It is synthesised from arginine and oxygen by the NO synthase and works through
activation of soluble guanylyl cyclase, which when activated produces another second messenger, cGMP. NO can
also act through covalent modification of proteins or their metal co-factors; some have a redox mechanism and are
reversible. It is toxic in high concentrations and causes damage during stroke, but is the cause of many other
functions like relaxation of blood vessels, apoptosis and erections.
Redox signaling
In addition to nitric oxide, other electronically activated species are also signal-transducing agents in a process called
redox signaling. Examples include superoxide, hydrogen peroxide, carbon monoxide, and hydrogen sulfide. Redox
signaling also includes active modulation of electronic flows in semiconductive biological macromolecules.
[10]
Cellular responses
Gene activations
[]
and metabolism alterations
[]
are examples of cellular responses to extracellular stimulation that
require signal transduction. Gene activation leads to further cellular effects, since the products of responding genes
include instigators of activation; transcription factors produced as a result of a signal transduction cascade can
activate even more genes. Hence, an initial stimulus can trigger the expression of a large number of genes, leading to
physiological events like the increased uptake of glucose from the blood stream
[]
and the migration of neutrophils to
sites of infection. The set of genes and their activation order to certain stimuli is referred to as a genetic program.
[]
Signal transduction
315
Mammalian cells require stimulation for cell division and survival; in the absence of growth factor, apoptosis ensues.
Such requirements for extracellular stimulation are necessary for controlling cell behavior in unicellular and
multicellular organisms; signal transduction pathways are perceived to be so central to biological processes that a
large number of diseases are attributed to their disregulation.
Major pathways
Following are some major signaling pathways, demonstrating how ligands binding to their receptors can affect
second messengers and eventually result in altered cellular responses.
MAPK/ERK pathway: A pathway that couples intracellular responses to the binding of growth factors to cell
surface receptors. This pathway is very complex and includes many protein components.
[]
In many cell types,
activation of this pathway promotes cell division, and many forms of cancer are associated with aberrations in
it.
[11]
cAMP dependent pathway: In humans, cAMP works by activating protein kinase A (PKA, cAMP-dependent
protein kinase) (see picture), and thus, further effects mainly depend on cAMP-dependent protein kinase, which
vary based on the type of cell.
IP
3
/DAG pathway: PLC cleaves the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) yielding diacyl
glycerol (DAG) and inositol 1,4,5-triphosphate (IP
3
). DAG remains bound to the membrane, and IP
3
is released
as a soluble structure into the cytosol. IP
3
then diffuses through the cytosol to bind to IP
3
receptors, particular
calcium channels in the endoplasmic reticulum (ER). These channels are specific to calcium and only allow the
passage of calcium to move through. This causes the cytosolic concentration of Calcium to increase, causing a
cascade of intracellular changes and activity.
[]
In addition, calcium and DAG together works to activate PKC,
which goes on to phosphorylate other molecules, leading to altered cellular activity. End effects include taste,
manic depression, tumor promotion, etc.
[]
References
[1] http:/ / www. ncbi. nlm.nih. gov/ books/ NBK21517
[2] [2] Silverthorn (2007). Human Physiology. 4th ed.
[4] A molecular model for receptor activation (http:/ / www. bio-balance. com/ JMGM_article. pdf)
[10] Forman, H.J., Signal transduction and reactive species. Free Radic. Biol. Med. 47:1237-1238; 2009
External links
Netpath - A curated resource of signal transduction pathways in humans (http:/ / www. netpath. org/ )
Signal Transduction - The Virtual Library of Biochemistry and Cell Biology (http:/ / www. biochemweb. org/
signaling. shtml)
TRANSPATH(R) (http:/ / www. gene-regulation. com/ cgi-bin/ pub/ databases/ transpath/ search. cgi) - A
database about signal transduction pathways
Science's STKE - Signal Transduction Knowledge Environment (http:/ / stke. sciencemag. org/ ), from the journal
Science, published by AAAS.
Signal Transduction (http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2011/ MB_cgi?mode=& term=Signal+
Transduction) at the US National Library of Medicine Medical Subject Headings (MeSH)
UCSD-Nature Signaling Gateway (http:/ / www. signaling-gateway. org/ ), from Nature Publishing Group
LitInspector (http:/ / www. litinspector. org) - Signal transduction pathway mining in PubMed abstracts
Huaxian Chen, et al. A Cell Based Immunocytochemical Assay For Monitoring Kinase Signaling Pathways And
Drug Efficacy (PDF) (http:/ / www. licor. com. / bio/ PDF/ AppNote_AnalBiochem. pdf) Analytical Biochemistry
338 (2005) 136-142
www.Redoxsignaling.com (http:/ / www. redoxsignaling. com)
Signal transduction
316
Signaling PAthway Database (http:/ / www. grt. kyushu-u. ac. jp/ spad/ ) - Kyushu University
Cell cycle - Homo sapiens (human) (http:/ / www. genome. jp/ kegg/ pathway/ hsa/ hsa04110. html) - KEGG
PATHWAY (http:/ / www. genome. jp/ kegg/ pathway. html)
Pathway Interaction Database (http:/ / pid. nci. nih. gov/ ) - NCI
Diabetes mellitus
Diabetes mellitus
Classification and external resources
Universal blue circle symbol for diabetes.
[1]
ICD-10
E10
[2]
E14
[3]
ICD-9
250
[4]
MedlinePlus
001214
[5]
eMedicine
med/546
[6]
emerg/134
[7]
MeSH
C18.452.394.750
[8]
Diabetes mellitus, or simply diabetes, is a group of metabolic diseases in which a person has high blood sugar,
either because the pancreas does not produce enough insulin, or because cells do not respond to the insulin that is
produced.
[]
This high blood sugar produces the classical symptoms of polyuria (frequent urination), polydipsia
(increased thirst) and polyphagia (increased hunger).
There are three main types of diabetes mellitus (DM).
Type1 DM results from the body's failure to produce insulin, and currently requires the person to inject insulin or
wear an insulin pump. This form was previously referred to as "insulin-dependent diabetes mellitus" (IDDM) or
"juvenile diabetes".
Type2 DM results from insulin resistance, a condition in which cells fail to use insulin properly, sometimes
combined with an absolute insulin deficiency. This form was previously referred to as non insulin-dependent
diabetes mellitus (NIDDM) or "adult-onset diabetes".
The third main form, gestational diabetes occurs when pregnant women without a previous diagnosis of diabetes
develop a high blood glucose level. It may precede development of type2 DM.
Diabetes mellitus
317
Other forms of diabetes mellitus include congenital diabetes, which is due to genetic defects of insulin secretion,
cystic fibrosis-related diabetes, steroid diabetes induced by high doses of glucocorticoids, and several forms of
monogenic diabetes.
Untreated, diabetes can cause many complications. Acute complications include diabetic ketoacidosis and nonketotic
hyperosmolar coma. Serious long-term complications include cardiovascular disease, chronic renal failure, and
diabetic retinopathy (retinal damage). Adequate treatment of diabetes is thus important, as well as blood pressure
control and lifestyle factors such as stopping smoking and maintaining a healthy body weight.
All forms of diabetes have been treatable since insulin became available in 1921, and type2 diabetes may be
controlled with medications. Insulin and some oral medications can cause hypoglycemia (low blood sugars), which
can be dangerous if severe. Both types 1 and 2 are chronic conditions that cannot be cured. Pancreas transplants have
been tried with limited success in type1 DM; gastric bypass surgery has been successful in many with morbid
obesity and type2 DM. Gestational diabetes usually resolves after delivery.
Classification
Comparison of type 1 and 2 diabetes
[]
Feature Type 1 diabetes Type 2 diabetes
Onset Sudden Gradual
Age at onset Mostly in children Mostly in adults
Body habitus
Thin or normal
[9]
Often obese
Ketoacidosis Common Rare
Autoantibodies Usually present Absent
Endogenous insulin Low or absent Normal,
decreased
or increased
Concordance
in identical twins
50% 90%
Prevalence ~10% ~90%
Diabetes mellitus is classified into four broad categories: type1, type2, gestational diabetes and "other specific
types".
[]
The "other specific types" are a collection of a few dozen individual causes.
[]
The term "diabetes", without
qualification, usually refers to diabetes mellitus. The rare disease diabetes insipidus has similar symptoms as diabetes
mellitus, but without disturbances in the sugar metabolism (insipidus means "without taste" in Latin) and does not
involve the same disease mechanisms.
The term "type1 diabetes" has replaced several former terms, including childhood-onset diabetes, juvenile diabetes,
and insulin-dependent diabetes mellitus (IDDM). Likewise, the term "type2 diabetes" has replaced several former
terms, including adult-onset diabetes, obesity-related diabetes, and noninsulin-dependent diabetes mellitus
(NIDDM). Beyond these two types, there is no agreed-upon standard nomenclature.
Diabetes mellitus
318
Type 1 diabetes
Type1 diabetes mellitus is characterized by loss of the insulin-producing beta cells of the islets of Langerhans in the
pancreas, leading to insulin deficiency. This type can be further classified as immune-mediated or idiopathic. The
majority of type1 diabetes is of the immune-mediated nature, in which beta cell loss is a T-cell-mediated
autoimmune attack.
[]
There is no known preventive measure against type1 diabetes, which causes approximately
10% of diabetes mellitus cases in North America and Europe. Most affected people are otherwise healthy and of a
healthy weight when onset occurs. Sensitivity and responsiveness to insulin are usually normal, especially in the
early stages. Type1 diabetes can affect children or adults, but was traditionally termed "juvenile diabetes" because a
majority of these diabetes cases were in children.
"Brittle" diabetes, also known as unstable diabetes or labile diabetes, is a term that was traditionally used to describe
to dramatic and recurrent swings in glucose levels, often occurring for no apparent reason in insulin-dependent
diabetes. This term, however, has no biologic basis and should not be used.
[]
There are many reasons for type1
diabetes to be accompanied by irregular and unpredictable hyperglycemias, frequently with ketosis, and sometimes
serious hypoglycemias, including an impaired counterregulatory response to hypoglycemia, occult infection,
gastroparesis (which leads to erratic absorption of dietary carbohydrates), and endocrinopathies (e.g., Addison's
disease).
[]
These phenomena are believed to occur no more frequently than in 1% to 2% of persons with type1
diabetes.
[]
Type 2 diabetes
Type2 diabetes mellitus is characterized by insulin resistance, which may be combined with relatively reduced
insulin secretion.
[]
The defective responsiveness of body tissues to insulin is believed to involve the insulin receptor.
However, the specific defects are not known. Diabetes mellitus cases due to a known defect are classified separately.
Type2 diabetes is the most common type.
In the early stage of type2, the predominant abnormality is reduced insulin sensitivity. At this stage, hyperglycemia
can be reversed by a variety of measures and medications that improve insulin sensitivity or reduce glucose
production by the liver.
Gestational diabetes
Gestational diabetes mellitus (GDM) resembles type2 diabetes in several respects, involving a combination of
relatively inadequate insulin secretion and responsiveness. It occurs in about 2%5% of all pregnancies and may
improve or disappear after delivery. Gestational diabetes is fully treatable, but requires careful medical supervision
throughout the pregnancy. About 20%50% of affected women develop type2 diabetes later in life.
Though it may be transient, untreated gestational diabetes can damage the health of the fetus or mother. Risks to the
baby include macrosomia (high birth weight), congenital cardiac and central nervous system anomalies, and skeletal
muscle malformations. Increased fetal insulin may inhibit fetal surfactant production and cause respiratory distress
syndrome. Hyperbilirubinemia may result from red blood cell destruction. In severe cases, perinatal death may
occur, most commonly as a result of poor placental perfusion due to vascular impairment. Labor induction may be
indicated with decreased placental function. A Caesarean section may be performed if there is marked fetal distress
or an increased risk of injury associated with macrosomia, such as shoulder dystocia.
A 2008 study completed in the U.S. found the number of American women entering pregnancy with pre-existing
diabetes is increasing. In fact, the rate of diabetes in expectant mothers has more than doubled in the past six
years.
[10]
This is particularly problematic as diabetes raises the risk of complications during pregnancy, as well as
increasing the potential for the children of diabetic mothers to become diabetic in the future.
Diabetes mellitus
319
Other types
Prediabetes indicates a condition that occurs when a person's blood glucose levels are higher than normal but not
high enough for a diagnosis of type2 DM. Many people destined to develop type2 DM spend many years in a state
of prediabetes which has been termed "America's largest healthcare epidemic."
[11]:1011
Latent autoimmune diabetes of adults (LADA) is a condition in which type1 DM develops in adults. Adults with
LADA are frequently initially misdiagnosed as having type2 DM, based on age rather than etiology.
Some cases of diabetes are caused by the body's tissue receptors not responding to insulin (even when insulin levels
are normal, which is what separates it from type2 diabetes); this form is very uncommon. Genetic mutations
(autosomal or mitochondrial) can lead to defects in beta cell function. Abnormal insulin action may also have been
genetically determined in some cases. Any disease that causes extensive damage to the pancreas may lead to diabetes
(for example, chronic pancreatitis and cystic fibrosis). Diseases associated with excessive secretion of
insulin-antagonistic hormones can cause diabetes (which is typically resolved once the hormone excess is removed).
Many drugs impair insulin secretion and some toxins damage pancreatic beta cells. The ICD-10 (1992) diagnostic
entity, malnutrition-related diabetes mellitus (MRDM or MMDM, ICD-10 code E12), was deprecated by the World
Health Organization when the current taxonomy was introduced in 1999.
[]
Signs and symptoms
Overview of the most significant symptoms of diabetes
The classic symptoms of untreated diabetes
are loss of weight, polyuria (frequent
urination), polydipsia (increased thirst) and
polyphagia (increased hunger).
[12]
Symptoms may develop rapidly (weeks or
months) in type1 diabetes, while they
usually develop much more slowly and may
be subtle or absent in type2 diabetes.
Prolonged high blood glucose can cause
glucose absorption in the lens of the eye,
which leads to changes in its shape,
resulting in vision changes. Blurred vision is
a common complaint leading to a diabetes
diagnosis. A number of skin rashes that can
occur in diabetes are collectively known as
diabetic dermadromes.
Diabetic emergencies
People (usually with type1 diabetes) may
also present with diabetic ketoacidosis, a
state of metabolic dysregulation characterized by the smell of acetone, a rapid, deep breathing known as Kussmaul
breathing, nausea, vomiting and abdominal pain, and altered states of consciousness.
A rare but equally severe possibility is hyperosmolar nonketotic state, which is more common in type2 diabetes and
is mainly the result of dehydration.
Diabetes mellitus
320
Complications
All forms of diabetes increase the risk of long-term complications. These typically develop after many years
(1020), but may be the first symptom in those who have otherwise not received a diagnosis before that time. The
major long-term complications relate to damage to blood vessels. Diabetes doubles the risk of cardiovascular
disease.
[13]
The main "macrovascular" diseases (related to atherosclerosis of larger arteries) are ischemic heart
disease (angina and myocardial infarction), stroke and peripheral vascular disease.
Diabetes also damages the capillaries (causes microangiopathy).
[14]
Diabetic retinopathy, which affects blood vessel
formation in the retina of the eye, can lead to visual symptoms including reduced vision and potentially blindness.
Diabetic nephropathy, the impact of diabetes on the kidneys, can lead to scarring changes in the kidney tissue, loss of
small or progressively larger amounts of protein in the urine, and eventually chronic kidney disease requiring
dialysis.
Another risk is diabetic neuropathy, the impact of diabetes on the nervous system most commonly causing
numbness, tingling and pain in the feet, and also increasing the risk of skin damage due to altered sensation.
Together with vascular disease in the legs, neuropathy contributes to the risk of diabetes-related foot problems (such
as diabetic foot ulcers) that can be difficult to treat and occasionally require amputation. As well, proximal diabetic
neuropathy causes painful muscle wasting and weakness.
Several studies suggest
[]
a link between cognitive deficit and diabetes. Compared to those without diabetes, the
research showed that those with the disease have a 1.2 to 1.5-fold greater rate of decline in cognitive function, and
are at greater risk.
Causes
The cause of diabetes depends on the type.
Type1 diabetes is partly inherited, and then triggered by certain infections, with some evidence pointing at
Coxsackie B4 virus. A genetic element in individual susceptibility to some of these triggers has been traced to
particular HLA genotypes (i.e., the genetic "self" identifiers relied upon by the immune system). However, even in
those who have inherited the susceptibility, type1 DM seems to require an environmental trigger. The onset of
type1 diabetes is unrelated to lifestyle.
Type2 diabetes is due primarily to lifestyle factors and genetics.
[]
The following is a comprehensive list of other causes of diabetes:
[15]
Genetic defects of -cell function
Maturity onset diabetes of the young
Mitochondrial DNA mutations
Genetic defects in insulin processing or insulin action
Defects in proinsulin conversion
Insulin gene mutations
Insulin receptor mutations
Exocrine pancreatic defects
Chronic pancreatitis
Pancreatectomy
Pancreatic neoplasia
Cystic fibrosis
Hemochromatosis
Fibrocalculous pancreatopathy
Endocrinopathies
Growth hormone excess (acromegaly)
Cushing syndrome
Hyperthyroidism
Pheochromocytoma
Glucagonoma
Infections
Cytomegalovirus infection
Coxsackievirus B
Drugs
Glucocorticoids
Thyroid hormone
-adrenergic agonists
Statins
[16]
Diabetes mellitus
321
Pathophysiology
The fluctuation of blood sugar (red) and the
sugar-lowering hormone insulin (blue) in humans
during the course of a day with three meals - one
of the effects of a sugar-rich vs a starch-rich meal
is highlighted.
Mechanism of insulin release in normal
pancreatic beta cells - insulin production is more
or less constant within the beta cells. Its release is
triggered by food, chiefly food containing
absorbable glucose.
Insulin is the principal hormone that regulates uptake of glucose from
the blood into most cells (primarily muscle and fat cells, but not central
nervous system cells). Therefore, deficiency of insulin or the
insensitivity of its receptors plays a central role in all forms of diabetes
mellitus.
Humans are capable of digesting some carbohydrates, in particular
those most common in food; starch, and some disaccharides such as
sucrose, are converted within a few hours to simpler forms, most
notably the monosaccharide glucose, the principal carbohydrate energy
source used by the body. The rest are passed on for processing by gut
flora largely in the colon. Insulin is released into the blood by beta
cells (-cells), found in the islets of Langerhans in the pancreas, in
response to rising levels of blood glucose, typically after eating. Insulin
is used by about two-thirds of the body's cells to absorb glucose from
the blood for use as fuel, for conversion to other needed molecules, or
for storage.
Insulin is also the principal control signal for conversion of glucose to
glycogen for internal storage in liver and muscle cells. Lowered
glucose levels result both in the reduced release of insulin from the
-cells and in the reverse conversion of glycogen to glucose when
glucose levels fall. This is mainly controlled by the hormone glucagon,
which acts in the opposite manner to insulin. Glucose thus forcibly
produced from internal liver cell stores (as glycogen) re-enters the
bloodstream; muscle cells lack the necessary export mechanism.
Normally, liver cells do this when the level of insulin is low (which
normally correlates with low levels of blood glucose).
Higher insulin levels increase some anabolic ("building up") processes, such as cell growth and duplication, protein
synthesis, and fat storage. Insulin (or its lack) is the principal signal in converting many of the bidirectional
processes of metabolism from a catabolic to an anabolic direction, and vice versa. In particular, a low insulin level is
the trigger for entering or leaving ketosis (the fat-burning metabolic phase).
If the amount of insulin available is insufficient, if cells respond poorly to the effects of insulin (insulin insensitivity
or resistance), or if the insulin itself is defective, then glucose will not have its usual effect, so it will not be absorbed
properly by those body cells that require it, nor will it be stored appropriately in the liver and muscles. The net effect
is persistent high levels of blood glucose, poor protein synthesis, and other metabolic derangements, such as
acidosis.
When the glucose concentration in the blood is raised to about 9-10mmol/L (except certain conditions, such as
pregnancy), beyond its renal threshold (i.e. when glucose level surpasses the transport maximum of glucose
reabsorption), reabsorption of glucose in the proximal renal tubuli is incomplete, and part of the glucose remains in
the urine (glycosuria). This increases the osmotic pressure of the urine and inhibits reabsorption of water by the
kidney, resulting in increased urine production (polyuria) and increased fluid loss. Lost blood volume will be
replaced osmotically from water held in body cells and other body compartments, causing dehydration and increased
thirst.
Diabetes mellitus
322
Diagnosis
Diabetes diagnostic criteria
[][17]
Condition 2 hour glucose Fasting glucose HbA
1c
mmol/l(mg/dl) mmol/l(mg/dl) %
Normal <7.8 (<140) <6.1 (<110) <6.0
Impaired fasting glycaemia <7.8 (<140) 6.1(110) & <7.0(<126) 6.06.4
Impaired glucose tolerance 7.8 (140) <7.0 (<126) 6.06.4
Diabetes mellitus 11.1 (200) 7.0 (126) 6.5
Diabetes mellitus is characterized by recurrent or persistent hyperglycemia, and is diagnosed by demonstrating any
one of the following:
[]
Fasting plasma glucose level 7.0mmol/l (126mg/dl)
Plasma glucose 11.1mmol/l (200mg/dL) two hours after a 75g oral glucose load as in a glucose tolerance test
Symptoms of hyperglycemia and casual plasma glucose 11.1mmol/l (200mg/dl)
Glycated hemoglobin (Hb A1C) 6.5%.
[18]
A positive result, in the absence of unequivocal hyperglycemia, should be confirmed by a repeat of any of the above
methods on a different day. It is preferable to measure a fasting glucose level because of the ease of measurement
and the considerable time commitment of formal glucose tolerance testing, which takes two hours to complete and
offers no prognostic advantage over the fasting test.
[19]
According to the current definition, two fasting glucose
measurements above 126mg/dl (7.0mmol/l) is considered diagnostic for diabetes mellitus.
People with fasting glucose levels from 110 to 125mg/dl (6.1 to 6.9mmol/l) are considered to have impaired fasting
glucose.
[20]
Patients with plasma glucose at or above 140mg/dL (7.8mmol/L), but not over 200mg/dL
(11.1mmol/L), two hours after a 75g oral glucose load are considered to have impaired glucose tolerance. Of these
two prediabetic states, the latter in particular is a major risk factor for progression to full-blown diabetes mellitus, as
well as cardiovascular disease.
[21]
Glycated hemoglobin is better than fasting glucose for determining risks of cardiovascular disease and death from
any cause.
[22]
Management
Diabetes mellitus is a chronic disease, for which there is no known cure except in very specific situations.
Management concentrates on keeping blood sugar levels as close to normal ("euglycemia") as possible, without
causing hypoglycemia. This can usually be accomplished with diet, exercise, and use of appropriate medications
(insulin in the case of type1 diabetes; oral medications, as well as possibly insulin, in type2 diabetes).
Patient education, understanding, and participation is vital, since the complications of diabetes are far less common
and less severe in people who have well-managed blood sugar levels.
[23][24]
The goal of treatment is an HbA1C level
of 6.5%, but should not be lower than that, and may be set higher.
[]
Attention is also paid to other health problems
that may accelerate the deleterious effects of diabetes. These include smoking, elevated cholesterol levels, obesity,
high blood pressure, and lack of regular exercise.
[]
Specialised footwear is widely used to reduce the risk of
ulceration, or re-ulceration, in at-risk diabetic feet. Evidence for the efficacy of this remains equivocal, however.
[25]
Diabetes mellitus
323
Lifestyle
There are roles for patient education, dietetic support, sensible exercise, with the goal of keeping both short-term and
long-term blood glucose levels within acceptable bounds. In addition, given the associated higher risks of
cardiovascular disease, lifestyle modifications are recommended to control blood pressure.
[26]
Medications
Oral medications
Metformin is generally recommended as a first line treatment for type2 diabetes, as there is good evidence that it
decreases mortality.
[]
Routine use of aspirin, however, has not been found to improve outcomes in uncomplicated
diabetes.
[27]
Insulin
Type1 diabetes is typically treated with a combinations of regular and NPH insulin, or synthetic insulin analogs.
When insulin is used in type2 diabetes, a long-acting formulation is usually added initially, while continuing oral
medications.
[]
Doses of insulin are then increased to effect.
