BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.
Chapter 1
BIOCHEMISTRY & MEDICINE
BIOCHEMISTRY
➢ Biochemistry is the science concerned with
studying the various molecules that occur in living
cells and organisms, the individual chemical
reactions and their enzyme catalysts, and the
expression and regulation of each metabolic
process. Because life depends on biochemical
reactions, biochemistry has become the basic
language of all biologic sciences.
➢ Biochemistry makes significant contributions to
the fields of cell biology, physiology, immunology,
microbiology, pharmacology, and toxicology, as
well as the fields of inflammation, cell injury, and
cancer. These close relationships emphasize that
life, as we know it, depends on biochemical
reactions and processes.
➢ Biochemical studies have illuminated many
aspects of health and disease, and conversely, the
study of various aspects of health and disease has
opened up new areas of biochemistry
HISTORY
• Medieval Period – ALCHEMY was the forerunner of
chemistry, based on the supposed transformation of
matter. It was concerned particularly with attempts
to convert base metals into gold or to find a
universal elixir.
• 1780 – ANTOINE LAVOISIER proposed that
the combustion of a candle is similar to animal
respiration, as both need O₂
• 1828 – FREIDRICH WOHLER disproved the
Theory of Vitalism (proposes that life is sustained
and explained by an unmeasurable, intelligent force
or energy) by synthesizing urea from ammonium
cyanate.
• 1810s-1830s – a major substance from animals and
plants was identified, composed of C, H, O and N.
• 1838 – The term PROTEIN, meaning the most
important thing, was first used
• 1860 – LOUIS PASTEUR discovered that
fermentation can only occur in intact cells
• 1850s-1890s – Carbohydrates, lipids, and nucleic
acids were recognized
• 1870s – The term BIOCHEMISTRY was coined
• 1884 – EMIL FISCHER introduced the Lock and
Key model of enzyme action
• 1899 – EDUARD BÜCHNER discovered that
fermentation can occur in cell-free extracts; his
discovery is considered the starting point of
biochemistry.
Kevin C. Bolinget
• 1900 – ARCHIBALD GARROD studied patients
with relatively rare disorders of alkaptonuria, albinism,
cystinuria, and pentosuria and established that these
conditions were genetically determined, calling them
“inborn errors of metabolism”
➢ Alkaptonuria – problem in phenylalanine and
tyrosine metabolism; homogentisate dioxygenase
deficiency; accumulation of homogentisic acid; aka
“black urine disease”; s/s: black pigments in the
conjunctiva, cardiac and joint problems; tx:
decrease intake of phenylalanine and tyrosine by
decreasing protein intake; intake of vitamin C to
help the renal excretion of excess homogentisic
acid.
➢ Albinism – reduced amount (or none at all) of
melanin production; s/s: problems with eyesight,
nystagmus, photophobia, pale hair, skin, and eyes
➢ Cystinuria – buildup of amino acid cystine (can
form crystals), a building block of most proteins,
in the kidneys and bladder
➢ Pentosuria – high levels of the pentose sugar
xylitol in the urine; associated with a deficiency
of L-xylulose reductase, necessary for xylitol
metabolism
• 1950 – STANLEY MILLER & HAROLD UREY
showed that a variety of organic molecules including
the amino acids could form in an early reducing
atmosphere.
• 1953 – JAMES WATSON, FRANCIS CRICK &
ROSALIND FRANKLIN discovered the double
helical structure of DNA
BIOMEDICAL IMPORTANCE
❖ Biochemistry and medicine have stimulated
mutual advances
• Biochemistry, medicine, and other health care
disciplines are intimately related. Health in all
species depends on a harmonious balance of the
biochemical reactions occurring in the body,
while disease reflects abnormalities in
biomolecules, biochemical reactions, or
biochemical processes.
• Advances in biochemical knowledge have
illuminated many areas of medicine, and the study
of diseases has often revealed previously
unsuspected aspects of biochemistry.
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❖ Most diseases have biochemical basis
• Biochemical approaches are often fundamental in