[]
Support
In countries using a general practitioner system, such as the United Kingdom, care may take place mainly outside
hospitals, with hospital-based specialist care used only in case of complications, difficult blood sugar control, or
research projects. In other circumstances, general practitioners and specialists share care of a patient in a team
approach. Home telehealth support can be an effective management technique.
[]
Epidemiology
Prevalence of diabetes worldwide in 2000 (per
1,000 inhabitants) - world average was 2.8%.
Disability-adjusted life year for diabetes mellitus
per 100,000inhabitants in 2004
Globally, as of 2010[28], an estimated 285million people had diabetes,
with type2 making up about 90% of the cases.
[]
Its incidence is
increasing rapidly, and by 2030, this number is estimated to almost
double.
[]
Diabetes mellitus occurs throughout the world, but is more
common (especially type2) in the more developed countries. The
greatest increase in prevalence is, however, expected to occur in Asia
and Africa, where most patients will probably be found by 2030.
[]
The
increase in incidence in developing countries follows the trend of
urbanization and lifestyle changes, perhaps most importantly a
"Western-style" diet. This has suggested an environmental (i.e.,
dietary) effect, but there is little understanding of the mechanism(s) at
present, though there is much speculation, some of it most
compellingly presented.
[]
Australia
Indigenous populations in first world countries have a higher
prevalence and increasing incidence of diabetes than their corresponding nonindigenous populations. In Australia,
the age-standardised prevalence of self-reported diabetes in indigenous Australians is almost four times that of
nonindigenous Australians.
[29]
Preventative community health programs, such as Sugar Man (diabetes education),
are showing some success in tackling this problem.
Diabetes mellitus
324
China
Almost one Chinese adult in ten has diabetes. A 2010 study estimated that more than 92 million Chinese adults have
the disease, with another 150 million showing early symptoms.
[]
The incidence of the disease is increasing rapidly; a
2009 study found a 30% increase in 7 years.
[30]
India
India has more diabetics than any other country in the world, according to the International Diabetes Foundation,
[]
although more recent data suggest that China has even more.
[]
The disease affects more than 50 million Indians -
7.1% of the nation's adults - and kills about 1 million Indians a year.
[]
The average age on onset is 42.5 years.
[]
The
high incidence is attributed to a combination of genetic susceptibility plus adoption of a high-calorie, low-activity
lifestyle by India's growing middle class.
[31]
United Kingdom
About 3.8million people in the United Kingdom have diabetes mellitus, but the charity Diabetes U.K. have made
predictions that that could become high as 6.2million by 2035/2036. Diabetes U.K. have also predicted that the
National Health Service could be spending as much as 16.9billion pounds on diabetes mellitus by 2035, a figure that
means the NHS could be spending as much as 17% of its budget on diabetes treatment by 2035.
[32][33][34]
United States
Diabetes rates at county levels 2004 - 2009.
For at least 20 years, diabetes rates in North America have been
increasing substantially. In 2010, nearly 26million people have
diabetes in the United States, of whom 7million people remain
undiagnosed. Another 57million people are estimated to have
prediabetes.
[35][36]
The Centers for Disease Control and Prevention (CDC) has termed the
change an epidemic.
[37]
The National Diabetes Information
Clearinghouse estimates diabetes costs $132billion in the United
States alone every year. About 5%10% of diabetes cases in North
America are type1, with the rest being type2. The fraction of type1 in
other parts of the world differs. Most of this difference is not currently understood. The American Diabetes
Association (ADA) cites the 2003 assessment of the National Center for Chronic Disease Prevention and Health
Promotion (Centers for Disease Control and Prevention) that one in three Americans born after 2000 will develop
diabetes in their lifetimes.
[38][]
According to the ADA, about 18.3% (8.6million) of Americans age 60 and older have diabetes.
[]
Diabetes mellitus
prevalence increases with age, and the numbers of older persons with diabetes are expected to grow as the elderly
population increases in number. The National Health and Nutrition Examination Survey (NHANES III)
demonstrated, in the population over 65 years old, 18% to 20% have diabetes, with 40% having either diabetes or its
precursor form of impaired glucose tolerance.
[]
Diabetes mellitus
325
History
Diabetes was one of the first diseases described,
[39]
with an Egyptian manuscript from c. 1500 BCE mentioning "too
great emptying of the urine".
[]
The first described cases are believed to be of type1 diabetes.
[]
Indian physicians
around the same time identified the disease and classified it as madhumeha or "honey urine", noting the urine would
attract ants.
[]
The term "diabetes" or "to pass through" was first used in 230BCE by the Greek Appollonius of
Memphis.
[]
The disease was considered as rare during the time of the Roman empire, with Galen commenting he had
only seen two cases during his career.
[]
This is possibly due the diet and life-style of the ancient people, or because
the clinical symptoms were observed during the advanced stage of the disease. Galen named the disease "diarrhea of
the urine" (diarrhea urinosa). The earliest surviving work with a detailed reference to diabetes is that of Aretaeus of
Cappadocia (2nd or early 3rd century CE). He described the symptoms and the course of the disease, which he
attributed to the moisture and coldness, reflecting the beliefs of the "Pneumatic School". He hypothesized a
correlation of diabetes with other diseases and he discussed differential diagnosis from the snakebite which also
provokes excessive thirst. His work remained unknown in the West until the middle of the 16th century when, in
1552, the first Latin edition was published in Venice.
[40]
Type1 and type2 diabetes where identified as separate conditions for the first time by the Indian physicians
Sushruta and Charaka in 400-500CE with type1 associated with youth and type2 with being overweight.
[]
The term
"mellitus" or "from honey" was added by the Briton John Rolle in the late 1700s to separate the condition from
diabetes insipidus, which is also associated with frequent urination.
[]
Effective treatment was not developed until the
early part of the 20th century, when Canadians Frederick Banting and Charles Herbert Best isolated and purified
insulin in 1921 and 1922.
[]
This was followed by the development of the long-acting insulin NPH in the 1940s.
[]
Etymology
The word diabetes (pron.: /da.bitiz/ or /da.bits/) comes from Latin diabts, which in turn comes from
Ancient Greek (diabts) which literally means "a passer through; a siphon."
[41]
Ancient Greek physician
Aretaeus of Cappadocia (fl. 1st century CE) used that word, with the intended meaning "excessive discharge of
urine", as the name for the disease.
[][]
Ultimately, the word comes from Greek (diabainein), meaning "to
pass through,"
[41]
which is composed of - (dia-), meaning "through" and (bainein), meaning "to go".
[]
The word "diabetes" is first recorded in English, in the form diabete, in a medical text written around 1425.
The word mellitus (/mlats/ or /mlts/) comes from the classical Latin word melltus, meaning "mellite"
[42]
(i.e.
sweetened with honey;
[42]
honey-sweet
[]
). The Latin word comes from mell-, which comes from mel, meaning
"honey";
[42][]
sweetness;
[]
pleasant thing,
[]
and the suffix -tus,
[42]
whose meaning is the same as that of the English
suffix "-ite".
[43]
It was Thomas Willis who in 1675 added "mellitus" to the word "diabetes" as a designation for the
disease, when he noticed the urine of a diabetic had a sweet taste (glycosuria).
[]
This sweet taste had been noticed in
urine by the ancient Greeks, Chinese, Egyptians, Indians, and Persians.
Society and culture
The 1989 "St. Vincent Declaration"
[44][45]
was the result of international efforts to improve the care accorded to
those with diabetes. Doing so is important not only in terms of quality of life and life expectancy, but also
economicallyexpenses due to diabetes have been shown to be a major drain on healthand productivity-related
resources for healthcare systems and governments.
Several countries established more and less successful national diabetes programmes to improve treatment of the
disease.
[]
Diabetic patients with neuropathic symptoms such as numbness or tingling in feet or hands are twice as likely to be
unemployed as those without the symptoms.
[]
Diabetes mellitus
326
In other animals
In animals, diabetes is most commonly encountered in dogs and cats. Middle-aged animals are most commonly
affected. Female dogs are twice as likely to be affected as males, while according to some sources, male cats are also
more prone than females. In both species, all breeds may be affected, but some small dog breeds are particularly
likely to develop diabetes, such as Miniature Poodles.
[]
The symptoms may relate to fluid loss and polyuria, but the
course may also be insidious. Diabetic animals are more prone to infections. The long-term complications recognised
in humans are much rarer in animals. The principles of treatment (weight loss, oral antidiabetics, subcutaneous
insulin) and management of emergencies (e.g. ketoacidosis) are similar to those in humans.
[]
References
[2] http:/ / apps.who.int/ classifications/ icd10/ browse/ 2010/ en#/ E10
[3] http:/ / apps.who.int/ classifications/ icd10/ browse/ 2010/ en#/ E14
[4] http:/ / www. icd9data. com/ getICD9Code.ashx?icd9=250
[5] http:/ / www. nlm. nih.gov/ medlineplus/ ency/ article/ 001214. htm
[6] http:/ / www. emedicine. com/ med/ topic546.htm
[7] http:/ / www. emedicine. com/ emerg/ topic134. htm#
[8] http:/ / www. nlm. nih.gov/ cgi/ mesh/ 2013/ MB_cgi?mode=& term=Diabetes& field=entry#TreeC18. 452. 394. 750
[15] [15] Unless otherwise specified, reference is: Table 20-5 in 8th edition.
[28] http:/ / en. wikipedia. org/ w/ index. php?title=Diabetes_mellitus& action=edit
[33] http:/ / managingdiabetes. co. uk/ articles/ diabetesnhsspending. php
[34] http:/ / www.nhs. uk/ news/ 2012/ 04april/ Pages/ nhs-diabetes-costs-cases-rising. aspx
[41] Oxford English Dictionary. diabetes. Retrieved 2011-06-10.
[42] Oxford English Dictionary. mellite. Retrieved 2011-06-10.
[43] Oxford English Dictionary. -ite. Retrieved 2011-06-10.
Further reading
Polonsky, K. S. (2012). "The Past 200 Years in Diabetes". New England Journal of Medicine 367 (14):
13321340. doi: 10.1056/NEJMra1110560 (http:/ / dx. doi. org/ 10. 1056/ NEJMra1110560). PMID 23034021
(http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 23034021).
External links
Diabetes (http:/ / www. dmoz. org/ Health/ Conditions_and_Diseases/ Endocrine_Disorders/ Pancreas/ Diabetes/ )
at the Open Directory Project
American Diabetes Association (http:/ / www. diabetes. org/ )
IDF Diabetes Atlas (http:/ / www. diabetesatlas. org/ )
National Diabetes Education Program (http:/ / ndep. nih. gov/ )
327
Informational Macromolecules
328
DNA synthesis and repair
DNA replication
DNA replication. The double helix is unwound
and each strand acts as a template for the next
strand. Bases are matched to synthesize the new
partner strands.
DNA replication is a biological process that occurs in all living
organisms and copies their DNA. DNA replication during mitosis is
the basis for biological inheritance. The process of DNA replication
starts when one double-stranded DNA molecule produces two
identical copies of the molecule. Each strand of the original
double-stranded DNA molecule serves as template for the production
of the complementary strand, a process referred to as semiconservative
replication. Cellular proofreading and error-checking mechanisms
ensure near perfect fidelity for DNA replication.
[1][1]
In a cell, DNA replication begins at specific locations, or origin of
replication, in the genome.
[]
Unwinding of DNA at the origin, and
synthesis of new strands, forms a replication fork. A number of
proteins are associated with the fork and assist in the initiation and
continuation of DNA synthesis. Most prominently, DNA polymerase
synthesizes the new DNA by adding matching nucleotides to the
template strand.
DNA replication can also be performed in vitro (artificially, outside a
cell). DNA polymerases isolated from cells and artificial DNA primers
can be used to initiate DNA synthesis at known sequences in a
template DNA molecule. The polymerase chain reaction (PCR), a
common laboratory technique, cyclically apply such artificial
synthesis to amplify a specific target DNA fragment from a pool of
DNA.
Background on DNA structure
DNA usually exists as a double-stranded structure, with both strands coiled together to form the characteristic
double-helix. Each single strand of DNA is a chain of four types of nucleotides. Nucleotides in DNA contain a
deoxyribose sugar, a phosphate, and a nucleobase. The four types of nucleotide correspond to the four nucleobases
adenine, cytosine, guanine, and thymine, commonly notated as A,C, G and T. These nucleotides form
phosphodiester bonds, creating the phosphate-deoxyribose backbone of the DNA double helix with the nucleobases
pointing inward. Nucleotides (bases) are matched between strands through hydrogen bonds to form base pairs.
Adenine pairs with thymine (two hydrogen bonds), and cytosine pairs with guanine (three hydrogen bonds) because
a purine must pair with a pyrimidine.
DNA strands have a directionality, and the different ends of a single strand are called the "3' (three-prime) end" and
the "5' (five-prime) end" with the direction of the naming going 5 prime to the 3 prime region. The strands of the
helix are anti-parallel with one being 5 prime to 3 then the opposite strand 3 prime to 5. These terms refer to the
carbon atom in deoxyribose to which the next phosphate in the chain attaches. Directionality has consequences in
DNA synthesis, because DNA polymerase can synthesize DNA in only one direction by adding nucleotides to the 3'
DNA replication
329
end of a DNA strand.
The pairing of bases in DNA through hydrogen bonding means that the information contained within each strand is
redundant. The nucleotides on a single strand can be used to reconstruct nucleotides on a newly synthesized partner
strand.
[2]
DNA polymerase
DNA polymerases adds nucleotides to the 3' end
of a strand of DNA.
[3]
If a mismatch is
accidentally incorporated, the polymerase is
inhibited from further extension. Proofreading
removes the mismatched nucleotide and extension
continues.
DNA polymerases are a family of enzymes that carry out all forms of
DNA replication.
[4]
However, a DNA polymerase can only extend an
existing DNA strand paired with a template strand; it cannot begin the
synthesis of a new strand. To begin synthesis, a short fragment of
DNA or RNA, called a primer, must be created and paired with the
template DNA strand.
DNA polymerase then synthesizes a new strand of DNA by extending
the 3' end of an existing nucleotide chain, adding new nucleotides
matched to the template strand one at a time via the creation of
phosphodiester bonds. The energy for this process of DNA
polymerization comes from two of the three total phosphates attached
to each unincorporated base. (Free bases with their attached phosphate
groups are called nucleoside triphosphates.) When a nucleotide is
being added to a growing DNA strand, two of the phosphates are
removed and the energy produced creates a phosphodiester bond that
attaches the remaining phosphate to the growing chain. The energetics
of this process also help explain the directionality of synthesisif
DNA were synthesized in the 3' to 5' direction, the energy for the
process would come from the 5' end of the growing strand rather than
from free nucleotides.
In general, DNA polymerases are extremely accurate, making less
than one mistake for every 10
7
nucleotides added.
[]
Even so, some
DNA polymerases also have proofreading ability; they can remove
nucleotides from the end of a strand in order to correct mismatched
bases. If the 5' nucleotide needs to be removed during proofreading,
the triphosphate end is lost. Hence, the energy source that usually
provides energy to add a new nucleotide is also lost.
The rate of DNA replication in a living cell was first measured as the rate of phage T4 DNA elongation in
phage-infected E. coli.
[5]
During the period of exponential DNA increase at 30C, the rate was 749 nucleotides per
second. The mutation rate per base pair per replication during phage T4 DNA synthesis is 1.7 per 10
8
.
[6]
Thus DNA
replication is both impressively fast and accurate.
DNA replication
330
Replication process
DNA Replication, like all biological polymerization processes, proceeds in three enzymatically catalyzed and
coordinated steps: initiation, elongation and termination.
Replication origin
For a cell to divide, it must first replicate its DNA.
[7]
This process is initiated at particular points in the DNA, known
as "origins", which are targeted by proteins that initiate DNA synthesis.
[]
Origins contain DNA sequences recognized
by replication initiator proteins (e.g., DnaA in E. coli and the Origin Recognition Complex in yeast).
[8]
Sequences
used by initiator proteins tend to be "AT-rich" (rich in adenine and thymine bases), because A-T base pairs have two
hydrogen bonds (rather than the three formed in a C-G pair). AT-rich sequences are easier to unzip since less energy
is required to break relatively fewer hydrogen bonds.
[9]
Once the origin has been located, these initiators recruit other
proteins and form the pre-replication complex, which unzips, or separates, the DNA strands at the origin.
Extensions
All known DNA replication systems require a free 3' hydroxyl group before synthesis can be initiated (Important
note: DNA is read in 3' to 5' direction whereas a new strand is synthesised in the 5' to 3' directionthis is entirely
logical but is often confused). Four distinct mechanisms for synthesis have been described.
1. All cellular life forms and many DNA viruses, phages and plasmids use a primase to synthesize a short RNA
primer with a free 3 OH group which is subsequently elongated by a DNA polymerase.
2. The retroelements (including retroviruses) employ a transfer RNA that primes DNA replication by providing a
free 3 OH that is used for elongation by the reverse transcriptase.
3. In the adenoviruses and the 29 family of bacteriophages, the 3' OH group is provided by the side chain of an
amino acid of the genome attached protein (the terminal protein) to which nucleotides are added by the DNA
polymerase to form a new strand.
4. In the single stranded DNA viruses a group that includes the circoviruses, the geminiviruses, the parvoviruses
and others and also the many phages and plasmids that use the rolling circle replication (RCR) mechanism, the
RCR endonuclease creates a nick the genome strand (single stranded viruses) or one of the DNA strands
(plasmids). The 5 end of the nicked strand is transferred to a tyrosine residue on the nuclease and the free 3 OH
group is then used by the DNA polymerase for new strand synthesis.
The first is the best known of these mechanisms and is used by the cellular organisms. In this mechanism, once the
two strands are separated, primase adds an RNA primers to the template strands. The leading strand receives one
RNA primer while the lagging strand receives several. The leading strand is extended from the primer in one motion
by DNA polymerase, while the lagging strand is extended discontinuously from each primer, forming Okazaki
fragments. RNase removes the primer RNA fragments, and another DNA Polymerase enters to fill the gaps. When
this is complete, a single nick on the leading strand and several nicks on the lagging strand can be found. Ligase
works to fill these nicks in, thus completing the newly replicated DNA molecule.
The primase used in this process differs significantly between bacteria and archaea/eukaryotes. Bacteria use a
primase belonging to the DnaG protein superfamily which contains a catalytic domain of the TOPRIM fold type.
The TOPRIM fold contains an / core with four conserved strands in a Rossmann-like topology. This structure is
also found in the catalytic domains of topoisomerase Ia, topoisomerase II, the OLD-family nucleases and DNA
repair proteins related to the RecR protein.
The primase used by archaea and eukaryotes in contrast contains a highly derived version of the RNA recognition
motif (RRM). This primase is structurally similar to many viral RNA dependent RNA polymerases, reverse
transcriptases, cyclic nucleotide generating cyclases and DNA polymerases of the A/B/Y families that are involved
in DNA replication and repair. All these proteins share a catalytic mechanism of di-metal-ion-mediated nucleotide
DNA replication
331
transfer, whereby two acidic residues located at the end of the first strand and between the second and third strands
of the RRM-like unit respectively, chelate two divalent cations.
As DNA synthesis continues, the original DNA strands continue to unwind on each side of the bubble, forming a
replication fork with two prongs. In bacteria, which have a single origin of replication on their circular chromosome,
this process eventually creates a "theta structure" (resembling the Greek letter theta: -). In contrast, eukaryotes have
longer linear chromosomes and initiate replication at multiple origins within these.
[10]
DNA replication proteins
List of major DNA replication enzymes in the Replisome:
[11]
Enzyme Function in DNA replication
DNA Helicase Also known as helix destabilizing enzyme. Unwinds the DNA double helix at the Replication Fork.
DNA Polymerase Builds a new duplex DNA strand by adding nucleotides in the 5' to 3' direction. Also performs proof-reading and
error correction.
DNA clamp A protein which prevents DNA polymerase III from dissociating from the DNA parent strand.
Single-Strand Binding (SSB)
Proteins
Bind to ssDNA and prevent the DNA double helix from re-annealing after DNA helicase unwinds it thus
maintaining the strand separation.
Topoisomerase Relaxes the DNA from its super-coiled nature.
DNA Gyrase Relieves strain of unwinding by DNA helicase; this is a specific type of topisomerase
DNA Ligase Re-anneals the semi-conservative strands and joins Okazaki Fragments of the lagging strand.
Primase Provides a starting point of RNA (or DNA) for DNA polymerase to begin synthesis of the new DNA strand.
Telomerase Lengthens telomeric DNA by adding repetitive nucleotide sequences to the ends of eukaryotic chromosomes.
Replication fork
Scheme of the replication fork.
a: template, b: leading strand, c: lagging strand, d:
replication fork, e: primer, f: Okazaki fragments
The replication fork is a structure that forms within the nucleus during
DNA replication. It is created by helicases, which break the hydrogen
bonds holding the two DNA strands together. The resulting structure
has two branching "prongs", each one made up of a single strand of
DNA. These two strands serve as the template for the leading and
lagging strands, which will be created as DNA polymerase matches
complementary nucleotides to the templates; the templates may be
properly referred to as the leading strand template and the lagging
strand templates
Leading strand
The leading strand is the template strand of the DNA double helix so that the replication fork moves along it in the 3'
to 5' direction.
DNA replication
332
Many enzymes are involved in the DNA replication fork.
This allows the newly synthesized strand
complementary to the original strand to be
synthesized 5' to 3' in the same direction as
the movement of the replication fork.
On the leading strand, a polymerase "reads"
the DNA and adds nucleotides to it
continuously. This polymerase is DNA
polymerase III (DNA Pol III) in prokaryotes
and presumably Pol
[][12]
in yeasts. In
human cells the leading and lagging strands
are synthesized by Pol and Pol within
the nucleus and Pol in the mitochondria. Pol can substitute for Pol in special circumstances.
[]
Lagging strand
The lagging strand is the strand of the template DNA double helix that is oriented so that the replication fork moves
along it in a 5' to 3' manner. Because of its orientation, opposite to the working orientation of DNA polymerase III,
which moves on a template in a 3' to 5' manner, replication of the lagging strand is more complicated than that of the
leading strand.
On the lagging strand, primase "reads" the DNA and adds RNA to it in short, separated segments. In eukaryotes,
primase is intrinsic to Pol .
[13]
DNA polymerase III or Pol lengthens the primed segments, forming Okazaki
fragments. Primer removal in eukaryotes is also performed by Pol .
[14]
In prokaryotes, DNA polymerase I "reads"
the fragments, removes the RNA using its flap endonuclease domain (RNA primers are removed by 5'-3'
exonuclease activity of polymerase I [weaver, 2005]), and replaces the RNA nucleotides with DNA nucleotides (this
is necessary because RNA and DNA use slightly different kinds of nucleotides). DNA ligase joins the fragments
together.
Dynamics at the replication fork
The assembled human DNA clamp, a trimer of the
protein PCNA.
As helicase unwinds DNA at the replication fork, the DNA ahead
is forced to rotate. This process results in a build-up of twists in
the DNA ahead.
[15]
This build-up would form a resistance that
would eventually halt the progress of the replication fork. DNA
Gyrase is an enzyme that temporarily breaks the strands of DNA,
relieving the tension caused by unwinding the two strands of the
DNA helix; DNA Gyrase achieves this by adding negative
supercoils to the DNA helix.
[16]
Bare single-stranded DNA tends to fold back on itself and form
secondary structures; these structures can interfere with the
movement of DNA polymerase. To prevent this, single-strand
binding proteins bind to the DNA until a second strand is
synthesized, preventing secondary structure formation.
[17]
Clamp proteins form a sliding clamp around DNA, helping the
DNA polymerase maintain contact with its template, thereby
assisting with processivity. The inner face of the clamp enables
DNA to be threaded through it. Once the polymerase reaches the end of the template or detects double-stranded
DNA replication
333
DNA, the sliding clamp undergoes a conformational change that releases the DNA polymerase. Clamp-loading
proteins are used to initially load the clamp, recognizing the junction between template and RNA primers.
[1]:274-5
Regulation
The cell cycle of eukaryotic cells.
Eukaryotes
Within eukaryotes, DNA replication is controlled within the context of the
cell cycle. As the cell grows and divides, it progresses through stages in the
cell cycle; DNA replication occurs during the S phase (synthesis phase). The
progress of the eukaryotic cell through the cycle is controlled by cell cycle
checkpoints. Progression through checkpoints is controlled through complex
interactions between various proteins, including cyclins and cyclin-dependent
kinases.
[18]
The G1/S checkpoint (or restriction checkpoint) regulates whether eukaryotic
cells enter the process of DNA replication and subsequent division. Cells that
do not proceed through this checkpoint remain in the G0 stage and do not
replicate their DNA.
Replication of chloroplast and mitochondrial genomes occurs independent of the cell cycle, through the process of
D-loop replication.
Bacteria
Most bacteria do not go through a well-defined cell cycle but instead continuously copy their DNA; during rapid
growth, this can result in the concurrent occurrences of multiple rounds of replication.
[19]
In E. coli, the
best-characterized bacteria, DNA replication is regulated through several mechanisms, including: the
hemimethylation and sequestering of the origin sequence, the ratio of ATP to ADP, and the levels of protein DnaA.
All these control the process of initiator proteins binding to the origin sequences.
Because E. coli methylates GATC DNA sequences, DNA synthesis results in hemimethylated sequences. This
hemimethylated DNA is recognized by the protein SeqA, which binds and sequesters the origin sequence; in
addition, DnaA (required for initiation of replication) binds less well to hemimethylated DNA. As a result, newly
replicated origins are prevented from immediately initiating another round of DNA replication.
[20]
ATP builds up when the cell is in a rich medium, triggering DNA replication once the cell has reached a specific
size. ATP competes with ADP to bind to DnaA, and the DnaA-ATP complex is able to initiate replication. A certain
number of DnaA proteins are also required for DNA replication each time the origin is copied, the number of
binding sites for DnaA doubles, requiring the synthesis of more DnaA to enable another initiation of replication.
Termination
Eukaryotes initiate DNA replication at multiple points in the chromosome, so replication forks meet and terminate at
many points in the chromosome; these are not known to be regulated in any particular way. Because eukaryotes have
linear chromosomes, DNA replication is unable to reach the very end of the chromosomes, but ends at the telomere
region of repetitive DNA close to the end. This shortens the telomere of the daughter DNA strand. This is a normal
process in somatic cells. As a result, cells can only divide a certain number of times before the DNA loss prevents
further division. (This is known as the Hayflick limit.) Within the germ cell line, which passes DNA to the next
generation, telomerase extends the repetitive sequences of the telomere region to prevent degradation. Telomerase
can become mistakenly active in somatic cells, sometimes leading to cancer formation.
Additionally, to aid termination, the progress of the DNA replication fork must stop or be blocked. Essentially, there
are two methods that organisms do this, firstly, it is to have a termination site sequence in the DNA, and secondly, it
is to have a protein which binds to this sequence to physically stop DNA replication proceeding. This is named the
DNA replication
334
DNA replication terminus site-binding protein or in other words, Ter protein.
Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet
each other on the opposite end of the parental chromosome. E coli regulate this process through the use of
termination sequences that, when bound by the Tus protein, enable only one direction of replication fork to pass
through. As a result, the replication forks are constrained to always meet within the termination region of the
chromosome.
[21]
Polymerase chain reaction
Researchers commonly replicate DNA in vitro using the polymerase chain reaction (PCR). PCR uses a pair of
primers to span a target region in template DNA, and then polymerizes partner strands in each direction from these
primers using a thermostable DNA polymerase. Repeating this process through multiple cycles produces
amplification of the targeted DNA region. At the start of each cycle, the mixture of template and primers is heated,
separating the newly synthesized molecule and template. Then, as the mixture cools, both of these become templates
for annealing of new primers, and the polymerase extends from these. As a result, the number of copies of the target
region doubles each round, increasing exponentially.
[]
References
[1] Imperfect DNA replication results in mutations.
[2] Alberts, B., et.al., Molecular Biology of the Cell, Garland Science, 4th ed., 2002, pp. 238-240 ISBN 0-8153-3218-1
[3] Allison, Lizabeth A. Fundamental Molecular Biology. Blackwell Publishing. 2007. p.112 ISBN 978-1-4051-0379-4
[4] Chapter 27, Section 2: DNA Polymerases Require a Template and a Primer (http:/ / www. ncbi. nlm. nih. gov/ books/ bv. fcgi?rid=stryer.
section.3769)
[6] Drake JW (1970) The Molecular Basis of Mutation. Holden-Day, San Francisco ISBN 0816224501 ISBN 978-0816224500
[7] Chapter 5: DNA Replication Mechanisms (http:/ / www. ncbi. nlm. nih. gov/ books/ bv. fcgi?rid=mboc4. section. 754)
[9] 12.1. General Features of Chromosomal Replication: Three Common Features of Replication Origins (http:/ / www. ncbi. nlm. nih. gov/
books/ bv.fcgi?rid=mcb.section. 3163#3179)
[11] [11] [ Chapter 7: DNA: Structure and Replication. pg 283-290 ]
[14] Distinguishing the pathways of primer removal during Eukaryotic Okazaki fragment maturation (http:/ / hdl. handle. net/ 1802/ 6537)
Contributor Author Rossi, Marie Louise. Date Accessioned: 2009-02-23T17:05:09Z. Date Available: 2009-02-23T17:05:09Z. Date Issued:
2009-02-23T17:05:09Z. Identifier Uri: http:/ / hdl.handle. net/ 1802/ 6537. Description: Dr. Robert A. Bambara, Faculty Advisor. Thesis
(PhD) - School of Medicine and Dentistry, University of Rochester. UR only until January 2010. UR only until January 2010.
[15] DNA Replication Mechanisms: DNA Topoisomerases Prevent DNA Tangling During Replication (http:/ / www. ncbi. nlm. nih. gov/ books/
bv. fcgi?rid=mboc4. section.754#787)
[16] DNA gyrase: structure and function. [Crit Rev Biochem Mol Biol. 1991] - PubMed - NCBI (http:/ / www. ncbi. nlm. nih. gov/ pubmed/
1657531)
[17] DNA Replication Mechanisms: Special Proteins Help to Open Up the DNA Double Helix in Front of the Replication Fork (http:/ / www.
ncbi.nlm. nih.gov/ books/ bv.fcgi?rid=mboc4. section.754#774)
[18] Intracellular Control of Cell-Cycle Events: S-Phase Cyclin-Cdk Complexes (S-Cdks) Initiate DNA Replication Once Per Cycle (http:/ /
www.ncbi. nlm. nih. gov/ books/ bv. fcgi?rid=mboc4. section. 3214#3215)
[21] 13.2.3. Termination of replication (http:/ / www.ncbi. nlm. nih. gov/ books/ bv. fcgi?rid=genomes. section. 8121#8156)
DNA repair
335
DNA repair
DNA damage resulting in multiple broken
chromosomes
DNA repair is a collection of processes by which a cell identifies and
corrects damage to the DNA molecules that encode its genome. In
human cells, both normal metabolic activities and environmental
factors such as UV light and radiation can cause DNA damage,
resulting in as many as 1 million individual molecular lesions per cell
per day.
[1]
Many of these lesions cause structural damage to the DNA
molecule and can alter or eliminate the cell's ability to transcribe the
gene that the affected DNA encodes. Other lesions induce potentially
harmful mutations in the cell's genome, which affect the survival of its
daughter cells after it undergoes mitosis. As a consequence, the DNA
repair process is constantly active as it responds to damage in the DNA
structure. When normal repair processes fail, and when cellular
apoptosis does not occur, irreparable DNA damage may occur,
including double-strand breaks and DNA crosslinkages (interstrand
crosslinks or ICLs).
[][]
The rate of DNA repair is dependent on many factors, including the
cell type, the age of the cell, and the extracellular environment. A cell
that has accumulated a large amount of DNA damage, or one that no
longer effectively repairs damage incurred to its DNA, can enter one of three possible states:
1. an irreversible state of dormancy, known as senescence
2. cell suicide, also known as apoptosis or programmed cell death
3. unregulated cell division, which can lead to the formation of a tumor that is cancerous
The DNA repair ability of a cell is vital to the integrity of its genome and thus to its normal functioning and that of
the organism. Many genes that were initially shown to influence life span have turned out to be involved in DNA
damage repair and protection.
[]
Failure to correct molecular lesions in cells that form gametes can introduce
mutations into the genomes of the offspring and thus influence the rate of evolution.
DNA damage
DNA damage, due to environmental factors and normal metabolic processes inside the cell, occurs at a rate of 1,000
to 1,000,000 molecular lesions per cell per day.
[1]
While this constitutes only 0.000165% of the human genome's
approximately 6 billion bases (3 billion base pairs), unrepaired lesions in critical genes (such as tumor suppressor
genes) can impede a cell's ability to carry out its function and appreciably increase the likelihood of tumor formation.
The vast majority of DNA damage affects the primary structure of the double helix; that is, the bases themselves are
chemically modified. These modifications can in turn disrupt the molecules' regular helical structure by introducing
non-native chemical bonds or bulky adducts that do not fit in the standard double helix. Unlike proteins and RNA,
DNA usually lacks tertiary structure and therefore damage or disturbance does not occur at that level. DNA is,
however, supercoiled and wound around "packaging" proteins called histones (in eukaryotes), and both
superstructures are vulnerable to the effects of DNA damage.
DNA repair
336
Sources of damage
DNA damage can be subdivided into two main types:
1. endogenous damage such as attack by reactive oxygen species produced from normal metabolic byproducts
(spontaneous mutation), especially the process of oxidative deamination
1. also includes replication errors
2. 2. exogenous damage caused by external agents such as
1. ultraviolet [UV 200-400 nm] radiation from the sun
2. other radiation frequencies, including x-rays and gamma rays
3. hydrolysis or thermal disruption
4. certain plant toxins
5. human-made mutagenic chemicals, especially aromatic compounds that act as DNA intercalating agents
6. viruses
[]
The replication of damaged DNA before cell division can lead to the incorporation of wrong bases opposite damaged
ones. Daughter cells that inherit these wrong bases carry mutations from which the original DNA sequence is
unrecoverable (except in the rare case of a back mutation, for example, through gene conversion).
Types of damage
There are five main types of damage to DNA due to endogenous cellular processes:
1. oxidation of bases [e.g. 8-oxo-7,8-dihydroguanine (8-oxoG)] and generation of DNA strand interruptions from
reactive oxygen species,
2. alkylation of bases (usually methylation), such as formation of 7-methylguanine, 1-methyladenine,
6-O-Methylguanine
3. hydrolysis of bases, such as deamination, depurination, and depyrimidination.
4. 4. "bulky adduct formation" (i.e., benzo[a]pyrene diol epoxide-dG adduct, aristolactam I-dA adduct)
5. mismatch of bases, due to errors in DNA replication, in which the wrong DNA base is stitched into place in a
newly forming DNA strand, or a DNA base is skipped over or mistakenly inserted.
Damage caused by exogenous agents comes in many forms. Some examples are:
1. UV-B light causes crosslinking between adjacent cytosine and thymine bases creating pyrimidine dimers. This is
called direct DNA damage.
2. UV-A light creates mostly free radicals. The damage caused by free radicals is called indirect DNA damage.
3. Ionizing radiation such as that created by radioactive decay or in cosmic rays causes breaks in DNA strands.
Low-level ionizing radiation may induce irreparable DNA damage (leading to replicational and transcriptional
errors needed for neoplasia or may trigger viral interactions) leading to pre-mature aging and cancer.
[2][3][4]
4. Thermal disruption at elevated temperature increases the rate of depurination (loss of purine bases from the DNA
backbone) and single-strand breaks. For example, hydrolytic depurination is seen in the thermophilic bacteria,
which grow in hot springs at 40-80 C.
[5][]
The rate of depurination (300 purine residues per genome per
generation) is too high in these species to be repaired by normal repair machinery, hence a possibility of an
adaptive response cannot be ruled out.
5. Industrial chemicals such as vinyl chloride and hydrogen peroxide, and environmental chemicals such as
polycyclic aromatic hydrocarbons found in smoke, soot and tar create a huge diversity of DNA adducts-
ethenobases, oxidized bases, alkylated phosphotriesters and Crosslinking of DNA just to name a few.
UV damage, alkylation/methylation, X-ray damage and oxidative damage are examples of induced damage.
Spontaneous damage can include the loss of a base, deamination, sugar ring puckering and tautomeric shift.
[6]
DNA repair
337
Nuclear versus mitochondrial DNA damage
In human cells, and eukaryotic cells in general, DNA is found in two cellular locations inside the nucleus and
inside the mitochondria. Nuclear DNA (nDNA) exists as chromatin during non-replicative stages of the cell cycle
and is condensed into aggregate structures known as chromosomes during cell division. In either state the DNA is
highly compacted and wound up around bead-like proteins called histones. Whenever a cell needs to express the
genetic information encoded in its nDNA the required chromosomal region is unravelled, genes located therein are
expressed, and then the region is condensed back to its resting conformation. Mitochondrial DNA (mtDNA) is
located inside mitochondria organelles, exists in multiple copies, and is also tightly associated with a number of
proteins to form a complex known as the nucleoid. Inside mitochondria, reactive oxygen species (ROS), or free
radicals, byproducts of the constant production of adenosine triphosphate (ATP) via oxidative phosphorylation,
create a highly oxidative environment that is known to damage mtDNA. A critical enzyme in counteracting the
toxicity of these species is superoxide dismutase, which is present in both the mitochondria and cytoplasm of
eukaryotic cells.
Senescence and apoptosis
Senescence, an irreversible state in which the cell no longer divides, is a protective response to the shortening of the
chromosome ends. The telomeres are long regions of repetitive noncoding DNA that cap chromosomes and undergo
partial degradation each time a cell undergoes division (see Hayflick limit).
[]
In contrast, quiescence is a reversible
state of cellular dormancy that is unrelated to genome damage (see cell cycle). Senescence in cells may serve as a
functional alternative to apoptosis in cases where the physical presence of a cell for spatial reasons is required by the
organism,
[]
which serves as a "last resort" mechanism to prevent a cell with damaged DNA from replicating
inappropriately in the absence of pro-growth cellular signaling. Unregulated cell division can lead to the formation of
a tumor (see cancer), which is potentially lethal to an organism. Therefore, the induction of senescence and apoptosis
is considered to be part of a strategy of protection against cancer.
[]
DNA damage and mutation
It is important to distinguish between DNA damage and mutation, the two major types of error in DNA. DNA
damages and mutation are fundamentally different. Damages are physical abnormalities in the DNA, such as single-
and double-strand breaks, 8-hydroxydeoxyguanosine residues, and polycyclic aromatic hydrocarbon adducts. DNA
damages can be recognized by enzymes, and, thus, they can be correctly repaired if redundant information, such as
the undamaged sequence in the complementary DNA strand or in a homologous chromosome, is available for
copying. If a cell retains DNA damage, transcription of a gene can be prevented, and, thus, translation into a protein
will also be blocked. Replication may also be blocked and/or the cell may die.
In contrast to DNA damage, a mutation is a change in the base sequence of the DNA. A mutation cannot be
recognized by enzymes once the base change is present in both DNA strands, and, thus, a mutation cannot be
repaired. At the cellular level, mutations can cause alterations in protein function and regulation. Mutations are
replicated when the cell replicates. In a population of cells, mutant cells will increase or decrease in frequency
according to the effects of the mutation on the ability of the cell to survive and reproduce. Although distinctly
different from each other, DNA damages and mutations are related because DNA damages often cause errors of
DNA synthesis during replication or repair; these errors are a major source of mutation.
Given these properties of DNA damage and mutation, it can be seen that DNA damages are a special problem in
non-dividing or slowly dividing cells, where unrepaired damages will tend to accumulate over time. On the other
hand, in rapidly dividing cells, unrepaired DNA damages that do not kill the cell by blocking replication will tend to
cause replication errors and thus mutation. The great majority of mutations that are not neutral in their effect are
deleterious to a cell's survival. Thus, in a population of cells comprising a tissue with replicating cells, mutant cells
will tend to be lost. However, infrequent mutations that provide a survival advantage will tend to clonally expand at
DNA repair
338
the expense of neighboring cells in the tissue. This advantage to the cell is disadvantageous to the whole organism,
because such mutant cells can give rise to cancer. Thus, DNA damages in frequently dividing cells, because they
give rise to mutations, are a prominent cause of cancer. In contrast, DNA damages in infrequently dividing cells are
likely a prominent cause of aging.
[]
DNA repair mechanisms
Single-strand and double-strand
DNA damage
Cells cannot function if DNA damage corrupts the integrity and accessibility of
essential information in the genome (but cells remain superficially functional when
so-called "non-essential" genes are missing or damaged). Depending on the type of
damage inflicted on the DNA's double helical structure, a variety of repair
strategies have evolved to restore lost information. If possible, cells use the
unmodified complementary strand of the DNA or the sister chromatid as a template
to recover the original information. Without access to a template, cells use an
error-prone recovery mechanism known as translesion synthesis as a last resort.
Damage to DNA alters the spatial configuration of the helix, and such alterations
can be detected by the cell. Once damage is localized, specific DNA repair
molecules bind at or near the site of damage, inducing other molecules to bind and
form a complex that enables the actual repair to take place.
Direct reversal
Cells are known to eliminate three types of damage to their DNA by chemically
reversing it. These mechanisms do not require a template, since the types of
damage they counteract can occur in only one of the four bases. Such direct
reversal mechanisms are specific to the type of damage incurred and do not involve
breakage of the phosphodiester backbone. The formation of pyrimidine dimers
upon irradiation with UV light results in an abnormal covalent bond between
adjacent pyrimidine bases. The photoreactivation process directly reverses this
damage by the action of the enzyme photolyase, whose activation is obligately
dependent on energy absorbed from blue/UV light (300500nm wavelength) to
promote catalysis.
[]
Another type of damage, methylation of guanine bases, is
directly reversed by the protein methyl guanine methyl transferase (MGMT), the
bacterial equivalent of which is called ogt. This is an expensive process because each MGMT molecule can be used
only once; that is, the reaction is stoichiometric rather than catalytic.
[7]
A generalized response to methylating agents
in bacteria is known as the adaptive response and confers a level of resistance to alkylating agents upon sustained
exposure by upregulation of alkylation repair enzymes.
[]
The third type of DNA damage reversed by cells is certain
methylation of the bases cytosine and adenine.
DNA repair
339
Single-strand damage
Structure of the base-excision repair enzyme uracil-DNA
glycosylase. The uracil residue is shown in yellow.
When only one of the two strands of a double helix has
a defect, the other strand can be used as a template to
guide the correction of the damaged strand. In order to
repair damage to one of the two paired molecules of
DNA, there exist a number of excision repair
mechanisms that remove the damaged nucleotide and
replace it with an undamaged nucleotide
complementary to that found in the undamaged DNA
strand.
[7]
1. Base excision repair (BER), which repairs damage
to a single base caused by oxidation, alkylation,
hydrolysis, or deamination. The damaged base is
removed by a DNA glycosylase. The "missing
tooth" is then recognized by an enzyme called AP
endonuclease, which cuts the Phosphodiester bond. The missing part is then resynthesized by a DNA polymerase,
and a DNA ligase performs the final nick-sealing step.
2. Nucleotide excision repair (NER), which recognizes bulky, helix-distorting lesions such as pyrimidine dimers and
6,4 photoproducts. A specialized form of NER known as transcription-coupled repair deploys NER enzymes to
genes that are being actively transcribed.
3. Mismatch repair (MMR), which corrects errors of DNA replication and recombination that result in mispaired
(but undamaged) nucleotides.
Double-strand breaks
Double-strand breaks, in which both strands in the double helix are severed, are particularly hazardous to the cell
because they can lead to genome rearrangements. Three mechanisms exist to repair double-strand breaks (DSBs):
non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous
recombination.
[7]
PVN Acharya noted that double-strand breaks and a "cross-linkage joining both strands at the
same point is irreparable because neither strand can then serve as a template for repair. The cell will die in the next
mitosis or in some rare instances, mutate."
[][]
DNA repair
340
DNA ligase, shown above repairing chromosomal damage, is
an enzyme that joins broken nucleotides together by catalyzing
the formation of an internucleotide ester bond between the
phosphate backbone and the deoxyribose nucleotides.
In NHEJ, DNA Ligase IV, a specialized DNA ligase that
forms a complex with the cofactor XRCC4, directly joins
the two ends.
[8]
To guide accurate repair, NHEJ relies on
short homologous sequences called microhomologies
present on the single-stranded tails of the DNA ends to be
joined. If these overhangs are compatible, repair is usually
accurate.
[][9][][10]
NHEJ can also introduce mutations during
repair. Loss of damaged nucleotides at the break site can
lead to deletions, and joining of nonmatching termini forms
translocations. NHEJ is especially important before the cell
has replicated its DNA, since there is no template available
for repair by homologous recombination. There are
"backup" NHEJ pathways in higher eukaryotes.
[]
Besides its
role as a genome caretaker, NHEJ is required for joining
hairpin-capped double-strand breaks induced during V(D)J
recombination, the process that generates diversity in B-cell
and T-cell receptors in the vertebrate immune system.
[11]
Homologous recombination requires the presence of an
identical or nearly identical sequence to be used as a
template for repair of the break. The enzymatic machinery
responsible for this repair process is nearly identical to the
machinery responsible for chromosomal crossover during
meiosis. This pathway allows a damaged chromosome to be repaired using a sister chromatid (available in G2 after
DNA replication) or a homologous chromosome as a template. DSBs caused by the replication machinery attempting
to synthesize across a single-strand break or unrepaired lesion cause collapse of the replication fork and are typically
repaired by recombination.
Topoisomerases introduce both single- and double-strand breaks in the course of changing the DNA's state of
supercoiling, which is especially common in regions near an open replication fork. Such breaks are not considered
DNA damage because they are a natural intermediate in the topoisomerase biochemical mechanism and are
immediately repaired by the enzymes that created them.
A team of French researchers bombarded Deinococcus radiodurans to study the mechanism of double-strand break
DNA repair in that organism. At least two copies of the genome, with random DNA breaks, can form DNA
fragments through annealing. Partially overlapping fragments are then used for synthesis of homologous regions
through a moving D-loop that can continue extension until they find complementary partner strands. In the final step
there is crossover by means of RecA-dependent homologous recombination.
[]
Translesion synthesis
Translesion synthesis (TLS) is a DNA damage tolerance process that allows the DNA replication machinery to
replicate past DNA lesions such as thymine dimers or AP sites.
[]
It involves switching out regular DNA polymerases
for specialized translesion polymerases (i.e. DNA polymerase IV or V, from the Y Polymerase family), often with
larger active sites that can facilitate the insertion of bases opposite damaged nucleotides. The polymerase switching
is thought to be mediated by, among other factors, the post-translational modification of the replication processivity
factor PCNA. Translesion synthesis polymerases often have low fidelity (high propensity to insert wrong bases) on
undamaged templates relative to regular polymerases. However, many are extremely efficient at inserting correct
bases opposite specific types of damage. For example, Pol mediates error-free bypass of lesions induced by UV
irradiation, whereas Pol introduces mutations at these sites. Pol is known to add the first adenine across the T^T
DNA repair
341
photodimer using Watson-Crick base pairing and the second adenine will be added in its syn conformation using
Hoogsteen base pairing. From a cellular perspective, risking the introduction of point mutations during translesion
synthesis may be preferable to resorting to more drastic mechanisms of DNA repair, which may cause gross
chromosomal aberrations or cell death. In short, the process involves specialized polymerases either bypassing or
repairing lesions at locations of stalled DNA replication. For example Human DNA polymerase eta can bypass
complex DNA lesions like guanine-thymine intra-strand crosslink, G[8,5-Me]T, although can cause targeted and
semi-targeted mutations.
[]
Paromita Raychaudhury and Ashis Basu
[]
studied the toxicity and mutagenesis of the
same lesion in E.coli by replicating a G[8,5-Me]T-modified plasmid in Escherichia coli with specific DNA
polymerase knockouts. Viability was very low in a strain lacking pol II, pol IV, and pol V, the three SOS-inducible
DNA polymerases, indicating that translesion synthesis is conducted primarily by these specialized DNA
polymerases. A bypass platform is provided to these polymerases by Proliferating cell nuclear antigen (PCNA).
Under normal circumstances, PCNA bound to polymerases replicates the DNA. At a site of lesion, PCNA is
ubiquitinated, or modified, by the RAD6/RAD18 proteins to provide a platform for the specialized polymerases to
bypass the lesion and resume DNA replication.