illuminating the causes of diseases and in
designing appropriate therapies, and various
biochemical laboratory tests represent an integral
component of diagnosis and monitoring of
treatment.
• By the International Human Genome Sequencing
Consortium and by Celera Genomics, over 90%
of the genome had been sequenced in mid-2000s.
Results of the HGP and of research in related
areas will have a profound influence on the future
of biology, medicine, and other health sciences.
Genomic research on model organisms such as
yeast, the fruit fly Drosophila melanogaster, and the
round worm Caenorhabditis elegans provides
insight into understanding human diseases.
• Examples of disturbances in human biochemistry
responsible for diseases or other debilitating
conditions include electrolyte imbalance,
defective nutrient ingestion or absorption,
hormonal imbalances, toxic chemicals or biologic
agents, and DNA-based genetic disorders. To
address these challenges, biochemical research
continues to be interwoven with studies in
disciplines such as genetics, cell biology,
immunology, nutrition, pathology, and
pharmacology.
GLOSSARY
• Bioinformatics: The discipline concerned with the
collection, storage, and analysis of biologic data,
mainly DNA and protein sequences
• Biophysics: The application of physics and its
techniques to biology and medicine.
• Biotechnology: The field in which biochemical,
engineering, and other approaches are combined to
develop biological products of use in medicine and
industry.
• Gene Therapy: Applies to the use of genetically
engineered genes to treat various diseases.
• Genomics: The genome is the complete set of genes
of an organism, and genomics is the in-depth study of
the structures and functions of genomes.
• Glycomics: The glycome is the total complement of
simple and complex carbohydrates in an organism.
Glycomics is the systematic study of the structures
and functions of glycomes such as the human
glycome.
• Lipidomics: The lipidome is the complete
complement of lipids found in an organism.
Lipidomics is the in-depth study of the structures
and functions of all members of the lipidome and of
Kevin C. Bolinget
BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.
their interactions, in both health and disease.
Metabolomics: The metabolome is the complete
complement of metabolites (small molecules
involved in metabolism) present in an organism.
Metabolomics is the in-depth study of their
structures, functions, and changes in various
metabolic states.
• Molecular Diagnostics: Refers to the use of
molecular approaches such as DNA probes to assist
in the diagnosis of various biochemical, genetic,
immunologic, microbiologic, and other medical
conditions.
• Nanotechnology: The development and application
to medicine and to other areas of devices such as
nanoshells which are only a few nanometers in size
(10−9 m = 1 nm). Nutrigenomics: The systematic
study of the effects of nutrients on genetic
expression and of the effects of genetic variations on
the metabolism of nutrients.
• Pharmacogenomics: The use of genomic
information and technologies to optimize the
discovery and development of new drugs and drug
targets.
• Proteomics: The proteome is the complete
complement of proteins of an organism. Proteomics
is the systematic study of the structures and
functions of proteomes and their variations in health
and disease.
• Stem Cell Biology: Stem cells are undifferentiated
cells that have the potential to self-renew and to
differentiate into any of the adult cells of an
organism. Stem cell biology concerns the biology of
stem cells and their potential for treating various
diseases.
• Synthetic Biology: The field that combines
biomolecular techniques with engineering
approaches to build new biological functions and
systems.
• Systems Biology: The field concerns complex
biologic systems studied as integrated entities.
Transcriptomics: The comprehensive study of the
transcriptome, the complete set of RNA transcripts
produced by the genome during a fixed period of
time.
2
 BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.