[12][13]
After translesion synthesis, extension is required. This
extension can be carried out by a replicative polymerase if the TLS is error-free, as in the case of Pol , yet if TLS
results in a mismatch, a specialized polymerase is needed to extend it; Pol . Pol is unique in that it can extend
terminal mismatches, whereas more processive polymerases cannot. So when a lesion is encountered, the replication
fork will stall, PCNA will switch from a processive polymerase to a TLS polymerase such as Pol to fix the lesion,
then PCNA may switch to Pol to extend the mismatch, and last PCNA will switch to the processive polymerase to
continue replication.
Global response to DNA damage
Cells exposed to ionizing radiation, ultraviolet light or chemicals are prone to acquire multiple sites of bulky DNA
lesions and double-strand breaks. Moreover, DNA damaging agents can damage other biomolecules such as proteins,
carbohydrates, lipids, and RNA. The accumulation of damage, to be specific, double-strand breaks or adducts
stalling the replication forks, are among known stimulation signals for a global response to DNA damage.
[14]
The
global response to damage is an act directed toward the cells' own preservation and triggers multiple pathways of
macromolecular repair, lesion bypass, tolerance, or apoptosis. The common features of global response are induction
of multiple genes, cell cycle arrest, and inhibition of cell division.
DNA damage checkpoints
After DNA damage, cell cycle checkpoints are activated. Checkpoint activation pauses the cell cycle and gives the
cell time to repair the damage before continuing to divide. DNA damage checkpoints occur at the G1/S and G2/M
boundaries. An intra-S checkpoint also exists. Checkpoint activation is controlled by two master kinases, ATM and
ATR. ATM responds to DNA double-strand breaks and disruptions in chromatin structure,
[15]
whereas ATR
primarily responds to stalled replication forks. These kinases phosphorylate downstream targets in a signal
transduction cascade, eventually leading to cell cycle arrest. A class of checkpoint mediator proteins including
BRCA1, MDC1, and 53BP1 has also been identified.
[16]
These proteins seem to be required for transmitting the
checkpoint activation signal to downstream proteins.
An important downstream target of ATM and ATR is p53, as it is required for inducing apoptosis following DNA
damage.
[17]
The cyclin-dependent kinase inhibitor p21 is induced by both p53-dependent and p53-independent
mechanisms and can arrest the cell cycle at the G1/S and G2/M checkpoints by deactivating cyclin/cyclin-dependent
kinase complexes.
[18]
DNA repair
342
The prokaryotic SOS response
The SOS response is the changes in gene expression in Escherichia coli and other bacteria in response to extensive
DNA damage. The prokaryotic SOS system is regulated by two key proteins: LexA and RecA. The LexA
homodimer is a transcriptional repressor that binds to operator sequences commonly referred to as SOS boxes. In
Escherichia coli it is known that LexA regulates transcription of approximately 48 genes including the lexA and
recA genes.
[]
The SOS response is known to be widespread in the Bacteria domain, but it is mostly absent in some
bacterial phyla, like the Spirochetes.
[]
The most common cellular signals activating the SOS response are regions of
single-stranded DNA (ssDNA), arising from stalled replication forks or double-strand breaks, which are processed
by DNA helicase to separate the two DNA strands.
[14]
In the initiation step, RecA protein binds to ssDNA in an ATP
hydrolysis driven reaction creating RecAssDNA filaments. RecAssDNA filaments activate LexA autoprotease
activity, which ultimately leads to cleavage of LexA dimer and subsequent LexA degradation. The loss of LexA
repressor induces transcription of the SOS genes and allows for further signal induction, inhibition of cell division
and an increase in levels of proteins responsible for damage processing.
In Escherichia coli, SOS boxes are 20-nucleotide long sequences near promoters with palindromic structure and a
high degree of sequence conservation. In other classes and phyla, the sequence of SOS boxes varies considerably,
with different length and composition, but it is always highly conserved and one of the strongest short signals in the
genome.
[]
The high information content of SOS boxes permits differential binding of LexA to different promoters
and allows for timing of the SOS response. The lesion repair genes are induced at the beginning of SOS response.
The error-prone translesion polymerases, for example, UmuCD'2 (also called DNA polymerase V), are induced later
on as a last resort.
[]
Once the DNA damage is repaired or bypassed using polymerases or through recombination, the
amount of single-stranded DNA in cells is decreased, lowering the amounts of RecA filaments decreases cleavage
activity of LexA homodimer, which then binds to the SOS boxes near promoters and restores normal gene
expression.
Eukaryotic transcriptional responses to DNA damage
Eukaryotic cells exposed to DNA damaging agents also activate important defensive pathways by inducing multiple
proteins involved in DNA repair, cell cycle checkpoint control, protein trafficking and degradation. Such genome
wide transcriptional response is very complex and tightly regulated, thus allowing coordinated global response to
damage. Exposure of yeast Saccharomyces cerevisiae to DNA damaging agents results in overlapping but distinct
transcriptional profiles. Similarities to environmental shock response indicates that a general global stress response
pathway exist at the level of transcriptional activation. In contrast, different human cell types respond to damage
differently indicating an absence of a common global response. The probable explanation for this difference between
yeast and human cells may be in the heterogeneity of mammalian cells. In an animal different types of cells are
distributed among different organs that have evolved different sensitivities to DNA damage.
[19]
In general global response to DNA damage involves expression of multiple genes responsible for postreplication
repair, homologous recombination, nucleotide excision repair, DNA damage checkpoint, global transcriptional
activation, genes controlling mRNA decay, and many others. A large amount of damage to a cell leaves it with an
important decision: undergo apoptosis and die, or survive at the cost of living with a modified genome. An increase
in tolerance to damage can lead to an increased rate of survival that will allow a greater accumulation of mutations.
Yeast Rev1 and human polymerase are members of [Y family translesion DNA polymerases present during global
response to DNA damage and are responsible for enhanced mutagenesis during a global response to DNA damage in
eukaryotes.
[14]
DNA repair
343
DNA repair and aging
Pathological effects of poor DNA repair
DNA repair rate is an important determinant of
cell pathology
Experimental animals with genetic deficiencies in DNA repair often
show decreased life span and increased cancer incidence.
[]
For
example, mice deficient in the dominant NHEJ pathway and in
telomere maintenance mechanisms get lymphoma and infections more
often, and, as a consequence, have shorter lifespans than wild-type
mice.
[]
In similar manner, mice deficient in a key repair and
transcription protein that unwinds DNA helices have premature onset
of aging-related diseases and consequent shortening of lifespan.
[]
However, not every DNA repair deficiency creates exactly the
predicted effects; mice deficient in the NER pathway exhibited
shortened life span without correspondingly higher rates of mutation.
[]
If the rate of DNA damage exceeds the capacity of the cell to repair it, the accumulation of errors can overwhelm the
cell and result in early senescence, apoptosis, or cancer. Inherited diseases associated with faulty DNA repair
functioning result in premature aging,
[]
increased sensitivity to carcinogens, and correspondingly increased cancer
risk (see below). On the other hand, organisms with enhanced DNA repair systems, such as Deinococcus
radiodurans, the most radiation-resistant known organism, exhibit remarkable resistance to the double-strand
break-inducing effects of radioactivity, likely due to enhanced efficiency of DNA repair and especially NHEJ.
[]
Longevity and caloric restriction
Most life span influencing genes affect the rate of
DNA damage
A number of individual genes have been identified as influencing
variations in life span within a population of organisms. The effects of
these genes is strongly dependent on the environment, in particular, on
the organism's diet. Caloric restriction reproducibly results in extended
lifespan in a variety of organisms, likely via nutrient sensing pathways
and decreased metabolic rate. The molecular mechanisms by which
such restriction results in lengthened lifespan are as yet unclear (see
[]
for some discussion); however, the behavior of many genes known to
be involved in DNA repair is altered under conditions of caloric
restriction.
For example, increasing the gene dosage of the gene SIR-2, which
regulates DNA packaging in the nematode worm Caenorhabditis
elegans, can significantly extend lifespan.
[]
The mammalian homolog
of SIR-2 is known to induce downstream DNA repair factors involved
in NHEJ, an activity that is especially promoted under conditions of
caloric restriction.
[]
Caloric restriction has been closely linked to the
rate of base excision repair in the nuclear DNA of rodents,
[]
although
similar effects have not been observed in mitochondrial DNA.
[]
It is interesting to note that the C. elegans gene AGE-1, an upstream effector of DNA repair pathways, confers
dramatically extended life span under free-feeding conditions but leads to a decrease in reproductive fitness under
conditions of caloric restriction.
[]
This observation supports the pleiotropy theory of the biological origins of aging,
which suggests that genes conferring a large survival advantage early in life will be selected for even if they carry a
corresponding disadvantage late in life.
DNA repair
344
Medicine and DNA repair modulation
Hereditary DNA repair disorders
Defects in the NER mechanism are responsible for several genetic disorders, including:
Xeroderma pigmentosum: hypersensitivity to sunlight/UV, resulting in increased skin cancer incidence and
premature aging
Cockayne syndrome: hypersensitivity to UV and chemical agents
Trichothiodystrophy: sensitive skin, brittle hair and nails
Mental retardation often accompanies the latter two disorders, suggesting increased vulnerability of developmental
neurons.
Other DNA repair disorders include:
Werner's syndrome: premature aging and retarded growth
Bloom's syndrome: sunlight hypersensitivity, high incidence of malignancies (especially leukemias).
Ataxia telangiectasia: sensitivity to ionizing radiation and some chemical agents
All of the above diseases are often called "segmental progerias" ("accelerated aging diseases") because their victims
appear elderly and suffer from aging-related diseases at an abnormally young age, while not manifesting all the
symptoms of old age.
Other diseases associated with reduced DNA repair function include Fanconi's anemia, hereditary breast cancer and
hereditary colon cancer.
DNA repair and cancer
Because of inherent limitations in the DNA repair mechanisms, if humans lived long enough, they would all
eventually develop cancer.
[][20]
There are at least 34 Inherited human DNA repair gene mutations that increase
cancer risk. Many of these mutations cause DNA repair to be less effective than normal. In particular, Hereditary
nonpolyposis colorectal cancer (HNPCC) is strongly associated with specific mutations in the DNA mismatch repair
pathway. BRCA1 and BRCA2, two famous genes whose mutations confer a hugely increased risk of breast cancer on
carriers, are both associated with a large number of DNA repair pathways, especially NHEJ and homologous
recombination.
Cancer therapy procedures such as chemotherapy and radiotherapy work by overwhelming the capacity of the cell to
repair DNA damage, resulting in cell death. Cells that are most rapidly dividing most typically cancer cells are
preferentially affected. The side-effect is that other non-cancerous but rapidly dividing cells such as stem cells in the
bone marrow are also affected. Modern cancer treatments attempt to localize the DNA damage to cells and tissues
only associated with cancer, either by physical means (concentrating the therapeutic agent in the region of the tumor)
or by biochemical means (exploiting a feature unique to cancer cells in the body).
DNA repair and evolution
The basic processes of DNA repair are highly conserved among both prokaryotes and eukaryotes and even among
bacteriophage (viruses that infect bacteria); however, more complex organisms with more complex genomes have
correspondingly more complex repair mechanisms.
[]
The ability of a large number of protein structural motifs to
catalyze relevant chemical reactions has played a significant role in the elaboration of repair mechanisms during
evolution. For an extremely detailed review of hypotheses relating to the evolution of DNA repair, see.
[]
The fossil record indicates that single-cell life began to proliferate on the planet at some point during the
Precambrian period, although exactly when recognizably modern life first emerged is unclear. Nucleic acids became
the sole and universal means of encoding genetic information, requiring DNA repair mechanisms that in their basic
form have been inherited by all extant life forms from their common ancestor. The emergence of Earth's oxygen-rich
DNA repair
345
atmosphere (known as the "oxygen catastrophe") due to photosynthetic organisms, as well as the presence of
potentially damaging free radicals in the cell due to oxidative phosphorylation, necessitated the evolution of DNA
repair mechanisms that act specifically to counter the types of damage induced by oxidative stress.
Rate of evolutionary change
On some occasions, DNA damage is not repaired, or is repaired by an error-prone mechanism that results in a change
from the original sequence. When this occurs, mutations may propagate into the genomes of the cell's progeny.
Should such an event occur in a germ line cell that will eventually produce a gamete, the mutation has the potential
to be passed on to the organism's offspring. The rate of evolution in a particular species (or, in a particular gene) is a
function of the rate of mutation. As a consequence, the rate and accuracy of DNA repair mechanisms have an
influence over the process of evolutionary change.
[]
References
[1] [1] Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL, Darnell J. (2004). Molecular Biology of the Cell, p963. WH
Freeman: New York, NY. 5th ed.
[2] [2] Acharya, PVN; The Effect of Ionizing Radiation on the Formation of Age-Correlated Oligo Deoxyribo Nucleo Phospheryl Peptides in
Mammalian Cells; 10th International Congress of Gerontology, Jerusalem. Abstract No. 1; January 1975. Work done while employed by
Dept. of Pathology, University of Wisconsin, Madison.
[3] [3] Acharya, PVN; Implicatons of The Action of Low-Level Ionizing Radiation on the Inducement of Irreparable DNA Damage Leading to
Mammalian Aging and Chemical Carcinogenesis.; 10th International Congress of Biochemistry, Hamburg, Germany. Abstract No. 01-1-079;
July 1976. Work done while employed by Dept. of Pathology, University of Wisconsin, Madison.
[4] [4] Acharya, PV Narasimh; Irreparable DNA-Damage by Industrial Pollutants in Pre-mature Aging, Chemical Carcinogenesis and Cardiac
Hypertrophy: Experiments and Theory; 1st International Meeting of Heads of Clinical Biochemistry Laboratories, Jerusalem, Israel. April
1977. Work conducted at Industrial Safety Institute and Behavioral Cybernetics Laboratory, University of Wisconsin, Madison.
[6] DNA Lesions That Require Repair: http:/ / www. ncbi.nlm. nih. gov/ books/ bv. fcgi?highlight=lesion& rid=mcb. table. 3236
[7] [7] Watson JD, Baker TA, Bell SP, Gann A, Levine M, Losick R. (2004). Molecular Biology of the Gene, ch. 9 and 10. Peason Benjamin
Cummings; CSHL Press. 5th ed.
[14] [14] Friedberg EC, Walker GC, Siede W, Wood RD, Schultz RA, Ellenberger T. (2006). DNA Repair and Mutagenesis, part 3. ASM Press. 2nd
ed.
[19] Fry RC, Begley TJ, Samson LD. (2004). Genome-wide responses to DNA-damaging agents. Annu Rev Microbiol. 59 pp 35777
External links
Books about DNA repair:
Online books (http:/ / onlinebooks. library. upenn. edu/ webbin/ ftl?st=& su=DNA+ repair& library=OLBP),
Resources in your library (http:/ / onlinebooks. library. upenn. edu/ webbin/ ftl?st=& su=DNA+ repair),
Resources in other libraries (http:/ / onlinebooks. library. upenn. edu/ webbin/ ftl?st=& su=DNA+ repair&
library=0CHOOSE0)
Roswell Park Cancer Institute DNA Repair Lectures (http:/ / asajj. roswellpark. org/ huberman/ DNA_Repair/
DNA_Repair. htm)
A comprehensive list of Human DNA Repair Genes (http:/ / www. cgal. icnet. uk/ DNA_Repair_Genes. html)
3D structures of some DNA repair enzymes (http:/ / www. biochem. ucl. ac. uk/ bsm/ xtal/ teach/ repair/ tibs3.
html)
Human DNA repair diseases (http:/ / www. scielo. br/ scielo. php?pid=S0100-84551997000400032&
script=sci_arttext& tlng=en)
DNA repair special interest group (http:/ / tango01. cit. nih. gov/ sig/ home. taf?_function=main&
SIGInfo_SIGID=32)
DNA Repair (http:/ / users. rcn. com/ jkimball. ma. ultranet/ BiologyPages/ D/ DNArepair. html)
DNA Damage and DNA Repair (http:/ / www. benbest. com/ lifeext/ aging. html#dna)
Segmental Progeria (http:/ / www. benbest. com/ lifeext/ aging. html#progeria)
DNA repair
346
DNA-damage repair; the good, the bad, and the ugly (https:/ / www. researchgate. net/ publication/
5565866_DNA-damage_repair_the_good_the_bad_and_the_ugly)
Oncogenes
Oncogene
An oncogene is a gene that has the potential to cause cancer.
[1]
In
tumor cells, they are often mutated or expressed at high levels.
[2]
Most normal cells undergo a programmed form of death (apoptosis).
Activated oncogenes can cause those cells that ought to die to survive
and proliferate instead.
[3]
Most oncogenes require an additional step,
such as mutations in another gene, or environmental factors, such as
viral infection, to cause cancer. Since the 1970s, dozens of oncogenes
have been identified in human cancer. Many cancer drugs target the
proteins encoded by oncogenes.
[2][4][5][6]
History
The term "oncogene" was coined in 1969 by National Cancer Institute
scientists, Robert Huebner and George Todaro
[7]
The first confirmed oncogene was discovered in 1970 and was termed
src (pronounced sarc as in sarcoma). Src was in fact first discovered as an oncogene in a chicken retrovirus.
Experiments performed by Dr G. Steve Martin of the University of California, Berkeley demonstrated that the Src
was indeed the oncogene of the virus. The first nucleotide sequence of v-src was sequenced 1980 by A.P.
Czernilofsky et al.
[8]
In 1976 Drs. Dominique Stehelin, J. Michael Bishop and Harold E. Varmus of the University of California, San
Francisco demonstrated that oncogenes were activated proto-oncogenes, found in many organisms including
humans. For this discovery Bishop and Varmus were awarded the Nobel Prize in Physiology or Medicine in 1989.
[9]
Proto-oncogene
A proto-oncogene is a normal gene that can become an oncogene due to mutations or increased expression. The
resultant protein may be termed an oncoprotein.
[10]
Proto-oncogenes code for proteins that help to regulate cell
growth and differentiation. Proto-oncogenes are often involved in signal transduction and execution of mitogenic
signals, usually through their protein products. Upon activation, a proto-oncogene (or its product) becomes a
tumor-inducing agent, an oncogene.
[11]
Examples of proto-oncogenes include RAS, WNT, MYC, ERK, and TRK.
The MYC gene is implicated in Burkitt's Lymphoma, which starts when a chromosomal translocation moves an
enhancer sequence within the vicinity of the MYC gene. The MYC gene codes for widely used transcription factors.
When the enhancer sequence is wrongly placed, these transcription factors are produced at much higher rates.
Another example of an oncogene is the Bcr-Abl gene found on the Philadelphia Chromosome, a piece of genetic
material seen in Chronic Myelogenous Leukemia caused by the translocation of pieces from chromosomes 9 and 22.
Bcr-Abl codes for a receptor tyrosine kinase, which is constitutively active, leading to uncontrolled cell proliferation.
(More information about the Philadelphia Chromosome below)
Oncogenes
347
Activation
From proto-oncogene to oncogene
The proto-oncogene can become an oncogene by a relatively small
modification of its original function. There are three basic methods of
activation:
1. A mutation within a proto-oncogene, or within a regulatory region
(for example the promoter region), can cause a change in the protein
structure, causing
an increase in protein (enzyme) activity
a loss of regulation
2. 2. An increase in the amount of a certain protein (protein concentration), caused by
an increase of protein expression (through misregulation)
an increase of protein (mRNA) stability, prolonging its existence and thus its activity in the cell
gene duplication (one type of chromosome abnormality), resulting in an increased amount of protein in the cell
3. A chromosomal translocation (another type of chromosome abnormality)
There are 2 different types of chromosomal translocations that can occur:
1. 1. translocation events which relocate a proto-oncogene to a new chromosomal site that leads to higher
expression
2. 2. translocation events that lead to a fusion between a proto-oncogene and a 2nd gene (this creates a fusion
protein with increased cancerous/oncogenic activity)
the expression of a constitutively active hybrid protein. This type of mutation in a dividing stem cell in the
bone marrow leads to adult leukemia
Philadelphia Chromosome is an example of this type of translocation event. This chromosome was
discovered in 1960 by Peter Nowell and David Hungerford, and it is a fusion of parts of DNA from
chromosome 22 and chromosome 9. The broken end of chromosome 22 contains the "BCR" gene, which
fuses with a fragment of chromosome 9 that contains the "ABL1" gene. When these two chromosome
fragments fuse the genes also fuse creating a new gene: "BCR-ABL". This fused gene encodes for a protein
that displays high protein tyrosine kinase activity (this activity is due to the "ABL1" half of the protein).
The unregulated expression of this protein activates other proteins that are involved in cell cycle and cell
division which can cause a cell to grow and divide uncontrollably (the cell becomes cancerous). As a result,
the Philadelphia Chromosome is associated with Chronic Myelogenous Leukemia (as mentioned before) as
well as other forms of Leukemia.
[12]
The expression of oncogenes can be regulated by microRNAs (miRNAs), small RNAs 21-25 nucleotides in length
that control gene expression by downregulating them.
[]
Mutations in such microRNAs (known as oncomirs) can lead
to activation of oncogenes.
[]
Antisense messenger RNAs could theoretically be used to block the effects of
oncogenes.
Oncogenes
348
Classification
There are several systems for classifying oncogenes,
[13][14]
but there is not yet a widely accepted standard. They are
sometimes grouped both spatially (moving from outside the cell inwards) and chronologically (parallelling the
"normal" process of signal transduction). There are several categories that are commonly used:
Category Examples Cancers Gene functions
Growth factors, or
mitogens
c-Sis
glioblastomas, fibrosarcomas,
osteosarcomas, breast carcinomas, and
melanomas
[15]
induces cell proliferation.
Receptor tyrosine
kinases
epidermal growth factor receptor
(EGFR), platelet-derived growth factor
receptor (PDGFR), and vascular
endothelial growth factor receptor
(VEGFR), HER2/neu
Breast cancer, gastrointestinal stromal
tumours, non-small-cell lung cancer and
pancreatic cancer
[16]
transduce signals for cell growth
and differentiation.
Cytoplasmic tyrosine
kinases
Src-family, Syk-ZAP-70 family, and
BTK family of tyrosine kinases, the Abl
gene in CML - Philadelphia chromosome
colorectal and breast cancers, melanomas,
ovarian cancers, gastric cancers, head and
neck cancers, pancreatice cancer, lung
cancer, brain cancers, and blood
cancers
[17]
mediate the responses to, and the
activation receptors of cell
proliferation, migration,
differentiation, and survival
[18]
Cytoplasmic
Serine/threonine kinases
and their regulatory
subunits
Raf kinase, and cyclin-dependent kinases
(through overexpression).
malignant melanoma, papillary thyroid
cancer, colorectal cancer, and ovarian
cancer
[19]
Involved in organism
development, cell cycle
regulation, cell proliferation,
differentiation, cells survival,
and apoptosis
[20]
Regulatory GTPases Ras protein
adenocarcinomas of the pancreas and
colon, thyroid tumors, and myeloid
leukemia
[21]
involved in signalling a major
pathway leading to cell
proliferation.
[22]
Transcription factors myc gene
malignant T-cell lymphomas and acute
myleoid leukemias, breast cancer,
pancreatic cancer, retinoblastoma, and
small cell lung cancer
[23]
-They regulate transcription of
genes that induce cell
proliferation.
More detailed information for the above Table:
Growth factors are usually secreted by specialized cells to induce cell proliferation in themselves, nearby
cells, or distant cells. An oncogene may cause a cell to secrete growth factors even though it does not
normally do so. It will thereby induce its own uncontrolled proliferation (autocrine loop), and proliferation
of neighboring cells. It may also cause production of growth hormones in other parts of the body.
Receptor Tyrosine Kinases add phosphate groups to other proteins to turn them on or off. Receptor kinases
add phosphate groups to receptor proteins at the surface of the cell (which receive protein signals from
outside the cell and transmit them to the inside of the cell). Tyrosine kinases add phosphate groups to the
amino acid tyrosine in the target protein. They can cause cancer by turning the receptor permanently on
(constitutively), even without signals from outside the cell.
Ras is a small GTPase that hydrolyses GTP into GDP and phosphate. Ras is activated by growth factor
signaling (i.e., EGF, TGFbeta) and acting like a binary switch (on/off) in growth signaling pathways.
Downstream effectors of Ras include Raf, MEK, MEKK, MAPK, ERK, most of which in turn regulate
genes that mediate cell proliferation.
Oncogenes
349
References
[1] [1] Wilbur, Beth, editor. The World of the Cell, Becker, W.M., et al., 7th ed. San Francisco, CA; 2009.
[2] Kimball's Biology Pages. (http:/ / users.rcn. com/ jkimball. ma. ultranet/ BiologyPages/ O/ Oncogenes. html) "Oncogenes" Free full text
[3] The Nobel Prize in Physiology or Medicine 2002. (http:/ / nobelprize. org/ nobel_prizes/ medicine/ laureates/ 2002/ illpres/ implications.
html) Illustrated presentation.
[6] The Nobel Prize in Physiology or Medicine 1989 (http:/ / nobelprize. org/ nobel_prizes/ medicine/ laureates/ 1989/ press. html) to J. Michael
Bishop and Harold E. Varmus for their discovery of "the cellular origin of retroviral oncogenes".
[7] The Emperor of All Maladies, Siddhartha Mukherjee, 2011, p. 363, endnote.
[8] [8] (A.P. Czernilofsky et al., 1980, Nature Vol 287, pp 198-203).
[9] Nobel Prize in Physiology or Medicine for 1989 jointly to J. Michael Bishop and Harold E. Varmus for their discovery of "the cellular origin
of retroviral oncogenes". (http:/ / nobelprize. org/ nobel_prizes/ medicine/ laureates/ 1989/ press. html) Press Release.
[10] [10] Chapter 20 - NEOPLASMS OF THE THYROID - in: 8th edition.
[13] THE Medical Biochemistry Page (http:/ / web.indstate. edu/ thcme/ mwking/ oncogene. html#classes)
[14] Classification of Oncogene Function (http:/ / www.qub. ac. uk/ cm/ pat/ education/ Carcinogenesis/ tsld014. htm)
External links
Drosophila Oncogenes and Tumor Suppressors - The Interactive Fly (http:/ / www. sdbonline. org/ fly/ aignfam/
tumorsup. htm)
350
RNA synthesis and processing
Transcription
Simplified diagram of mRNA synthesis and processing. Enzymes not shown.
Transcription is the first step of gene
expression, in which a particular
segment of DNA is copied into RNA
by the enzyme, RNA polymerase. Both
RNA and DNA are nucleic acids,
which use base pairs of nucleotides as
a complementary language that can be
converted back and forth from DNA to
RNA by the action of the correct
enzymes. During transcription, a DNA
sequence is read by an RNA
polymerase, which produces a
complementary, antiparallel RNA
strand. As opposed to DNA
replication, transcription results in an
RNA complement that includes uracil
(U) in all instances where thymine (T)
would have occurred in a DNA
complement. Also unlike DNA
replication where DNA is synthesised,
transcription does not involve an RNA
primer to initiate RNA synthesis.
Transcription proceeds in 5 or 6 steps, each moving like a wave along the DNA.
1. One or more sigma factors initiate transcription of a gene by enabling binding of RNA polymerase to promoter
DNA.
2. Helicase enzymes move a transcription bubble, like the slider of a zipper, which splits the double helix DNA
molecule into two strands of unpaired DNA nucleotides, by breaking the hydrogen bonds between
complementary DNA nucleotides.
3. 3. RNA polymerase adds matching RNA nucleotides that are paired with complementary DNA nucleotides of one
DNA strand.
4. 4. RNA sugar-phosphate backbone forms with assistance from RNA polymerase to form an RNA strand.
5. 5. Hydrogen bonds of the untwisted RNA + DNA helix break, freeing the newly synthesized RNA strand.
6. If the cell has a nucleus, the RNA is further processed (addition of a 3'UTR poly-A tail and a 5'UTR cap) and
exits to the cytoplasm through the nuclear pore complex.
Transcription is the first step leading to gene expression. The stretch of DNA transcribed into an RNA molecule is
called a transcription unit and encodes at least one gene. If the gene transcribed encodes a protein, the result of
transcription is messenger RNA (mRNA), which will then be used to create that protein via the process of
translation. Alternatively, the transcribed gene may encode for either non-coding RNA genes (such as microRNA,
lincRNA, etc.) or ribosomal RNA (rRNA) or transfer RNA (tRNA), other components of the protein-assembly
Transcription
351
process, or other ribozymes.
[1]
A DNA transcription unit encoding for a protein contains not only the sequence that will eventually be directly
translated into the protein (the coding sequence) but also regulatory sequences that direct and regulate the synthesis
of that protein. The regulatory sequence before (i.e., upstream from) the coding sequence is called the five prime
untranslated region (5'UTR), and the sequence following (downstream from) the coding sequence is called the three
prime untranslated region (3'UTR).
[1]
Transcription has some proofreading mechanisms, but they are fewer and less effective than the controls for copying
DNA; therefore, transcription has a lower copying fidelity than DNA replication.
[]
As in DNA replication, DNA is read from 3'UTR 5'UTR during transcription. Meanwhile, the complementary
RNA is created from the 5'UTR 3'UTR direction. This means its 5' end is created first in base pairing. Although
DNA is arranged as two antiparallel strands in a double helix, only one of the two DNA strands, called the template
strand, is used for transcription. This is because RNA is only single-stranded, as opposed to double-stranded DNA.
The other DNA strand is called the coding (lagging) strand, because its sequence is the same as the newly created
RNA transcript (except for the substitution of uracil for thymine). The use of only the 3'UTR 5'UTR strand
eliminates the need for the Okazaki fragments seen in DNA replication.
[1]
Transcription is divided into five stages: pre-initiation, initiation, promoter clearance, elongation and termination.
[1]
Major steps
Pre-initiation
In eukaryotes, RNA polymerase, and therefore the initiation of transcription, requires the presence of a core
promoter sequence in the DNA. Promoters are regions of DNA that promote transcription and, in eukaryotes, are
found at -30, -75, and -90 base pairs upstream from the transcription start site (abbreviated to TSS). Core promoters
are sequences within the promoter that are essential for transcription initiation. RNA polymerase is able to bind to
core promoters in the presence of various specific transcription factors.
[citation needed]
The most characterized type of core promoter in eukaryotes is a short DNA sequence known as a TATA box, found
25-30 base pairs upstream from the TSS.
[citation needed]
The TATA box, as a core promoter, is the binding site for a
transcription factor known as TATA-binding protein (TBP), which is itself a subunit of another transcription factor,
called Transcription Factor II D (TFIID). After TFIID binds to the TATA box via the TBP, five more transcription
factors and RNA polymerase combine around the TATA box in a series of stages to form a preinitiation complex.
One transcription factor, Transcription factor II H, has two components with helicase activity and so is involved in
the separating of opposing strands of double-stranded DNA to form the initial transcription bubble. However, only a
low, or basal, rate of transcription is driven by the preinitiation complex alone. Other proteins known as activators
and repressors, along with any associated coactivators or corepressors, are responsible for modulating transcription
rate.
[citation needed]
Thus, preinitiation complex contains:
[citation needed]
1. 1. Core Promoter Sequence
2. 2. Transcription Factors
3. 3. RNA Polymerase
4. 4. Activators and Repressors.
The transcription preinitiation in archaea is, in essence, homologous to that of eukaryotes, but is much less
complex.
[2]
The archaeal preinitiation complex assembles at a TATA-box binding site; however, in archaea, this
complex is composed of only RNA polymerase II, TBP, and TFB (the archaeal homologue of eukaryotic
transcription factor II B (TFIIB)).
[3][4]
Transcription
352
Initiation
Simple diagram of transcription initiation. RNAP = RNA polymerase
In bacteria, transcription begins with
the binding of RNA polymerase to the
promoter in DNA. RNA polymerase is
a core enzyme consisting of five
subunits: 2 subunits, 1 subunit, 1 '
subunit, and 1 subunit. At the start of
initiation, the core enzyme is
associated with a sigma factor that aids
in finding the appropriate -35 and -10
base pairs downstream of promoter sequences.
[5]
When the sigma factor and RNA polymerase combine, they form a
holoenzyme.
Transcription initiation is more complex in eukaryotes. Eukaryotic RNA polymerase does not directly recognize the
core promoter sequences. Instead, a collection of proteins called transcription factors mediate the binding of RNA
polymerase and the initiation of transcription. Only after certain transcription factors are attached to the promoter
does the RNA polymerase bind to it. The completed assembly of transcription factors and RNA polymerase bind to
the promoter, forming a transcription initiation complex. Transcription in the archaea domain is similar to
transcription in eukaryotes.
[6]
Promoter clearance
After the first bond is synthesized, the RNA polymerase must clear the promoter. During this time there is a
tendency to release the RNA transcript and produce truncated transcripts. This is called abortive initiation and is
common for both eukaryotes and prokaryotes.
[7]
Abortive initiation continues to occur until the factor rearranges,
resulting in the transcription elongation complex (which gives a 35 bp moving footprint). The factor is released
before 80 nucleotides of mRNA are synthesized.
[8]
Once the transcript reaches approximately 23 nucleotides, it no
longer slips and elongation can occur. This, like most of the remainder of transcription, is an energy-dependent
process, consuming adenosine triphosphate (ATP).
[citation needed]
Promoter clearance coincides with phosphorylation of serine 5 on the carboxy terminal domain of RNAP II in
eukaryotes, which is phosphorylated by TFIIH.
[citation needed]
Elongation
Simple diagram of transcription elongation
One strand of the DNA, the template
strand (or noncoding strand), is used as
a template for RNA synthesis. As
transcription proceeds, RNA
polymerase traverses the template
strand and uses base pairing
complementarity with the DNA template to create an RNA copy. Although RNA polymerase traverses the template
strand from 3' 5', the coding (non-template) strand and newly formed RNA can also be used as reference points,
so transcription can be described as occurring 5' 3'. This produces an RNA molecule from 5' 3', an exact copy
of the coding strand (except that thymines are replaced with uracils, and the nucleotides are composed of a ribose
(5-carbon) sugar where DNA has deoxyribose (one less oxygen atom) in its sugar-phosphate backbone).
[citation
needed]
Unlike DNA replication, mRNA transcription can involve multiple RNA polymerases on a single DNA template and
multiple rounds of transcription (amplification of particular mRNA), so many mRNA molecules can be rapidly
Transcription
353
produced from a single copy of a gene.
[citation needed]
Elongation also involves a proofreading mechanism that can replace incorrectly incorporated bases. In eukaryotes,
this may correspond with short pauses during transcription that allow appropriate RNA editing factors to bind. These
pauses may be intrinsic to the RNA polymerase or due to chromatin structure.
[citation needed]
Termination
Bacteria use two different strategies for transcription termination.1.Rho-independent transcription 2.Rho-dependent
transcription. In Rho-independent transcription termination,also called intrinsic termination, RNA transcription stops
when the newly synthesized RNA molecule forms a G-C-rich hairpin loop followed by a run of Us. When the
hairpin forms, the mechanical stress breaks the weak rU-dA bonds, now filling the DNA-RNA hybrid. This pulls the
poly-U transcript out of the active site of the RNA polymerase, in effect, terminating transcription. In the
"Rho-dependent" type of termination, a protein factor called "Rho" destabilizes the interaction between the template
and the mRNA, thus releasing the newly synthesized mRNA from the elongation complex.
[9]
Transcription termination in eukaryotes is less understood but involves cleavage of the new transcript followed by
template-independent addition of As at its new 3' end, in a process called polyadenylation.
[10]
Measuring and detecting transcription
Electron micrograph of transcription of ribosomal
RNA. The forming ribosomal RNA strands are
visible as branches from the main DNA
strand.
[citation needed]
Transcription can be measured and detected in a variety of ways:
[citation
needed]
Nuclear Run-on assay: measures the relative abundance of newly
formed transcripts
RNase protection assay and ChIP-Chip of RNAP: detect active
transcription sites
RT-PCR: measures the absolute abundance of total or nuclear RNA
levels, which may however differ from transcription rates
DNA microarrays: measures the relative abundance of the global
total or nuclear RNA levels; however, these may differ from
transcription rates
In situ hybridization: detects the presence of a transcript
MS2 tagging: by incorporating RNA stem loops, such as MS2, into
a gene, these become incorporated into newly synthesized RNA.
The stem loops can then be detected using a fusion of GFP and the
MS2 coat protein, which has a high affinity, sequence-specific
interaction with the MS2 stem loops. The recruitment of GFP to the
site of transcription is visualised as a single fluorescent spot. This new approach has revealed that transcription
occurs in discontinuous bursts, or pulses (see Transcriptional bursting). With the notable exception of in situ
techniques, most other methods provide cell population averages, and are not capable of detecting this
fundamental property of genes.
[11]
Northern blot: the traditional method, and until the advent of RNA-Seq, the most quantitative
RNA-Seq: applies next-generation sequencing techniques to sequence whole transcriptomes, which allows the
measurement of relative abundance of RNA, as well as the detection of additional variations such as fusion genes,
post-translational edits and novel splice sites
Transcription
354
Transcription factories
Active transcription units are clustered in the nucleus, in discrete sites called transcription factories or euchromatin.
Such sites can be visualized by allowing engaged polymerases to extend their transcripts in tagged precursors
(Br-UTP or Br-U) and immuno-labeling the tagged nascent RNA. Transcription factories can also be localized using
fluorescence in situ hybridization or marked by antibodies directed against polymerases. There are ~10,000 factories
in the nucleoplasm of a HeLa cell, among which are ~8,000 polymerase II factories and ~2,000 polymerase III
factories. Each polymerase II factory contains ~8 polymerases. As most active transcription units are associated with
only one polymerase, each factory usually contains ~8 different transcription units. These units might be associated
through promoters and/or enhancers, with loops forming a cloud around the factor.
[12]
History
A molecule that allows the genetic material to be realized as a protein was first hypothesized by Franois Jacob and
Jacques Monod. Severo Ochoa won a Nobel Prize in Physiology or Medicine in 1959 for developing a process for
synthesizing RNA in vitro with polynucleotide phosphorylase, which was useful for cracking the genetic code. RNA
synthesis by RNA polymerase was established in vitro by several laboratories by 1965; however, the RNA
synthesized by these enzymes had properties that suggested the existence of an additional factor needed to terminate
transcription correctly.
[citation needed]
In 1972, Walter Fiers became the first person to actually prove the existence of the terminating enzyme.
Roger D. Kornberg won the 2006 Nobel Prize in Chemistry "for his studies of the molecular basis of eukaryotic
transcription".
[13]
Reverse transcription
Scheme of reverse transcription
Some viruses (such as HIV, the cause of
AIDS), have the ability to transcribe RNA
into DNA. HIV has an RNA genome that is
duplicated into DNA. The resulting DNA
can be merged with the DNA genome of the
host cell. The main enzyme responsible for
synthesis of DNA from an RNA template is
called reverse transcriptase. In the case of
HIV, reverse transcriptase is responsible for
synthesizing a complementary DNA strand
(cDNA) to the viral RNA genome. An
associated enzyme, ribonuclease H, digests
the RNA strand, and reverse transcriptase
synthesises a complementary strand of DNA
to form a double helix DNA structure. This
cDNA is integrated into the host cell's
genome via another enzyme (integrase) causing the host cell to generate viral proteins that reassemble into new viral
particles. In HIV, subsequent to this, the host cell undergoes programmed cell death, apoptosis of T cells.
[14]
However, in other retroviruses, the host cell remains intact as the virus buds out of the cell.
Some eukaryotic cells contain an enzyme with reverse transcription activity called telomerase. Telomerase is a
reverse transcriptase that lengthens the ends of linear chromosomes. Telomerase carries an RNA template from
which it synthesizes DNA repeating sequence, or "junk" DNA. This repeated sequence of DNA is important
Transcription
355
because, every time a linear chromosome is duplicated, it is shortened in length. With "junk" DNA at the ends of
chromosomes, the shortening eliminates some of the non-essential, repeated sequence rather than the
protein-encoding DNA sequence farther away from the chromosome end. Telomerase is often activated in cancer
cells to enable cancer cells to duplicate their genomes indefinitely without losing important protein-coding DNA
sequence. Activation of telomerase could be part of the process that allows cancer cells to become immortal. The
immortalizing factor of cancer via telomere lengthening due to telomerase has been proven to occur in 90% of all
carcinogenic tumors in vivo with the remaining 10% using an alternative telomere maintenance route called ALT or
Alternative Lengthening of Telomeres.
[15]
Inhibitors
Transcription inhibitors can be used as antibiotics against, for example, pathogenic bacteria (antibacterials) and fungi
(antifungals). An example of such an antibacterial is rifampicin, which inhibits prokaryotic DNA transcription into
mRNA by inhibiting DNA-dependent RNA polymerase by binding its beta-subunit. 8-Hydroxyquinoline is an
antifungal transcription inhibitor.
[16]
The effects of histone methylation may also work to inhibit the action of
transcription.
References
[1] Eldra P. Solomon, Linda R. Berg, Diana W. Martin. Biology, 8th Edition, International Student Edition. Thomson Brooks/Cole. ISBN
978-0-495-30978-8
[9] Richardson J. Rho-dependent termination and ATPases in transcript termination. Biochimica et Biophysica Acta (BBA) - Gene Structure and
Expression. 2002;1577(2):251-260. Available at: http:/ / dx. doi. org/ 10. 1016/ S0167-4781(02)00456-6 [Accessed March 5, 2011].
[10] Lykke-Andersen S, Jensen TH. Overlapping pathways dictate termination of RNA polymerase II transcription. Biochimie.
2007;89(10):1177-82. Available at: http:/ / dx.doi.org/ 10. 1016/ j. biochi. 2007. 05. 007 [Accessed August 5, 2010].
[11] [11] Raj, A. and van Oudenaarden, A. (2008). Nature, nurture, or chance: stochastic gene expression and its consequences. Cell 135, 216-26.
[15] ALT and Telomerase (http:/ / www. nature.com/ nrg/ journal/ v11/ n5/ abs/ nrg2763. html) from Nature. Retrieved May 2010
[16] 8-Hydroxyquinoline info (http:/ / www.sigmaaldrich.com/ catalog/ ProductDetail. do?D7=0&
N5=SEARCH_CONCAT_PNO|BRAND_KEY& N4=H6878|SIAL& N25=0& QS=ON& F=SPEC) from SIGMA-ALDRICH. Retrieved Feb
2012
External links
Interactive Java simulation of transcription initiation. (http:/ / cmol. nbi. dk/ models/ dynamtrans/ dynamtrans.
html) From Center for Models of Life (http:/ / cmol. nbi. dk/ ) at the Niels Bohr Institute.
Interactive Java simulation of transcription interference--a game of promoter dominance in bacterial virus. (http:/
/ cmol. nbi. dk/ models/ dna/ rnap. html) From Center for Models of Life (http:/ / cmol. nbi. dk/ ) at the Niels
Bohr Institute.
Biology animations about this topic under Chapter 15 and Chapter 18 (http:/ / highered. mcgraw-hill. com/ sites/
dl/ free/ 0072437316/ 120060/ ravenanimation. html)
Virtual Cell Animation Collection, Introducing Transcription (http:/ / vcell. ndsu. nodak. edu/ animations/
transcription/ index. htm)
Easy to use DNA transcription site (http:/ / dnatorna. com)
Regulation of gene expression
356
Regulation of gene expression
Gene modulation redirects here. For information on therapeutic regulation of gene expression, see therapeutic
gene modulation.
For vocabulary, see Glossary of gene expression terms
Diagram showing at which stages in the
DNA-mRNA-protein pathway expression can be
controlled
Regulation of gene expression includes a wide range of mechanisms
that are used by cells to increase or decrease the production of specific
gene products (protein or RNA), and is informally termed gene
regulation. Sophisticated programs of gene expression are widely used
in biology, for example to trigger developmental pathways, respond to
environmental stimuli, or adapt to new food sources. Virtually any step
of gene expression can be modulated, from transcriptional initiation, to
RNA processing, and to the post-translational modification of a
protein.
Gene regulation is essential for viruses, prokaryotes and eukaryotes as
it increases the versatility and adaptability of an organism by allowing
the cell to express protein when needed. Although as early as 1951
Barbara McClintock showed interaction between two genetic loci, Activator (Ac) and Dissociator (Ds), in the color
formation of maize seeds, the first discovery of a gene regulation system is widely considered to be the identification
in 1961 of the lac operon, discovered by Jacques Monod, in which some enzymes involved in lactose metabolism are
expressed by the genome of E. coli only in the presence of lactose and absence of glucose.
Furthermore, in multicellular organisms, gene regulation drives the processes of cellular differentiation and
morphogenesis, leading to the creation of different cell types that possess different gene expression profiles, and
hence produce different proteins/have different ultrastructures that suit them to their functions (though they all
possess the genotype, which follows the same genome sequence).
Regulated stages of gene expression
Any step of gene expression may be modulated, from the DNA-RNA transcription step to post-translational
modification of a protein. The following is a list of stages where gene expression is regulated, the most extensively
utilised point is Transcription Initiation:
Chromatin domains
Transcription
Post-transcriptional modification
RNA transport
Translation
mRNA degradation
Regulation of gene expression
357
Modification of DNA
In eukaryotes, the accessibility of large regions of DNA can depend on its chromatin structure, which can be altered
as a result of histone modifications directed by DNA methylation, ncRNA, or DNA-binding protein. Hence these
modifications may up or down regulate the expression of gene. Certain of these modifications that regulate gene
expression are inheritable and are referred to as epigenetic regulation.
Structural
Transcription of DNA is dictated by its structure. In general, the density of its packing is indicative of the frequency
of transcription. Octameric protein complexes called nucleosomes are responsible for the amount of supercoiling of
DNA, and these complexes can be temporarily modified by processes such as phosphorylation or more permanently
modified by processes such as methylation. Such modifications are considered to be responsible for more or less
permanent changes in gene expression levels.
[]
Chemical
Methylation of DNA is a common method of gene silencing. DNA is typically methylated by methyltransferase
enzymes on cytosine nucleotides in a CpG dinucleotide sequence (also called "CpG islands" when densely
clustered). Analysis of the pattern of methylation in a given region of DNA (which can be a promoter) can be
achieved through a method called bisulfite mapping. Methylated cytosine residues are unchanged by the treatment,
whereas unmethylated ones are changed to uracil. The differences are analyzed by DNA sequencing or by methods
developed to quantify SNPs, such as Pyrosequencing (Biotage) or MassArray (Sequenom), measuring the relative
amounts of C/T at the CG dinucleotide. Abnormal methylation patterns are thought to be involved in oncogenesis.
[]
Histone acetylation is also an important process in transcription. Histone acetyltransferase enzymes (HATs) such as
CREB-binding protein also dissociate the DNA from the histone complex, allowing transcription to proceed. Often,
DNA methylation and histone deacetylation work together in gene silencing. The combination of the two seems to
be a signal for DNA to be packed more densely, lowering gene expression.
[citation needed]
Regulation of gene expression
358
Regulation of transcription
1: RNA Polymerase, 2: Repressor, 3: Promoter, 4: Operator, 5:
Lactose, 6: lacZ, 7: lacY, 8: lacA. Top: The gene is essentially
turned off. There is no lactose to inhibit the repressor, so the
repressor binds to the operator, which obstructs the RNA
polymerase from binding to the promoter and making lactase.
Bottom: The gene is turned on. Lactose is inhibiting the repressor,
allowing the RNA polymerase to bind with the promoter, and express
the genes, which synthesize lactase. Eventually, the lactase will
digest all of the lactose, until there is none to bind to the repressor.
The repressor will then bind to the operator, stopping the
manufacture of lactase.
Regulation of transcription thus controls when
transcription occurs and how much RNA is created.
Transcription of a gene by RNA polymerase can be
regulated by at least five mechanisms:
Specificity factors alter the specificity of RNA
polymerase for a given promoter or set of promoters,
making it more or less likely to bind to them (i.e.,
sigma factors used in prokaryotic transcription).
Repressors bind to the Operator, coding sequences
on the DNA strand that are close to or overlapping
the promoter region, impeding RNA polymerase's
progress along the strand, thus impeding the
expression of the gene.The image to the right
demonstrates regulation by a repressor in the lac
operon.
General transcription factors position RNA
polymerase at the start of a protein-coding sequence
and then release the polymerase to transcribe the
mRNA.
Activators enhance the interaction between RNA
polymerase and a particular promoter, encouraging
the expression of the gene. Activators do this by increasing the attraction of RNA polymerase for the promoter,
through interactions with subunits of the RNA polymerase or indirectly by changing the structure of the DNA.
Enhancers are sites on the DNA helix that are bound to by activators in order to loop the DNA bringing a
specific promoter to the initiation complex. Enhancers are much more common in eukaryote than prokaryotes,
where only a few examples exist (to date).