CHAPTER 2 • Influences water’s physical properties (e.g.

WATER & pH
WATER
➢ Predominant compound in the body
➢ Makes up 50% body weight in men, 60% in
women
• 55-75% ICF, 25-45% ECF
• ECF: 25% IV, 75% EV
PROPERTIES
✓ A water molecule is an irregular, slightly skewed
tetrahedron with oxygen at its center
✓ The two hydrogens and the unshared electrons of the
remaining two sp3-hybridized orbitals occupy the
corners of the tetrahedron
✓ Water is an excellent nucleophile (electron-rich)
✓ The 105° angle between the two hydrogen atoms
differs slightly from the ideal tetrahedral angle, 109.5°
✓ The strongly electronegative oxygen atom in a water
molecule attracts electrons away from the hydrogen
nuclei, leaving them with a partial positive charge, while
its two unshared electron pairs constitute a region of
local negative charge
❖ Water is an ideal biologic solvent
➢ Form strong dipoles
• Molecule with electrical
charge distributed
asymmetrically about its
structure
• Responsible for its high
dielectric constant (a
quantity measuring the ability of a substance
to store electrical energy in an electric field),
which is 78.5 at 25°C
• Water therefore greatly decreases the force
of attraction between charged and polar
species relative to water-free environments
with lower dielectric constants
• The strong dipole and high dielectric constant
enable water to dissolve large quantities of
charged compounds such as salts
➢ Form hydrogen bonds
• H-bonds are formed when a
partially unshielded hydrogen
nucleus covalently bound to an
electron-withdrawing oxygen
or nitrogen atom can interact
with an unshared electron pair
on another oxygen
• Favors self-association of
water molecules
high viscosity, high surface tension, and high
boiling point)
• Enables water to dissolve molecules that
contain functional groups which can
participate in hydrogen bonding
• Each molecule in liquid water associates
through hydrogen bonds with 3.5 others
❖ Interaction with water influences the
structure of biomolecules
➢ Biomolecules fold to position polar and charged
groups on their surfaces
• Most biomolecules are amphipathic (they
possess regions rich in charged or polar
functional groups as well as regions with
hydrophobic character)
• Proteins tend to fold with the R-groups of
amino acids with hydrophobic side chains in
the interior (e.g. phospholipid bilayer)
• This pattern maximizes the opportunities for
the formation of energetically favorable
charge-dipole, dipole-dipole, and hydrogen
bonding interactions between polar groups on
the biomolecule and water.
➢ Covalent and non-covalent bonds
• Make significant contributions to the
structure, stability, and functional competence
of macromolecules in living cells
• Covalent bonds – strongest force that holds
molecules together
• Noncovalent bonds – forces of lesser
magnitude
➢ Hydrophobic interactions (tendency of
nonpolar compounds to self-associate in an
aqueous environment)
• Water molecules adjacent to a hydrophobic
group are restricted in the number of
orientations (degrees of freedom) that permit
them to participate in the maximum number
of energetically favorable hydrogen bonds
➢ Electrostatic interactions
• Interactions between charged groups help
shape biomolecular structure
• Salt bridges – electrostatic interactions
between oppositely charged groups within or
between biomolecules; act over large
distances
➢ van der Waals Forces
• Arise from attractions between transient
dipoles generated by the rapid movement of
electrons of all neutral atoms.
• Weaker than hydrogen bonds but potentially
extremely numerous,

Kevin C. Bolinget
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• Decrease as the sixth power of the distance
separating atoms
➢ Multiple forces stabilize biomolecules
• Example: DNA double helix
o Individual DNA strand – covalent bonds
o Nucleotide bases – hydrogen bonds
o Purine and pyrimidine stacks – van der
Waals forces
❖ Water molecules exhibit a slight, but
important, tendency to dissociate
➢ Since water can act both as an acid and as a base,
its ionization may be represented as an
intermolecular proton transfer that forms a
hydronium ion and a hydroxide ion:
H2O + H2O  H3O + OH⁻
➢ Protons exist in solution not only as H3O⁺, but
also as multimers such as H5O2⁺ and H7O3⁺. The
proton is nevertheless routinely represented as
H⁺, even though it is in fact highly hydrated
➢ Since hydronium and hydroxide ions continuously
recombine to form water molecules, an individual
hydrogen or oxygen cannot be stated to be
present as an ion or as part of a water molecule
➢ Actual probability of a hydrogen atom in pure
water existing as a hydrogen ion is approximately
1.8 × 10–9
➢ K represents the dissociation constant,
whereas the constant KW is the ion product for
water
[H⁺] [OH⁻]
K =
[H2O]
[H] = 1.8×10–9 × 55.56 mol/L = 1.0×10–7 mol/L
[10–7] [10–7]
K = = 1.8×10–16 mol/L
[55.56
KW = (K) [H2O] = [H⁺] [OH⁻]
KW = (1.8×10–16 mol/L) (55.56 mol/L)
KW = 1.00×10–14 (mol/L)2
❖ pH is the negative logarithm of the hydrogen
ion concentration (pH = –log[H+])
➢ pH – introduced by Sörensen (1909), which is
known as the power (English), puissant (French),
or potennz (German) of the exponent
➢ Acids – proton donors
• Strong acids – completely dissociate (e.g. HCl,
H2SO4)
• Weak acids – partial dissociation (e.g. HOAc)
➢ Bases – proton acceptors
• Strong bases – completely dissociate even in
high pH (e.g. KOH, NaOH)
Kevin C. Bolinget
BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.
• Weak bases – (e.g. Ca(OH2))
➢ Sample problems: Compute for the pH
1. [H+] = 4.5×10–5 = -log (4.5×10–5); pH = 4.35
2. [H+] = 2.3×10–3 = -log (2.3×10–3); pH = 2.64
3. [OH–] = 3.2×10–4 = -log (3.2×10–4); pOH =
3.49; pH = 14 – 3.49 = 10.51
4. [OH–] = 2.2×10–3 = -log (2.2×10–3); pOH =
2.66; pH = 14 – 3.49 = 11.34
❖ Weak acids have great physiologic
significance
➢ Present in proteins and nucleic acids
➢ Relative strengths are expressed in terms of
dissociation constants, Ka
➢ Since the numeric values of Ka for weak acids are
negative exponential numbers, Ka is expressed as
pKa: pKa = −logKa
➢ pKa – used to express the relative strengths of
both acids and bases
• pH where the concentration of the acids is
equal to its base
• values depend on the properties of the
medium
• pKa of an acid group is the pH at which the
protonated and unprotonated species are
present at equal concentrations
• strong acids have low pKa values while weak
acids have high pKa values
❖ Henderson-Hasselbalch Equation describes
the behavior of weak acids and buffers
➢ Henderson-Hasselbalch equation has great
predictive value in protonic equilibria
[A⁻] (base)
pH = pKa + log
[HA] (acid)
❖ Solutions of weak acids and their salts buffer
changes in pH
➢ Buffering – ability to resist a change in pH
following addition of a strong acid or base;
exhibited by solutions of weak acids or bases and
their conjugates
➢ A solution of a weak acid and its conjugate base
buffers most effectively in the pH range pKa ± 1.0
pH unit.
4
 BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.