[]
Silencers are regions of DNA sequences that, when bound by particular transcription factors, can silence
expression of the gene.
Post-transcriptional regulation
After the DNA is transcribed and mRNA is formed, there must be some sort of regulation on how much the mRNA
is translated into proteins. Cells do this by modulating the capping, splicing, addition of a Poly(A) Tail, the
sequence-specific nuclear export rates, and, in several contexts, sequestration of the RNA transcript. These processes
occur in eukaryotes but not in prokaryotes. This modulation is a result of a protein or transcript that, in turn, is
regulated and may have an affinity for certain sequences.
Regulation of translation
The translation of mRNA can also be controlled by a number of mechanisms, mostly at the level of initiation.
Recruitment of the small ribosomal subunit can indeed be modulated by mRNA secondary structure, antisense RNA
binding, or protein binding. In both prokaryotes and eukaryotes, a large number of RNA binding proteins exist,
which often are directed to their target sequence by the secondary structure of the transcript, which may change
depending on certain conditions, such as temperature or presence of a ligand (aptamer). Some transcripts act as
ribozymes and self-regulate their expression.
Regulation of gene expression
359
Examples of gene regulation
Enzyme induction is a process in which a molecule (e.g., a drug) induces (i.e., initiates or enhances) the
expression of an enzyme.
The induction of heat shock proteins in the fruit fly Drosophila melanogaster.
The Lac operon is an interesting example of how gene expression can be regulated.
Viruses, despite having only a few genes, possess mechanisms to regulate their gene expression, typically into an
early and late phase, using collinear systems regulated by anti-terminators (lambda phage) or splicing modulators
(HIV).
Developmental biology
A large number of studied regulatory systems come from developmental biology. Examples include:
The colinearity of the Hox gene cluster with their nested antero-posterior patterning
It has been speculated that pattern generation of the hand (digits - interdigits) The gradient of Sonic hedgehog
(secreted inducing factor) from the zone of polarizing activity in the limb, which creates a gradient of active Gli3,
which activates Gremlin, which inhibits BMPs also secreted in the limb, resulting in the formation of an
alternating pattern of activity as a result of this reaction-diffusion system.
Somitogenesis is the creation of segments (somites) from a uniform tissue (Pre-somitic Mesoderm, PSM). They
are formed sequentially from anterior to posterior. This is achieved in amniotes possibly by means of two
opposing gradients, Retinoic acid in the anterior (wavefront) and Wnt and Fgf in the posterior, coupled to an
oscillating pattern (segmentation clock) composed of FGF + Notch and Wnt in antiphase.
[1]
Sex determination in the soma of a Drosophila requires the sensing of the ratio of autosomal genes to sex
chromosome-encoded genes, which results in the production of sexless splicing factor in females, resulting in the
female isoform of doublesex.
[2]
Circuitry
Up-regulation and down-regulation
Up-regulation is a process that occurs within a cell triggered by a signal (originating internal or external to the cell),
which results in increased expression of one or more genes and as a result the protein(s) encoded by those genes. On
the converse, down-regulation is a process resulting in decreased gene and corresponding protein expression.
Up-regulation occurs, for example, when a cell is deficient in some kind of receptor. In this case, more receptor
protein is synthesized and transported to the membrane of the cell and, thus, the sensitivity of the cell is brought
back to normal, reestablishing homeostasis.
Down-regulation occurs, for example, when a cell is overstimulated by a neurotransmitter, hormone, or drug for a
prolonged period of time, and the expression of the receptor protein is decreased in order to protect the cell (see
also tachyphylaxis).
Regulation of gene expression
360
Inducible vs. repressible systems
Gene Regulation can be summarized by the response of the respective system:
Inducible systems - An inducible system is off unless there is the presence of some molecule (called an inducer)
that allows for gene expression. The molecule is said to "induce expression". The manner by which this happens
is dependent on the control mechanisms as well as differences between prokaryotic and eukaryotic cells.
Repressible systems - A repressible system is on except in the presence of some molecule (called a corepressor)
that suppresses gene expression. The molecule is said to "repress expression". The manner by which this happens
is dependent on the control mechanisms as well as differences between prokaryotic and eukaryotic cells.
Theoretical circuits
Repressor/Inducer: an activation of a sensor results in the change of expression of a gene
negative feedback: the gene product downregulates its own production directly or indirectly, which can result in
keeping transcript levels constant/proportional to a factor
inhibition of run-away reactions when coupled with a positive feedback loop
creating an oscillator by taking advantage in the time delay of transcription and translation, given that the
mRNA and protein half-life is shorter
positive feedback: the gene product upregulates its own production directly or indirectly, which can result in
signal amplification
bistable switches when two genes inhibit each other and both have positive feedback
pattern generation
Methods
In general, most experiments investigating differential expression used whole cell extracts of RNA, called
steady-state levels, to determine which genes changed and by how much they did. These are, however, not
informative of where the regulation has occurred and may actually mask conflicting regulatory processess (see
post-transcriptional regulation), but it is still the most commonly analysed (QPCR and DNA microarray).
When studying gene expression, there are several methods to look at the various stages. In eukaryotes these include:
The local chromatin environment of the region can be determined by ChIP-chip analysis by pulling down RNA
Polymerase II, Histone 3 modifications, Trithorax-group protein, Polycomb-group protein, or any other
DNA-binding element to which a good antibody is available.
Epistatic interactions can be investigated by synthetic genetic array analysis
Due to post-transcriptional regulation, transcription rates and total RNA levels differ significantly. To measure the
transcription rates nuclear run-on assays can be done and newer high-throughput methods are being developed,
using thiol labelling instead of radioactivity.
[]
Only 5% of the RNA polymerised in the nucleus actually exits,
[]
and not only introns, abortive products, and
non-sense transcripts are degradated. Therefore, the differences in nuclear and cytoplasmic levels can be see by
separating the two fractions by gentle lysis.
[]
Alternative splicing can be analysed with a splicing array or with a tiling array (see DNA microarray).
All in vivo RNA is complexed as RNPs. The quantity of transcripts bound to specific protein can be also analysed
by RIP-Chip. For example, DCP2 will give an indication of sequestered protein; ribosome-bound gives and
indication of transcripts active in transcription (although it should be noted that a more dated method, called
polysome fractionation, is still popular in some labs)
Protein levels can be analysed by Mass spectrometry, which can be compared only to QPCR data, as microarray
data is relative and not absolute.
Regulation of gene expression
361
RNA and protein degradation rates are measured by means of transcription inhibitors (actinomycin D or
-amanitin) or translation inhibitors (Cycloheximide), respectively.
References
[1] [1] Dequant ML, Pourqui O. Segmental patterning of the vertebrate embryonic axis. Nat Rev Genet. 2008 May;9(5):370-82. PMID 18414404
[2] Gilbert SF (2003). Developmental biology, 7th ed., Sunderland, Mass: Sinauer Associates, 656. ISBN 0-87893-258-5.
Further reading
Latchman, David S. (2005). Gene regulation: a eukaryotic perspective (http:/ / books. google. com/
books?id=4x3ZzLNyfDsC). Psychology Press. ISBN978-0-415-36510-9.
External links
Gene Regulation Info -- manually curated lists of resources, reviews, community discussions (http:/ /
generegulation. info/ )
Cellular Darwinism (http:/ / www. scitopics. com/
Stochastic_gene_expression_and_cell_differentiation_during_embryo_development. html)
Regulation of Gene Expression (http:/ / www. nlm. nih. gov/ cgi/ mesh/ 2011/ MB_cgi?mode=&
term=Regulation+ of+ Gene+ Expression) at the US National Library of Medicine Medical Subject Headings
(MeSH)
362
Protein synthesis and modifications
Translation
Overview of the translation of eukaryotic messenger RNA
In molecular biology and genetics,
translation is the process through
which cellular ribosomes manufacture
proteins. It is part of the process of
gene expression. In translation,
messenger RNA (mRNA) produced by
transcription is decoded by the
ribosome to produce a specific amino
acid chain, or polypeptide, that will
later fold into an active protein. In
bacteria, translation occurs in the cell's
cytoplasm, where the large and small
subunits of the ribosome are located,
and bind to the mRNA. In eukaryotes,
translation occurs across the membrane
of the endoplasmic reticulum in a
process called vectorial synthesis. The
ribosome facilitates decoding by
inducing the binding of tRNAs with
complementary anticodon sequences to
that of the mRNA. The tRNAs carry
specific amino acids that are chained
together into a polypeptide as the
mRNA passes through and is "read" by
the ribosome in a fashion reminiscent
to that of a stock ticker and ticker tape.
In many instances, the entire ribosome/mRNA complex binds to the outer membrane of the rough endoplasmic
reticulum and releases the nascent protein polypeptide inside for later vesicle transport and secretion outside of the
cell. Many types of transcribed RNA, such as transfer RNA, ribosomal RNA, and small nuclear RNA, do not
undergo translation into proteins.
Translation
363
Diagram showing the translation of mRNA and the synthesis of proteins by a
ribosome
Translation proceeds in four phases:
initiation, elongation, translocation and
termination (all describing the growth of
the amino acid chain, or polypeptide that is
the product of translation). Amino acids are
brought to ribosomes and assembled into
proteins.
In activation, the correct amino acid is
covalently bonded to the correct transfer
RNA (tRNA). The amino acid is joined by
its carboxyl group to the 3' OH of the tRNA
by an ester bond. When the tRNA has an
amino acid linked to it, it is termed
"charged". Initiation involves the small
subunit of the ribosome binding to the 5' end
of mRNA with the help of initiation factors
(IF). Termination of the polypeptide happens when the A site of the ribosome faces a stop codon (UAA, UAG, or
UGA). No tRNA can recognize or bind to this codon. Instead, the stop codon induces the binding of a release factor
protein that prompts the disassembly of the entire ribosome/mRNA complex.
A number of antibiotics act by inhibiting translation; these include anisomycin, cycloheximide, chloramphenicol,
tetracycline, streptomycin, erythromycin, and puromycin, among others. Prokaryotic ribosomes have a different
structure from that of eukaryotic ribosomes, and thus antibiotics can specifically target bacterial infections without
any detriment to a eukaryotic host's cells.
Basic mechanisms
Tertiary structure of tRNA. CCA tail in orange,
Acceptor stem in purple, D arm in red, Anticodon
arm in blue with Anticodon in black, T arm in
green.
The basic process of protein production is addition of one amino acid
at a time to the end of a protein. This operation is performed by a
ribosome. The choice of amino acid type to add is determined by an
mRNA molecule. Each amino acid added is matched to a three
nucleotide subsequence of the mRNA. For each such triplet possible,
only one particular amino acid type is accepted. The successive amino
acids added to the chain are matched to successive nucleotide triplets
in the mRNA. In this way the sequence of nucleotides in the template
mRNA chain determines the sequence of amino acids in the generated
amino acid chain.
[1]
Addition of an amino acid occurs at the
C-terminus of the peptide and thus translation is said to be
amino-to-carboxyl directed.
[2]
The mRNA carries genetic information encoded as a ribonucleotide
sequence from the chromosomes to the ribosomes. The ribonucleotides
are "read" by translational machinery in a sequence of nucleotide
triplets called codons. Each of those triplets codes for a specific amino acid.
The ribosome molecules translate this code to a specific sequence of amino acids. The ribosome is a multisubunit
structure containing rRNA and proteins. It is the "factory" where amino acids are assembled into proteins. tRNAs are
Translation
364
small noncoding RNA chains (74-93 nucleotides) that transport amino acids to the ribosome. tRNAs have a site for
amino acid attachment, and a site called an anticodon. The anticodon is an RNA triplet complementary to the mRNA
triplet that codes for their cargo amino acid.
Aminoacyl tRNA synthetase (an enzyme) catalyzes the bonding between specific tRNAs and the amino acids that
their anticodon sequences call for. The product of this reaction is an aminoacyl-tRNA molecule. This
aminoacyl-tRNA travels inside the ribosome, where mRNA codons are matched through complementary base
pairing to specific tRNA anticodons. The ribosome has three sites for tRNA to bind. They are the aminoacyl site
(abbreviated A), the peptidyl site (abbreviated P) and the exit site (abbreviated E). With respect to the mRNA, the
three sites are oriented 5 to 3 E-P-A, because ribosomes moves toward the 3' end of mRNA. The A site binds the
incoming tRNA with the complementary codon on the mRNA. The P site holds the tRNA with the growing
polypeptide chain. The E site holds the tRNA without its amino acid. When an aminoacyl-tRNA initially binds to its
corresponding codon on the mRNA, it is in the A site. Then, a peptide bond forms between the amino acid of the
tRNA in the A site and the amino acid of the charged tRNA in the P site. The growing polypeptide chain is
transferred to the tRNA in the A site. Translocation occurs, moving the tRNA in the P site, now without an amino
acid, to the E site; the tRNA that was in the A site, now charged with the polypeptide chain, is moved to the P site.
The tRNA in the E site leaves and another aminoacyl-tRNA enters the A site to repeat the process.
[3]
After the new amino acid is added to the chain, the energy provided by the hydrolysis of a GTP bound to the
translocase EF-G (in prokaryotes) and eEF-2 (in eukaryotes) moves the ribosome down one codon towards the 3'
end. The energy required for translation of proteins is significant. For a protein containing n amino acids, the number
of high-energy phosphate bonds required to translate it is 4n-1
[citation needed]
. The rate of translation varies; it is
significantly higher in prokaryotic cells (up to 17-21 amino acid residues per second) than in eukaryotic cells (up to
6-9 amino acid residues per second).
[4]
Genetic code
Whereas other aspects such as the 3D structure, called tertiary structure, of protein can only be predicted using
sophisticated algorithms, the amino acid sequence, called primary structure, can be determined solely from the
nucleic acid sequence with the aid of a translation table.
This approach may not give the correct amino acid composition of the protein, in particular if unconventional amino
acids such as selenocysteine are incorporated into the protein, which is coded for by a conventional stop codon in
combination with a downstream hairpin (SElenoCysteine Insertion Sequence, or SECIS).
There are many computer programs capable of translating a DNA/RNA sequence into a protein sequence. Normally
this is performed using the Standard Genetic Code; many bioinformaticians have written at least one such program at
some point in their education. However, few programs can handle all the "special" cases, such as the use of the
alternative initiation codons. For instance, the rare alternative start codon CTG codes for Methionine when used as a
start codon, and for Leucine in all other positions.
Example: Condensed translation table for the Standard Genetic Code (from the NCBI Taxonomy webpage
[5]
).
AAs = FFLLSSSSYY**CC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGG
Starts = ---M---------------M---------------M----------------------------
Base1 = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGG
Base2 = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGG
Base3 = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG
Translation
365
Translation tables
Even when working with ordinary eukaryotic sequences such as the Yeast genome, it is often desired to be able to
use alternative translation tablesnamely for translation of the mitochondrial genes. Currently the following
translation tables are defined by the NCBI Taxonomy Group
[6]
for the translation of the sequences in GenBank:
1: The Standard
2: The Vertebrate Mitochondrial Code
3: The Yeast Mitochondrial Code
4: The Mold, Protozoan, and Coelenterate Mitochondrial Code and the Mycoplasma/Spiroplasma Code
5: The Invertebrate Mitochondrial Code
6: The Ciliate, Dasycladacean and Hexamita Nuclear Code
9: The Echinoderm and Flatworm Mitochondrial Code
10: The Euplotid Nuclear Codecbn dxh
11: The Bacterial and Plant Plastid Code
12: The Alternative Yeast Nuclear Code
13: The Ascidian Mitochondrial Code
14: The Alternative Flatworm Mitochondrial Code
15: Blepharisma Nuclear Code
16: Chlorophycean Mitochondrial Code
21: Trematode Mitochondrial Code
22: Scenedesmus obliquus mitochondrial Code
23: Thraustochytrium Mitochondrial Code
References
[5] http:/ / www. ncbi. nlm.nih. gov/ Taxonomy/ Utils/ wprintgc. cgi?mode=c
[6] http:/ / www. ncbi. nlm.nih. gov/ Taxonomy/
Further reading
Champe, Pamela C; Harvey, Richard A; Ferrier, Denise R (2004). Lippincott's Illustrated Reviews: Biochemistry
(3rd ed.). Hagerstwon, MD: Lippincott Williams & Wilkins. ISBN0-7817-2265-9.
Cox, Michael; Nelson, David R.; Lehninger, Albert L (2005). Lehninger principles of biochemistry (4th ed.). San
Francisco...: W.H. Freeman. ISBN0-7167-4339-6.
Malys N, McCarthy JEG (2010). "Translation initiation: variations in the mechanism can be anticipated". Cellular
and Molecular Life Sciences 68 (6): 9911003. doi: 10.1007/s00018-010-0588-z (http:/ / dx. doi. org/ 10. 1007/
s00018-010-0588-z). PMID 21076851 (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 21076851). Unknown
parameter |unused_data= ignored (help)
External links
Virtual Cell Animation Collection: Introducing Translation (http:/ / vcell. ndsu. nodak. edu/ animations/
translation/ index. htm)
Translate tool (from DNA or RNA sequence) (http:/ / web. expasy. org/ translate)
Posttranslational modification
366
Posttranslational modification
Posttranslational modification (PTM) is a step in protein biosynthesis. Proteins are created by ribosomes
translating mRNA into polypeptide chains. These polypeptide chains undergo PTM, (such as folding, cutting and
other processes), before becoming the mature protein product.
Posttranslational modification of insulin. At the top, the ribosome
translates a mRNA sequence into a protein, insulin, and passes the
protein through the endoplasmic reticulum, where it is cut, folded
and held in shape by disulfide (-S-S-) bonds. Then the protein passes
through the golgi apparatus, where it is packaged into a vesicle. In
the vesicle, more parts are cut off, and it turns into mature insulin.
A protein (also called a polypeptide) is a chain of
amino acids. During protein synthesis, 20 different
amino acids can be incorporated to become a protein.
After translation, the posttranslational modification of
amino acids extends the range of functions of the
protein by attaching it to other biochemical functional
groups (such as acetate, phosphate, various lipids and
carbohydrates), changing the chemical nature of an
amino acid (e.g. citrullination), or making structural
changes (e.g. formation of disulfide bridges).
Also, enzymes may remove amino acids from the
amino end of the protein, or cut the peptide chain in the
middle. For instance, the peptide hormone insulin is cut
twice after disulfide bonds are formed, and a propeptide
is removed from the middle of the chain; the resulting
protein consists of two polypeptide chains connected
by disulfide bonds. Also, most nascent polypeptides
start with the amino acid methionine because the "start"
codon on mRNA also codes for this amino acid. This
amino acid is usually taken off during post-translational
modification.
Other modifications, like phosphorylation, are part of
common mechanisms for controlling the behavior of a
protein, for instance activating or inactivating an
enzyme.
Post-translational modification of proteins is detected
by mass spectrometry or Eastern blotting.
Posttranslational modification
367
PTMs involving addition of functional groups
The genetic code diagram
[1]
showing the amino acid residues as target of
modification.
PTMs involving addition by an
enzyme in vivo
PTMs involving addition of hydrophobic
groups for membrane localization
myristoylation, attachment of myristate, a C
14
saturated acid
palmitoylation, attachment of palmitate, a C
16
saturated acid
isoprenylation or prenylation, the addition of
an isoprenoid group (e.g. farnesol and
geranylgeraniol)
farnesylation
geranylgeranylation
glypiation, glycosylphosphatidylinositol
(GPI) anchor formation via an amide bond to
C-terminal tail
PTMs involving addition of cofactors for
enhanced enzymatic activity
lipoylation, attachment of a lipoate (C
8
)
functional group
flavin moiety (FMN or FAD) may be
covalently attached
heme C attachment via thioether bonds with cysteins
phosphopantetheinylation, the addition of a 4'-phosphopantetheinyl moiety from coenzyme A, as in fatty acid,
polyketide, non-ribosomal peptide and leucine biosynthesis
retinylidene Schiff base formation
PTMs involving unique modifications of translation factors
diphthamide formation (on a histidine found in eEF2)
ethanolamine phosphoglycerol attachment (on glutamte found in eEF1)
[]
hypusine formation (on conserved lysine of eIF5A (eukaryotic) and aIF5A (archeal))
PTMs involving addition of smaller chemical groups
acylation, e.g. O-acylation (esters), N-acylation (amides), S-acylation (thioesters)
acetylation, the addition of an acetyl group, either at the N-terminus
[]
of the protein or at lysine residues.
[]
See
also histone acetylation.
[][]
The reverse is called deacetylation.
formylation
alkylation, the addition of an alkyl group, e.g. methyl, ethyl
methylation the addition of a methyl group, usually at lysine or arginine residues. The reverse is called
demethylation.
amide bond formation
Posttranslational modification
368
amidation at C-terminus
amino acid addition
arginylation, a tRNA-mediation addition
polyglutamylation, covalent linkage of glutamic acid residues to the N-terminus of tubulin and some other
proteins.
[]
(See tubulin polyglutamylase)
polyglycylation, covalent linkage of one to more than 40 glycine residues to the tubulin C-terminal tail
butyrylation
gamma-carboxylation dependent on Vitamin K
[2]
glycosylation, the addition of a glycosyl group to either arginine, asparagine, cysteine, hydroxylysine, serine,
threonine, tyrosine, or tryptophan resulting in a glycoprotein. Distinct from glycation, which is regarded as a
nonenzymatic attachment of sugars.
polysialylation, addition of polysialic acid, PSA, to NCAM
malonylation
hydroxylation
iodination (e.g. of thyroglobulin)
nucleotide addition such as ADP-ribosylation
oxidation
phosphate ester (O-linked) or phosphoramidate (N-linked) formation
phosphorylation, the addition of a phosphate group, usually to serine, threonine, and tyrosine (O-linked), or
histidine (N-linked)
adenylylation, the addition of an adenylyl moiety, usually to tyrosine (O-linked), or histidine and lysine
(N-linked)
propionylation
pyroglutamate formation
S-glutathionylation
S-nitrosylation
succinylation addition of a succinyl group to lysine
sulfation, the addition of a sulfate group to a tyrosine.
selenoylation (co-translational incorporation of selenium in selenoproteins)
PTMs involving non-enzymatic additions in vivo
glycation, the addition of a sugar molecule to a protein without the controlling action of an enzyme.
PTMs involving non-enzymatic additions in vitro
biotinylation, acylation of conserved lysine residues with a biotin appendage
pegylation
PTMs involving addition of other proteins or peptides
ISGylation, the covalent linkage to the ISG15 protein (Interferon-Stimulated Gene 15)
[3]
SUMOylation, the covalent linkage to the SUMO protein (Small Ubiquitin-related MOdifier)
[4]
ubiquitination, the covalent linkage to the protein ubiquitin.
Neddylation, the covalent linkage to Nedd
Pupylation, the covalent linkage to the Prokaryotic ubiquitin-like protein
Posttranslational modification
369
PTMs involving changing the chemical nature of amino acids
citrullination, or deimination, the conversion of arginine to citrulline
deamidation, the conversion of glutamine to glutamic acid or asparagine to aspartic acid
eliminylation, the conversion to an alkene by beta-elimination of phosphothreonine and phosphoserine, or
dehydration of threonine and serine, as well as by decarboxylation of cysteine
[5]
carbamylation, the conversion of lysine to homocitrulline
[6]
PTMs involving structural changes
disulfide bridges, the covalent linkage of two cysteine amino acids
proteolytic cleavage, cleavage of a protein at a peptide bond
racemization of proline by prolyl isomerase
Post-translational modification statistics
Recently, statistics of each post-translational modification experimentally and putatively detected have been
compiled using proteome-wide information from the Swiss-Prot database.
[7]
These statistics can be found at http:/ /
selene. princeton. edu/ PTMCuration/ .
Case examples
Cleavage and formation of disulfide bridges during the production of insulin
PTM of histones as regulation of transcription: RNA polymerase control by chromatin structure
PTM of RNA polymerase II as regulation of transcription
Cleavage of polypeptide chains
[8]
as crucial for lectin specificity
External links
dbPTM - database of protein post-translational modifications
[9]
List of posttranslational modifications in ExPASy
[10]
Browse SCOP domains by PTM
[11]
from the dcGO database
Statistics of each post-translational modification from the Swiss-Prot database
[12]
AutoMotif Server
[13]
- A Computational Protocol for Identification of Post-Translational Modifications in
Protein Sequences
[14]
Functional analyses for site-specific phosphorylation of a target protein in cells
[15]
Detection of Post-Translational Modifications after high-accuracy MSMS
[16]
References
[1] Gramatikoff K. in Abgent Catalog (2004-5) p.263
[4] Van G. Wilson (Ed.) (2004). Sumoylation: Molecular Biology and Biochemistry (http:/ / www. horizonpress. com/ hsp/ books/ sumo. html).
Horizon Bioscience. ISBN 0-9545232-8-8.
[8] http:/ / www. proteopedia.org/ wiki/ index. php/ 1tp8
[9] http:/ / dbptm. mbc. nctu.edu.tw
[10] http:/ / www.uniprot. org/ docs/ ptmlist
[11] http:/ / supfam. org/ SUPERFAMILY/ cgi-bin/ dcbo.cgi?type=KW;po=9991
[12] http:/ / selene.princeton. edu/ PTMCuration/
[13] http:/ / ams2.bioinfo. pl/
[14] http:/ / www.natureprotocols. com/ 2007/ 03/ 23/ automotif_server_a_computation. php
[15] http:/ / www.natureprotocols. com/ 2007/ 01/ 10/ functional_analyses_for_sitesp. php
[16] http:/ / www.detectorvision. com/ deltaMasses.html
Proteolysis
370
Proteolysis
Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. This generally occurs by the
hydrolysis of the peptide bond, and is most commonly achieved by cellular enzymes called proteases, but may also
occur by intramolecular digestion, as well as by non-enzymatic methods such as the action of mineral acids and heat.
Proteolysis in organisms serves many purposes; for example, digestive enzymes break down proteins in food to
provide amino acids for the organism, while proteolytic processing of polypeptide chain after its synthesis may be
necessary for the production of an active protein. It is also important in the regulation of some physiological and
cellular processes, as well as preventing the accumulation of unwanted or abnormal proteins in cells.
Post-translational proteolytic processing
Limited proteolysis of a polypeptide during or after translation in protein synthesis often occur for many proteins.
This may involve removal of the N-terminal methionine, signal peptide, and/or the conversion of an inactive or
non-functional protein to an active one. The precursor to the final functional form of protein is termed proprotein,
and these proproteins may be first synthesized as preproprotein. For example, albumin is first synthesized as
preproalbumin and contains an uncleaved signal peptide. This forms the proalbumin after the signal peptide is
cleaved, and a further processing to remove the N-terminal 6-residue propeptide yields the mature form of the
protein.
[]
Removal of N-terminal methionine
The initiating methonine (and in prokaryotes, fMet) may be removed during translation of the nascent protein. For E.
coli, fMet is efficiently removed if the second residue is small and uncharged, but not if the second residue is bulky
and charged.
[1]
In both prokaryotes and eukaryotes, the exposed N-terminal residue may determine the half-life of
the protein according to the N-end rule.
Removal of the signal sequence
Proteins that are to be targeted to a particular organelle or for secretion have an N-terminal signal peptide that directs
the protein to its final destination. This signal peptide is removed by proteolysis after their transport through a
membrane.
Cleavage of polyprotein
Some proteins and most eukaryotic polypeptide hormones are synthesized as a large precursor polypeptide known as
polyprotein that require proteolytic cleavage into individual smaller polypeptide chains. The polyprotein
pro-opiomelanocortin (POMC) contains many polypeptide hormones. The cleavage pattern of POMC however may
vary between different tissues, yielding different sets of polypeptide hormones from the same polyprotein.
Many viruses also produce their proteins initially as a single polypeptide chain that were translated from a
polycistronic mRNA. This polypeptide is subsequently cleaved into individual polypeptide chains.
[]
Cleavage of precursor proteins
Many proteins and hormones are synthesized in the form of their precursors - zymogens, proenzymes and
prehormones. These proteins are cleaved to form their final active structures. Insulin, for example, is synthesized as
preproinsulin which yields proinsulin after the signal peptide has been cleaved. To form the mature insulin, the
proinsulin is then cleaved at two positions to yield two polypeptide chains linked by 2 disulphide bonds. Proinsulin is
necessary for the folding of the polypeptide chain as the 2 polypeptide chains of insulin may not correctly assemble
into the correct form while its precursor proinsulin do.
Proteolysis
371
Proteases in particular are synthesized in the inactive form so that they may be safely stored in cells, and ready for
released in sufficient quantity when required. This is to ensure that the protease is only activated in the correct
location or context as inappropriate activation of these proteases can be very destructive for an organism. Proteolysis
of the zymogen yields an active protein; for example, when trypsinogen is cleaved to form trypsin, a slight
rearrangement of the protein structure occurs which completes the active site of the protease, thereby activating the
protein.
Proteolysis can therefore be a method of regulating biological processes by turning inactive proteins into active ones.
A good example is the blood clotting cascade whereby an initial event triggers a cascade of sequential proteolytic
activation of many specific proteases, resulting in blood coagulation. The complement system of the immune
response also involves a complex sequential proteolytic activation and interaction that result in an attack on invading
pathogens.
Protein degradation
Proteolytic cleavage breaks down proteins in food extracellularly into smaller peptides and amino acids so that they
may be absorbed and used by an organism. Proteins in cells are also constantly being broken down into amino acids.
This intracellular degradation of protein serves a number of functions - it removes damaged and abnormal protein
and prevent their accumulation, and it also serves to regulate cellular processes by removing enzymes and regulatory
proteins that are no longer needed. The amino acids may then be reused for protein synthesis.
Structure of a proteasome. Its active sites
are inside the tube (blue) where proteins
are degraded.
Lysosome and proteasome
The intracellular degradation of protein may be achieved in two ways -
proteolysis in lysosome, or a ubiquitin-dependent process which targets
unwanted proteins to proteasome. The autophagy-lysosomal pathway is
normally a non-selective process, but it may become selective upon starvation
whereby proteins with peptide sequence KFERQ or similar are selectively
broken down. The lysosome contains a large number of proteases such as
cathepsins.
The ubiquitin-mediated process is selective. Proteins marked for degradation
are covalently linked to ubiquitin. Many molecules of ubiquitin may be linked
in tandem to a protein destined for degradation. The polyubiquinated protein
is targeted to an ATP-dependent protease complex, the proteasome. The
ubiquitin is released and reused, while the targeted protein is degraded.
Rate of intracellular protein degradation
Different proteins are degraded at different rate. Abnormal proteins are
quickly degraded, while the rate of degradation of normal proteins may vary
widely depending on their functions. Enzymes at important metabolic control
points may be degraded much faster than those enzymes whose activity is
largely constant under all physiological conditions. One of the most rapidly
degraded protein is ornithine decarboxylase which has a half-life of 11
minutes. In contrast, other proteins like actin and myosin have half-life of a
month or more, while haemoglobin essentially lasts for the entire life-time of
erythrocyte.
[]
Proteolysis
372
The N-end rule may partially determine the half-life of a protein, and proteins with segments rich in proline,
glutamine, serine, and threonine (the so-called PEST proteins) have short half-life.
[2]
Other factors suspected to
affect degradation rate include the rate deamination of glutamine and asparagine and oxidation of cystein, histidine
and methionine, the absence of stabilizing ligands, the presence of attached carbohydrate or phosphate groups, the
presence of free -amino group, the negative charge of protein, and the flexibility and stability of the protein.
[]
The rate of proteolysis may also depend on the physiological state of the cell, such as its hormonal state as well as
nutritional status. In time of starvation, the rate of protein degradation increases.
Digestion
In human digestion, proteins in food are broken down into smaller peptide chains by digestive enzymes such as
pepsin, trypsin, chymotrypsin, and elastase, and into amino acids by various enzymes such as carboxypeptidase,
aminopeptidase and dipeptidase. It is necessary to break down proteins into small peptides (tripeptides and
dipeptides) and amino acids so they can be absorbed by the intestines, and the absorbed tripeptides and dipeptides
are also further broken into amino acids intracellularly before they enter the bloodstream.
[3]
Different enzymes have
different specificity for their substrate; trypsin for example cleaves the peptide bond after a positively charge residue
(arginine and lysine), chymotrypsin cleaves the bond after an aromatic residue (phenylalanine, tyrosine, and
tryptophan), elastase cleaves the bond after a small non-polar residue such as alanine or glycine.
In order to prevent inappropriate or premature activation of the digestive enzymes (they may, for example, trigger
pancreatic self-digestion), these enzymes are secreted as inactive zymogen. The precursor of pepsin, pepsinogen, is
secreted by the stomach, and is activated only in the acidic environment found in stomach. The pancreas secretes the
precursors of a number of proteases, such trypsin and chymotrypsin. The zymogen of trypsin is trypsinogen which is
activated by a very specific protease, enterokinase, which is secreted by the mucosa of the duodenum. The trypsin,
once activated, can also cleave other trypsinogen as well as the precursors of other proteases such as chymotrypsin
and carboxypeptidase.
In bacteria, a similar strategy of employing an inactive zymogen or prezymogen is used. Subtilisin which is produced
by Bacillus subtilis is produced as preprosubtilisin, and is released only if the signal peptide is cleaved and
autocatalytic proteolytic activation has occurred.
Proteolysis in cellular regulation
Proteolysis is also involved in the regulation of many cellular processes by activating or deactivating enzymes,
transcription factors, and receptors, for example in the biosynthesis of cholesterol,
[4]
or the mediation of thrombin
signalling through protease-activated receptors.
[5]
Some enzymes at important metabolic control points such as ornithine decarboxylase is regulated entirely by its rate
of synthesis and its rate of degradation. Other rapidly degraded proteins include the protein products of
proto-oncogenes which play central roles in the regulation of cell growth.
Proteolysis
373
Cell cycle regulation
Cyclins are a group of proteins that activate kinases involved in cell division. The degradation of cyclins is the key
step that governs the exit from mitosis and progress into the next cell cycle.
[6]
Cyclins accumulate in the course the
cell cycle, then abruptly disappear just before the anaphase of mitosis. The cyclins are removed via a
ubiquitin-mediated proteolytic pathway.
Apoptosis
Caspases are an important group of proteases involved in apoptosis. The precursors of caspase, procaspase, may be
activated by proteolysis through its association with a protein complex that forms apoptosome, or by granzyme B, or
via the death receptor pathways.
Regulatory domains in proteolysis
Protease may have one or more regulatory domains -
Calcium-binding domain - e.g. prothrombin, factor IX, X, VII, protein C in blood clotting cascade, calpain.
Kringle domain - e.g. in prothrombin it keeps the protease inactive.
Proteolysis and diseases
Abnormal proteolytic activity are associated with many diseases.
[7]
In pancreatitis, leakage of proteases and their
premature activation in the pancreas results in the self-digestion of the pancreas. People with diabetes mellitus may
have increased lysosomal activity and the degradation of some proteins can increase significantly. Chronic
inflammatory diseases such as rheumatoid arthritis may involve the release of lysosomal enzymes into extracellular
space which break down surrounding tissues. Abnormal proteolysis and generation of peptides that aggregate in cells
and their ineffective removal may result in many age-related neurological diseases such as Alzheimer.
[8]
Other diseases linked to aberrant proteolysis include muscular dystrophy, degenerative skin disorders, respiratory
and gastrointestinal diseases, and malignancy.
Non-enzymatic proteolysis
Chemicals may be used in laboratory to target specific residue and cleave its peptide bond so that protein may be
broken down into smaller polypeptides for analysis.
[9]
Cyanogen bromide is often used to cleave the peptide bond
after a methionine. Other methods may be used to specifically cleave tryptophanyl, aspartyl, cysteinyl, and
asparaginyl peptide bonds. Acids such as trifluoroacetic acid and formic acid may also be used.
Strong mineral acids can readily hydrolyse the peptide bonds in a protein. However, some proteins are remarkably
resistant to hydrolysis. One well-known example is ribonuclease A, and one method for its purification involves
treatment of crude extracts with hot sulphuric acid so that other proteins become degraded while ribonuclease A is
left intact.
[10]
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374
Laboratory applications
Proteolysis is also used in research and diagnostic applications:
Cleavage of fusion protein so that the fusion partner and protein tag used in protein expression and purification
may be removed. The proteases used have high degree of specificity, such as thrombin, enterokinase, and TEV
protease, so that only the targeted sequence may be cleaved.
Complete inactivation of undesirable enzymatic activity or removal of unwanted proteins. For example,
proteinase K, a broad spectrum proteinase stable in urea and SDS, is often used in the preparation of nucleic acids
to remove unwanted nuclease contaminants which may otherwise degrade the DNA or RNA.
[11]
Partial inactivation, or changing the functionality, of specific protein. For example, treatment of DNA polymerase
I with subtilisin yields the Klenow fragment which retains its polymerase function but lacks 5'-exonuclease
activity.
[12]
In-gel digestion of proteins after separation by gel electrophoresis for the identification by mass spectrometry.
Digestion of proteins in solution for proteome analysis by liquid chromatography-mass spectrometry (LC-MS).
Analysis of the stability of folded domain under a wide range of conditions.
[13]
Increasing success rate of crystallisation projects
[14]
Venoms
Certain types of venom, such as those produced by venomous snakes, can also cause proteolysis. These venoms are,
in fact, complex digestive fluids that begin their work outside of the body. Proteolytic venoms cause a wide range of
toxic effects,
[15]
including effects that are:
cytotoxic (cell-destroying)
hemotoxic (blood-destroying)
myotoxic (muscle-destroying)
hemorrhagic (bleeding)
References
[15] Hayes WK. 2005. Research on Biological Roles and Variation of Snake Venoms. (http:/ / www. llu. edu/ llu/ grad/ natsci/ hayes/
research-c-venom. html?PHPSESSID=55842bf3eeb83dcdfec66c45b91925fc) Loma Linda University.
External links
Proteolysis (http:/ / www. emedicinehealth. com/ script/ main/ srchcont_dict. asp?src=Proteolysis) at eMedicine
Dictionary
Proteolysis MAP from Center on Proteolytic Pathways (http:/ / www. proteolysis. org/ )
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375
Proteasome
Cartoon representation of a proteasome.
Its active sites are sheltered inside the
tube (blue). The caps (red; in this case,
11S regulatory particles) on the ends
regulate entry into the destruction
chamber, where the protein is degraded.
Top view of the proteasome above.
Proteasomes are protein complexes inside all eukaryotes and archaea,
and in some bacteria. In eukaryotes, they are located in the nucleus and
the cytoplasm.
[]
The main function of the proteasome is to degrade
unneeded or damaged proteins by proteolysis, a chemical reaction that
breaks peptide bonds. Enzymes that carry out such reactions are called
proteases. Proteasomes are part of a major mechanism by which cells
regulate the concentration of particular proteins and degrade misfolded
proteins. The degradation process yields peptides of about seven to
eight amino acids long, which can then be further degraded into shorter
amino acid sequences and used in synthesizing new proteins.
[]
Proteins
are tagged for degradation with a small protein called ubiquitin. The
tagging reaction is catalyzed by enzymes called ubiquitin ligases. Once
a protein is tagged with a single ubiquitin molecule, this is a signal to
other ligases to attach additional ubiquitin molecules. The result is a
polyubiquitin chain that is bound by the proteasome, allowing it to
degrade the tagged protein.
[]
In structure, the proteasome is a cylindrical complex containing a
"core" of four stacked rings forming a central pore. Each ring is
composed of seven individual proteins. The inner two rings are made
of seven subunits that contain three to seven protease active sites.
These sites are located on the interior surface of the rings, so that the
target protein must enter the central pore before it is degraded. The
outer two rings each contain seven subunits whose function is to
maintain a "gate" through which proteins enter the barrel. These
subunits are controlled by binding to "cap" structures or regulatory
particles that recognize polyubiquitin tags attached to protein
substrates and initiate the degradation process. The overall system of
ubiquitination and proteasomal degradation is known as the
ubiquitin-proteasome system.
The proteasomal degradation pathway is essential for many cellular
processes, including the cell cycle, the regulation of gene expression,
and responses to oxidative stress. The importance of proteolytic
degradation inside cells and the role of ubiquitin in proteolytic
pathways was acknowledged in the award of the 2004 Nobel Prize in
Chemistry to Aaron Ciechanover, Avram Hershko and Irwin Rose.
[]
Discovery
Before the discovery of the ubiquitin proteasome system, protein
degradation in cells was thought to rely mainly on lysosomes,
membrane-bound organelles with acidic and protease-filled interiors
Proteasome
376
that can degrade and then recycle exogenous proteins and aged or damaged organelles.
[]
However, work by Alfred
Goldberg in 1977 on ATP-dependent protein degradation in reticulocytes, which lack lysosomes, suggested the
presence of a second intracellular degradation mechanism.
[1]
This was shown in 1978 to be composed of several
distinct protein chains, a novelty among proteases at the time.
[]
Later work on modification of histones led to the
identification of an unexpected covalent modification of the histone protein by a bond between a lysine side chain of
the histone and the C-terminal glycine residue of ubiquitin, a protein that had no known function.
[]
It was then
discovered that a previously identified protein associated with proteolytic degradation, known as ATP-dependent
proteolysis factor 1 (APF-1), was the same protein as ubiquitin.
[]
The proteolytic activities of this system was
isolated as a multi-protein complex originally called the multi-catalytic proteinase complex by Sherwin Wilk and
Marion Orlowski.
[]
Later, the ATP-dependent proteolytic complex that was responsible for ubiquitin-dependent
protein degradation was discovered and was called the 26S proteasome.
[2][3]
Much of the early work leading up to the discovery of the ubiquitin proteasome system occurred in the late 1970s
and early 1980s at the Technion in the laboratory of Avram Hershko, where Aaron Ciechanover worked as a
graduate student. Hershko's year-long sabbatical in the laboratory of Irwin Rose at the Fox Chase Cancer Center
provided key conceptual insights, though Rose later downplayed his role in the discovery.
[]
The three shared the
2004 Nobel Prize in Chemistry for their work in discovering this system.
[]
Although electron microscopy data revealing the stacked-ring structure of the proteasome became available in the
mid-1980s,
[]
the first structure of the proteasome core particle was not solved by X-ray crystallography until 1994.
[]
As of 2006, no structure has been solved of the core particle in complex with the most common form of regulatory
cap.
Structure and organization
A schematic diagram of the proteasome 20S core
particle viewed from one side. The subunits that
make up the outer two rings are shown in green, and
the subunits that make up the inner two rings are
shown in blue.
The proteasome subcomponents are often referred to by their
Svedberg sedimentation coefficient (denoted S). The proteasome
most exclusively used in mammals is the cytosolic 26S
proteasome, which is about 2000 kilodaltons (kDa) in molecular
mass containing one 20S protein subunit and two 19S regulatory
cap subunits. The core is hollow and provides an enclosed cavity
in which proteins are degraded; openings at the two ends of the
core allow the target protein to enter. Each end of the core particle
associates with a 19S regulatory subunit that contains multiple
ATPase active sites and ubiquitin binding sites; it is this structure
that recognizes polyubiquitinated proteins and transfers them to
the catalytic core. An alternative form of regulatory subunit called
the 11S particle can associate with the core in essentially the same
manner as the 19S particle; the 11S may play a role in degradation
of foreign peptides such as those produced after infection by a
virus.
[]
20S core particle
The number and diversity of subunits contained in the 20S core
particle depends on the organism; the number of distinct and
specialized subunits is larger in multicellular than unicellular
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377
Top view of the same schematic, illustrating the
seven-fold symmetry of the rings.
organisms and larger in eukaryotes than in prokaryotes. All 20S
particles consist of four stacked heptameric ring structures that are
themselves composed of two different types of subunits;
subunits are structural in nature, whereas subunits are
predominantly catalytic. The outer two rings in the stack consist of
seven subunits each, which serve as docking domains for the
regulatory particles and the alpha subunits N-termini form a gate
that blocks unregulated access of substrates to the interior cavity.
[]
The inner two rings each consist of seven subunits and contain
the protease active sites that perform the proteolysis reactions.
Three distinct catalytic activities were identified in the purified
complex: chymotrypsin-like, trypsin-like and
peptidylglutamyl-peptide hydrolyzing.
[]
The size of the
proteasome is relatively conserved and is about 150 angstroms ()
by 115. The interior chamber is at most 53 wide, though the
entrance can be as narrow as 13, suggesting that substrate proteins must be at least partially unfolded to enter.
[]
In archaea such as Thermoplasma acidophilum, all the and all the subunits are identical, whereas eukaryotic
proteasomes such as those in yeast contain seven distinct types of each subunit. In mammals, the 1, 2, and 5
subunits are catalytic; although they share a common mechanism, they have three distinct substrate specificities
considered chymotrypsin-like, trypsin-like, and peptidyl-glutamyl peptide-hydrolyzing (PHGH).
[]
Alternative
forms denoted 1i, 2i, and 5i can be expressed in hematopoietic cells in response to exposure to pro-inflammatory
signals such as cytokines, in particular, interferon gamma. The proteasome assembled with these alternative subunits
is known as the immunoproteasome, whose substrate specificity is altered relative to the normal proteasome.
[]
19S regulatory particle
The 19S particle in eukaryotes consists of 19 individual proteins and is divisible into two subassemblies, a 9-subunit
base that binds directly to the ring of the 20S core particle, and a 10-subunit lid. Six of the nine base proteins are
ATPase subunits from the AAA Family, and an evolutionary homolog of these ATPases exists in archaea, called
PAN (Proteasome-Activating Nucleotidase).
[4]
The association of the 19S and 20S particles requires the binding of
ATP to the 19S ATPase subunits, and ATP hydrolysis is required for the assembled complex to degrade folded and
ubiquitinated proteins. Note that only the step of substrate unfolding requires energy from ATP hydrolysis, while
ATP-binding alone can support all the other steps required for protein degradation (e.g., complex assembly, gate
opening, translocation, and proteolysis).
[][]
In fact, ATP binding to the ATPases by itself supports the rapid
degradation of unfolded proteins. However, while ATP hydrolysis is required for unfolding only, it is not yet clear
whether this energy may be used in the coupling of some of these steps.
[][5]
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378
Cartoon representation of the 26S proteasome.
[]
In 2012, two independent efforts have elucidated the molecular
architecture of the 26S proteasome by single particle electron
microscopy.
[][]
More recently, a pseudo-atomic atomic model has been
built, again using cryo-EM.
[]
In the heart of the 19S, directly adjacent
to the 20S, are the AAA-ATPases that assemble to a heterohexameric
ring of the order Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5. This ring is a trimer
of dimers: Rpt1/Rpt2, Rpt6/Rpt3, and Rpt4/Rpt5 dimerize via their
N-terminal coiled-coils. These coiled-coils protrude from the
hexameric ring. The largest regulatory particle non-ATPases Rpn1 and
Rpn2 bind to the tips of Rpt1/2 and Rpt6/3, respectively. The ubiquitin
receptor Rpn13 binds to Rpn2 and completes the base cub-complex.
The lid covers one half of the AAA-ATPase hexamer
(Rpt6/Rpt3/Rpt4) and, unexpectedly, directly contacts the 20S via
Rpn6 and to lesser extent Rpn5. The subunits Rpn9, Rpn5, Rpn6,
Rpn7, Rpn3, and Rpn12, which are structurally related among
themselves and to subunits of the COP9 complex and the Eukaryotic
initiation factor 4 (hence called PCI subunits) assemble to a horse-shoe
like structure enclosing the Rpn8/Rpn11 heterodimer. Rpn11, the
deubiquinating enzyme, is placed at the mouth of the AAA-ATPase
hexamer, ideally positioned to remove ubiquitin moieties immediately
before translocation of substrates into the 20S. The second ubiquitin
receptor identified to date, Rpn10, is positioned at the periphery of the
lid, near subunits Rpn8 and Rpn9.
Regulation of the 20S by the 19S
The 19S regulatory particle is responsible for stimulating the 20S to degrade proteins. A primary function of the 19S
regulatory ATPases is to open the gate in the 20S that blocks the entry of substrates into the degradation chamber.
[6]
The mechanism by which the proteasomal ATPase open this gate has been recently elucidated.
[]
20S gate opening,
and thus substrate degradation, requires the C-termini of the proteasomal ATPases, which contains a specific motif
(i.e., HbYX motif). The ATPases C-termini bind into pockets in the top of the 20S, and tether the ATPase complex
to the 20S proteolytic complex, thus joining the substrate unfolding equipment with the 20S degradation machinery.
Binding of these C-termini into these 20S pockets by themselves stimulates opening of the gate in the 20S in much
the same way that a "key-in-a-lock" opens a door.
[]
The precise mechanism by which this "key-in-a-lock"
mechanism functions has been structurally elucidated.
[7]
11S regulatory particle
20S proteasomes can also associate with a second type of regulatory particle, the 11S regulatory particle, a
heptameric structure that does not contain any ATPases and can promote the degradation of short peptides but not of
complete proteins. It is presumed that this is because the complex cannot unfold larger substrates. This structure is
also known as PA28 or REG. The mechanisms by which it binds to the core particle through the C-terminal tails of
its subunits and induces -ring conformational changes to open the 20S gate suggest a similar mechanism for the
19S particle.
[]
The expression of the 11S particle is induced by interferon gamma and is responsible, in conjunction
with the immunoproteasome subunits, for the generation of peptides that bind to the major histocompatibility
complex.
[]
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379
Assembly
The assembly of the proteasome is a complex process due to the number of subunits that must associate to form an
active complex. The subunits are synthesized with N-terminal "propeptides" that are post-translationally modified
during the assembly of the 20S particle to expose the proteolytic active site. The 20S particle is assembled from two
half-proteasomes, each of which consists of a seven-membered pro- ring attached to a seven-membered ring. The
association of the rings of the two half-proteasomes triggers threonine-dependent autolysis of the propeptides to
expose the active site. These interactions are mediated mainly by salt bridges and hydrophobic interactions
between conserved alpha helices whose disruption by mutation damages the proteasome's ability to assemble.
[]
The
assembly of the half-proteasomes, in turn, is initiated by the assembly of the subunits into their heptameric ring,
forming a template for the association of the corresponding pro- ring. The assembly of subunits has not been
characterized.
[]
Only recently, the assembly process of the 19S regulatory particle has been elucidated to considerable extent. The
19S regulatory particle assembles as two distinct subcomponents, the base and the lid. Assembly of the base complex
is facilitated by four assembly chaperones, Hsm3/S5b, Nas2/p27, Rpn14/PAAF1, and Nas6/gankyrin (names for
yeast/mammals).
[]
These assembly chaperones bind to the AAA-ATPase subunits and their main function seems to
be to ensure proper assembly of the heterohexameric AAA-ATPase ring. To date it is still under debate whether the
base complex assembles separately, whether the assembly is templated by the 20S core particle, or whether
alternative assembly pathways exist. In addition to the four assembly chaperones, the deubiquitinating enzyme
Ubp6/Usp14 also promotes base assembly, but it is not essential.
[]
The lid assembles separately in a specific order
and does not require assembly chaperones.
[]
The protein degradation process
Ribbon diagram of ubiquitin, the highly conserved protein that serves
as a molecular tag targeting proteins for degradation by the
proteasome
Ubiquitination and targeting
Proteins are targeted for degradation by the proteasome
with covalent modification of a lysine residue that
requires the coordinated reactions of three enzymes. In
the first step, a ubiquitin-activating enzyme (known as
E1) hydrolyzes ATP and adenylylates a ubiquitin
molecule. This is then transferred to E1's active-site
cysteine residue in concert with the adenylylation of a
second ubiquitin.
[]
This adenylylated ubiquitin is then
transferred to a cysteine of a second enzyme,
ubiquitin-conjugating enzyme (E2). In the last step, a
member of a highly diverse class of enzymes known as
ubiquitin ligases (E3) recognizes the specific protein to
be ubiquitinated and catalyzes the transfer of ubiquitin
from E2 to this target protein. A target protein must be labeled with at least four ubiquitin monomers (in the form of
a polyubiquitin chain) before it is recognized by the proteasome lid.
[]
It is therefore the E3 that confers substrate
specificity to this system.
[]
The number of E1, E2, and E3 proteins expressed depends on the organism and cell type,
but there are many different E3 enzymes present in humans, indicating that there is a huge number of targets for the
ubiquitin proteasome system.
The mechanism by which a polyubiquitinated protein is targeted to the proteasome is not fully understood.
Ubiquitin-receptor proteins have an N-terminal ubiquitin-like (UBL) domain and one or more ubiquitin-associated
(UBA) domains. The UBL domains are recognized by the 19S proteasome caps and the UBA domains bind ubiquitin
Proteasome
380
via three-helix bundles. These receptor proteins may escort polyubiquitinated proteins to the proteasome, though the
specifics of this interaction and its regulation are unclear.
[]
The ubiquitin protein itself is 76 amino acids long and was named due to its ubiquitous nature, as it has a highly
conserved sequence and is found in all known eukaryotic organisms.
[]
The genes encoding ubiquitin in eukaryotes
are arranged in tandem repeats, possibly due to the heavy transcription demands on these genes to produce enough
ubiquitin for the cell. It has been proposed that ubiquitin is the slowest-evolving protein identified to date.
[]
Ubiquitin
contains seven lysine residues to which another ubiquitin can be ligated, resulting in different types of polyubiquitin
chains.
[8]
Chains in which each additional ubiquitin is linked to lysine 43 of the previous ubiquitin has a role in
proteasome targeting, while other types of chains may be involved in other processes.
[9][10]
The ubiquitination pathway
Unfolding and translocation
After a protein has been ubiquitinated, it is recognized
by the 19S regulatory particle in an ATP-dependent
binding step.
[]
The substrate protein must then enter the
interior of the 20S particle to come in contact with the
proteolytic active sites. Because the 20S particle's
central channel is narrow and gated by the N-terminal
tails of the ring subunits, the substrates must be at
least partially unfolded before they enter the core. The
passage of the unfolded substrate into the core is called
translocation and necessarily occurs after
deubiquitination.
[]
However, the order in which substrates are deubiquitinated and unfolded is not yet clear.
[]
Which
of these processes is the rate-limiting step in the overall proteolysis reaction depends on the specific substrate; for
some proteins, the unfolding process is rate-limiting, while deubiquitination is the slowest step for other proteins.
[]
The extent to which substrates must be unfolded before translocation is not known, but substantial tertiary structure,
and in particular nonlocal interactions such as disulfide bonds, are sufficient to inhibit degradation.
[]
The gate formed by the subunits prevents peptides longer than about four residues from entering the interior of the
20S particle. The ATP molecules bound before the initial recognition step are hydrolyzed before translocation. While
energy is needed for substrate unfolding, it is not required for translocation.
[][11]
The assembled 26S proteasome can
degrade unfolded proteins in the presence of a non-hydrolyzable ATP analog, but cannot degrade folded proteins,
indicating that energy from ATP hydrolysis is used for substrate unfolding.
[]
Passage of the unfolded substrate
through the opened gate occurs via facilitated diffusion if the 19S cap is in the ATP-bound state.
[]
The mechanism for unfolding of globular proteins is necessarily general, but somewhat dependent on the amino acid
sequence. Long sequences of alternating glycine and alanine have been shown to inhibit substrate unfolding
decreasing the efficiency of proteasomal degradation; this results in the release of partially degraded byproducts,
possibly due to the decoupling of the ATP hydrolysis and unfolding steps.
[]
Such glycine-alanine repeats are also
found in nature, for example in silk fibroin; in particular, certain Epstein-Barr virus gene products bearing this
sequence can stall the proteasome, helping the virus propagate by preventing antigen presentation on the major
histocompatibility complex.
[]
Proteasome
381
A cutaway view of the proteasome 20S core particle
illustrating the locations of the active sites. The
subunits are represented as green spheres and the
subunits as protein backbones colored by individual
polypeptide chain. The small pink spheres represent the
location of the active-site threonine residue in each
subunit. Light blue chemical structures are the inhibitor
bortezomib bound to the active sites.
Proteolysis
The mechanism of proteolysis by the subunits of the 20S core
particle is through a threonine-dependent nucleophilic attack. This
mechanism may depend on an associated water molecule for
deprotonation of the reactive threonine hydroxyl. Degradation
occurs within the central chamber formed by the association of the
two rings and normally does not release partially degraded
products, instead reducing the substrate to short polypeptides
typically 79 residues long, though they can range from 4 to 25
residues depending on the organism and substrate. The
biochemical mechanism that determines product length is not fully
characterized.
[]
Although the three catalytic subunits have a
common mechanism, they have slightly different substrate
specificities, which are considered chymotrypsin-like, trypsin-like,
and peptidyl-glutamyl peptide-hydrolyzing (PHGH)-like. These
variations in specificity are the result of interatomic contacts with
local residues near the active sites of each subunit. Each catalytic
subunit also possesses a conserved lysine residue required for
proteolysis.
[]
Although the proteasome normally produces very short peptide
fragments, in some cases these products are themselves
biologically active and functional molecules. Certain transcription
factors regulating the expression of specific genes, including one component of the mammalian complex NF-B, are
synthesized as inactive precursors whose ubiquitination and subsequent proteasomal degradation converts them to an
active form. Such activity requires the proteasome to cleave the substrate protein internally: rather than processively
degrading it from one terminus. It has been suggested that long loops on these proteins' surfaces serve as the
proteasomal substrates and enter the central cavity, while the majority of the protein remains outside.
[]
Similar
effects have been observed in yeast proteins; this mechanism of selective degradation is known as regulated
ubiquitin/proteasome dependent processing (RUP).
[]
Ubiquitin-independent degradation
Although most proteasomal substrates must be ubiquitinated before being degraded, there are some exceptions to
this general rule, especially when the proteasome plays a normal role in the post-translational processing of the
protein. The proteasomal activation of NF-B by processing p105 into p50 via internal proteolysis is one major
example.
[]
Some proteins that are hypothesized to be unstable due to intrinsically unstructured regions,
[]
are
degraded in a ubiquitin-independent manner. The most well-known example of a ubiquitin-independent proteasome
substrate is the enzyme ornithine decarboxylase.
[19]
Ubiquitin-independent mechanisms targeting key cell cycle
regulators such as p53 have also been reported, although p53 is also subject to ubiquitin-dependent degradation.
[]
Finally, structurally abnormal, misfolded, or highly oxidized proteins are also subject to ubiquitin-independent and
19S-independent degradation under conditions of cellular stress.
[]
Proteasome
382
Evolution
The assembled complex of hslV (blue) and hslU (red)
from E. coli. This complex of heat shock proteins is
thought to resemble the ancestor of the modern
proteasome.
The 20S proteasome is both ubiquitous and essential in
eukaryotes. Some prokaryotes, including many archaea and the
bacterial order Actinomycetales also share homologs of the 20S
proteasome, whereas most bacteria possess heat shock genes hslV
and hslU, whose gene products are a multimeric protease arranged
in a two-layered ring and an ATPase.
[]
The hslV protein has been
hypothesized to resemble the likely ancestor of the 20S
proteasome.
[]
In general, HslV is not essential in bacteria, and not
all bacteria possess it, whereas some protists possess both the 20S
and the hslV systems.
[]
Many bacteria also possess other homologs
of the proteasome and an associated ATPase, most notably ClpP
and ClpX. This redundancy explains why the HslUV system is not
essential.
Sequence analysis suggests that the catalytic subunits diverged
earlier in evolution than the predominantly structural subunits.
In bacteria that express a 20S proteasome, the subunits have high
sequence identity to archaeal and eukaryotic subunits, whereas the sequence identity is much lower. The
presence of 20S proteasomes in bacteria may result from lateral gene transfer, while the diversification of subunits
among eukaryotes is ascribed to multiple gene duplication events.
[]
Cell cycle control
Cell cycle progression is controlled by ordered action of cyclin-dependent kinases (CDKs), activated by specific
cyclins that demarcate phases of the cell cycle. Mitotic cyclins, which persist in the cell for only a few minutes, have
one of the shortest life spans of all intracellular proteins.
[]
After a CDK-cyclin complex has performed its function,
the associated cyclin is polyubiquitinated and destroyed by the proteasome, which provides directionality for the cell
cycle. In particular, exit from mitosis requires the proteasome-dependent dissociation of the regulatory component
cyclin B from the mitosis promoting factor complex.
[]
In vertebrate cells, "slippage" through the mitotic checkpoint
leading to premature M phase exit can occur despite the delay of this exit by the spindle checkpoint.
[]
Earlier cell cycle checkpoints such as post-restriction point check between G
1
phase and S phase similarly involve
proteasomal degradation of cyclin A, whose ubiquitination is promoted by the anaphase promoting complex (APC),
an E3 ubiquitin ligase.
[]
The APC and the Skp1/Cul1/F-box protein complex (SCF complex) are the two key
regulators of cyclin degradation and checkpoint control; the SCF itself is regulated by the APC via ubiquitination of
the adaptor protein, Skp2, which prevents SCF activity before the G1-S transition.
[]
Individual components of the 19S particle have their own regulatory roles. Gankyrin, a recently identified
oncoprotein, is one of the 19S subcomponents that also tightly binds the cyclin-dependent kinase CDK4 and plays a
key role in recognizing ubiquitinated p53, via its affinity for the ubiquitin ligase MDM2. Gankyrin is anti-apoptotic
and has been shown to be overexpressed in some tumor cell types such as hepatocellular carcinoma.
[]
Proteasome
383
Regulation of plant growth
In plants, signaling by auxins, or phytohormones that order the direction and tropism of plant growth, induces the
targeting of a class of transcription factor repressors known as Aux/IAA proteins for proteasomal degradation. These
proteins are ubiquitinated by SCFTIR1, or SCF in complex with the auxin receptor TIR1. Degradation of Aux/IAA
proteins derepresses transcription factors in the auxin-response factor (ARF) family and induces ARF-directed gene
expression.
[]
The cellular consequences of ARF activation depend on the plant type and developmental stage, but are
involved in directing growth in roots and leaf veins. The specific response to ARF derepression is thought to be
mediated by specificity in the pairing of individual ARF and Aux/IAA proteins.
[]
Apoptosis
Both internal and external signals can lead to the induction of apoptosis, or programmed cell death. The resulting
deconstruction of cellular components is primarily carried out by specialized proteases known as caspases, but the
proteasome also plays important and diverse roles in the apoptotic process. The involvement of the proteasome in
this process is indicated by both the increase in protein ubiquitination, and of E1, E2, and E3 enzymes that is
observed well in advance of apoptosis.
[][][]
During apoptosis, proteasomes localized to the nucleus have also been
observed to translocate to outer membrane blebs characteristic of apoptosis.
[]
Proteasome inhibition has different effects on apoptosis induction in different cell types. In general, the proteasome
is not required for apoptosis, although inhibiting it is pro-apoptotic in most cell types that have been studied.
Apoptosis is mediated through disrupting the regulated degradation of pro-growth cell cycle proteins.
[]
However,
some cell lines in particular, primary cultures of quiescent and differentiated cells such as thymocytes and
neurons are prevented from undergoing apoptosis on exposure to proteasome inhibitors. The mechanism for this
effect is not clear, but is hypothesized to be specific to cells in quiescent states, or to result from the differential
activity of the pro-apoptotic kinase JNK.
[]
The ability of proteasome inhibitors to induce apoptosis in rapidly
dividing cells has been exploited in several recently developed chemotherapy agents such as bortezomib and
salinosporamide A.
Response to cellular stress
In response to cellular stresses such as infection, heat shock, or oxidative damage heat shock proteins that
identify misfolded or unfolded proteins and target them for proteasomal degradation are expressed. Both Hsp27 and
Hsp90chaperone proteins have been implicated in increasing the activity of the ubiquitin-proteasome system,
though they are not direct participants in the process.
[]
Hsp70, on the other hand, binds exposed hydrophobic patches
on the surface of misfolded proteins and recruits E3 ubiquitin ligases such as CHIP to tag the proteins for
proteasomal degradation.
[]
The CHIP protein (carboxyl terminus of Hsp70-interacting protein) is itself regulated via
inhibition of interactions between the E3 enzyme CHIP and its E2 binding partner.
[]
Similar mechanisms exist to promote the degradation of oxidatively damaged proteins via the proteasome system. In
particular, proteasomes localized to the nucleus are regulated by PARP and actively degrade inappropriately
oxidized histones.
[]
Oxidized proteins, which often form large amorphous aggregates in the cell, can be degraded
directly by the 20S core particle without the 19S regulatory cap and do not require ATP hydrolysis or tagging with
ubiquitin.
[]
However, high levels of oxidative damage increases the degree of cross-linking between protein
fragments, rendering the aggregates resistant to proteolysis. Larger numbers and sizes of such highly oxidized
aggregates are associated with aging.
[]
Dysregulation of the ubiquitin proteasome system may contribute to several neural diseases. It may lead to brain
tumors such as astrocytomas.
[]
In some of the late-onset neurodegenerative diseases that share aggregation of
misfolded proteins as a common feature, such as Parkinson's disease and Alzheimer's disease, large insoluble
aggregates of misfolded proteins can form and then result in neurotoxicity, through mechanisms that are not yet well
understood. Decreased proteasome activity has been suggested as a cause of aggregation and Lewy body formation
Proteasome
384
in Parkinson's.
[]
This hypothesis is supported by the observation that yeast models of Parkinson's are more
susceptible to toxicity from -synuclein, the major protein component of Lewy bodies, under conditions of low
proteasome activity.
[]
Impaired proteasomal activity may underlie cognitive disorders such as the autism spectrum
disorders, and muscle and nerve diseases such as inclusion body myopathy.
[]
Role in the immune system
The proteasome plays a straightforward but critical role in the function of the adaptive immune system. Peptide
antigens are displayed by the major histocompatibility complex class I (MHC) proteins on the surface of
antigen-presenting cells. These peptides are products of proteasomal degradation of proteins originated by the
invading pathogen. Although constitutively expressed proteasomes can participate in this process, a specialized
complex composed of proteins, whose expression is induced by interferon gamma, are the primary producers of
peptides which are optimal in size and composition for MHC binding. These proteins whose expression increases
during the immune response include the 11S regulatory particle, whose main known biological role is regulating the
production of MHC ligands, and specialized subunits called 1i, 2i, and 5i with altered substrate specificity. The
complex formed with the specialized subunits is known as the immunoproteasome.
[]
Another 5i variant subunit,
5t, is expressed in the thymus, leading to a thymus-specific "thymoproteasome" whose function is as yet unclear.
[]
The strength of MHC class I ligand binding is dependent on the composition of the ligand C-terminus, as peptides
bind by hydrogen bonding and by close contacts with a region called the "B pocket" on the MHC surface. Many
MHC class I alleles prefer hydrophobic C-terminal residues, and the immunoproteasome complex is more likely to
generate hydrophobic C-termini.
[]
Due to its role in generating the activated form of NF-B, an anti-apoptotic and pro-inflammatory regulator of
cytokine expression, proteasomal activity has been linked to inflammatory and autoimmune diseases. Increased
levels of proteasome activity correlate with disease activity and have been implicated in autoimmune diseases
including systemic lupus erythematosus and rheumatoid arthritis.
[]
The proteasome is also involved in Intracellular antibody-mediated proteolysis of antibody bound virions. In this
neutralisation pathway, TRIM21 (a protein of the tripartite motif family) binds with immunoglobulin G to direct the
virion to the proteasome where it is degraded.
[]
Proteasome inhibitors
Chemical structure of bortezomib, a proteasome
inhibitor used in chemotherapy that is particularly
effective against multiple myeloma
Proteasome inhibitors have effective anti-tumor activity in cell
culture, inducing apoptosis by disrupting the regulated degradation
of pro-growth cell cycle proteins.
[]
This approach of selectively
inducing apoptosis in tumor cells has proven effective in animal
models and human trials. Bortezomib, a molecule developed by
Millennium Pharmaceuticals and marketed as Velcade, is the first
proteasome inhibitor to reach clinical use as a chemotherapy
agent.
[12]
Bortezomib is used in the treatment of multiple
myeloma.
[]
Notably, multiple myeloma has been observed to result
in increased proteasome levels in blood serum that decrease to
normal levels in response to successful chemotherapy.
[]
Studies in
animals have indicated that bortezomib may also have clinically
significant effects in pancreatic cancer.
[][]
Preclinical and early clinical studies have been started to examine
bortezomib's effectiveness in treating other B-cell-related cancers,
[]
particularly some types of non-Hodgkin's
lymphoma.
[]
Proteasome
385
Bortezomib bound to the core particle in a yeast
proteasome. The bortezomib molecule is in the center
colored by atom type (carbon = pink, nitrogen = blue,
oxygen = red, boron = yellow), surrounded by the local
protein surface. The blue patch is the catalytic
threonine residue whose activity is blocked by the
presence of bortezomib.
The molecule ritonavir, marketed as Norvir, was developed as a
protease inhibitor and used to target HIV infection. However, it
has been shown to inhibit proteasomes as well as free proteases; to
be specific, the chymotrypsin-like activity of the proteasome is
inhibited by ritonavir, while the trypsin-like activity is somewhat
enhanced.
[]
Studies in animal models suggest that ritonavir may
have inhibitory effects on the growth of glioma cells.
[]
Proteasome inhibitors have also shown promise in treating
autoimmune diseases in animal models. For example, studies in
mice bearing human skin grafts found a reduction in the size of
lesions from psoriasis after treatment with a proteasome inhibitor.
[]
Inhibitors also show positive effects in rodent models of asthma.
[]
Labeling and inhibition of the proteasome is also of interest in
laboratory settings for both in vitro and in vivo study of
proteasomal activity in cells. The most commonly used laboratory
inhibitors are lactacystin, a natural product synthesized by
Streptomyces bacteria,
[]
and peptide MG132. Fluorescent
inhibitors have also been developed to specifically label the active sites of the assembled proteasome.
[]
References
[12] United States Food and Drug Administration press release (http:/ / www. fda. gov/ bbs/ topics/ NEWS/ 2003/ NEW00905. html) 13 May
2003. Access date 29 December 2006. See also FDA Velcade information page (http:/ / www. fda. gov/ cder/ drug/ infopage/ velcade/ ).
External links
Glickman, Michael H.; Adir, Noam (2004). "The Proteasome and the Delicate Balance between Destruction and
Rescue" (http:/ / www. ncbi. nlm. nih. gov/ pmc/ articles/ PMC314468). PLoS Biology 2 (1): e13. doi:
10.1371/journal.pbio.0020013 (http:/ / dx. doi. org/ 10. 1371/ journal. pbio. 0020013). PMC 314468 (http:/ /
www. ncbi. nlm. nih. gov/ pmc/ articles/ PMC314468). PMID 14737189 (http:/ / www. ncbi. nlm. nih. gov/
pubmed/ 14737189).
The Yeast 26S Proteasome with list of subunits and pictures (http:/ / biochemie. web. med. uni-muenchen. de/
feldmann/ proteasome_units. html)
Ciechanover, A (2005). "Early work on the ubiquitin proteasome system, an interview with Aaron Ciechanover".
Cell Death and Differentiation 12 (9): 116777. doi: 10.1038/sj.cdd.4401691 (http:/ / dx. doi. org/ 10. 1038/ sj.
cdd. 4401691). PMID 16094393 (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 16094393).
Hershko, A (2005). "Early work on the ubiquitin proteasome system, an interview with Avram Hershko". Cell
Death and Differentiation 12 (9): 115861. doi: 10.1038/sj.cdd.4401709 (http:/ / dx. doi. org/ 10. 1038/ sj. cdd.
4401709). PMID 16094391 (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 16094391).
Adams, J (2005). "Early work on the ubiquitin proteasome system, an interview with Irwin Rose". Cell Death and
Differentiation 12 (9): 11626. doi: 10.1038/sj.cdd.4401700 (http:/ / dx. doi. org/ 10. 1038/ sj. cdd. 4401700).
PMID 16094392 (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 16094392).
Cvek, B; Dvorak, Z (2007). "Targeting of nuclear factor-kappaB and proteasome by dithiocarbamate complexes
with metals" (http:/ / www. benthamdirect. org/ pages/ content. php?CPD/ 2007/ 00000013/ 00000030/ 0010B.
SGM). Current pharmaceutical design 13 (30): 315567. doi: 10.2174/138161207782110390 (http:/ / dx. doi.
org/ 10. 2174/ 138161207782110390). PMID 17979756 (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 17979756).
3D proteasome structures in the EM Data Bank(EMDB) (http:/ / www. pdbe. org/ emsearch/ proteasome*)
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Properties of the twenty amino acids Source: http://en.wikipedia.org/w/index.php?oldid=551449465 Contributors: Aa77zz, Arcadian, BenJWoodcroft, Benlisquare, BrentN, Cacycle,
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