REGULATION PROPERTIES OF AMINO ACIDS

❖ Antidiuretic Hormone (ADH)/ vasopressin ❖ Genetic Code Specifies 20 L-α-Amino Acids
• Peptide hormone secreted by the hypothalamus • Essential versus non-essential amino acids
(in the paraventricular and supraoptic nucleus) Essential Non-essential
• Stored in the posterior pituitary Cannot be Naturally-occurring
• Secretion is stimulated when osmoreceptors in synthesized de novo
the hypothalamus detect increasing plasma Tryptophan (W) Glycine (G)
osmolarity (plasma gets too concentrated) Valine (V) Alanine (A)
• Causes the insertion of water channels into the Threonine (T) Serine (S)
membranes of cells lining the collecting ducts, Isoleucine (I) Tyrosine (Y)
allowing reabsorption to occur Leucine (L) Cysteine (C)
Lysine (K) Aspartic Acid (D)
Phenylalanine (F) Asparagine (N)
Methionine (M) Glutamic Acid (E)
Histidine (H) Glutamine (Q)
Arginine (R) Proline (P)
• Amino acids with aliphatic side chains
1. Glycine
2. Alanine
3. Valine
4. Leucine
5. Isoleucine
RELATED DISORDER • Amino acids with –OH side chains
❖ Diabetes Insipidus – passage of large volumes of 1. Serine
urine a day (>3 L), which is dilute (<300 mOsm/kg) 2. Threonine
1) Central (Neurogenic) – ADH secretion is 3. Tyrosine
decreased due to damage to the hypothalamus or • Amino acids with chains containing sulfur
pituitary gland 1. Cysteine
o Traumatic brain injury/ contusion 2. Methionine
o Surgery to remove the pituitary gland (as a • Amino acids with side chains containing an acidic
result of pituitary adenoma) group or their amides
2) Nephrogenic – sensitivity of kidneys to ADH is 1. Aspartic Acid
decreased (not genetic) 2. Asparagine
• Signs and Symptoms: polyuria, polydipsia, 3. Glutamic Acid
nocturia, dry skin (due to dehydration) 4. Glutamine
• Diagnosis: 24 hr. urine collection, Serum • Amino acids with side chains containing basic
Electrolytes, UA, Plasma ADH level, Miller-Moses groups
Test, CT/MRI 1. Arginine
• Treatment: hydration, synthetic ADH analog 2. Lysine
desmopressin (for central DI) 3. Histidine
• Amino acids with side chains containing aromatic
rings
Chapter 3 1. Phenylalanine
AMINO ACIDS & PEPTIDES 2. Tyrosine
3. Tryptophan
L-α-AMINO ACIDS 4. Histidine
➢ provide monomer units from which polypeptide • Amino acids with polar, uncharged side chains
chains of proteins are synthesized 1. Cysteine
➢ serve other several metabolic functions including the 2. Threonine
biosynthesis of urea, heme, nucleic acids, and 3. Methionine
hormones 4. Glutamine
5. Asparagine
6. Serine

Kevin C. Bolinget
5
 BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.

• Hydrophobic versus hydrophilic amino acids ❖ Stereochemistry of the Protein Amino Acids
Hydrophilic Hydrophobic • α-carbon of every amino acid is chiral (non-
Arginine Alanine superimposable on its mirror image), except
Asparagine Isoleucine glycine
Aspartic acid Leucine • All proteins share the absolute configuration of L-
Cysteine Methionine glyceraldehyde, although some are
Glutamic acid Phenylalanine dextrorotatory and some levorotatory
Glutamine Proline ❖ Non-protein L-α-amino acids
Glycine Tryptophan • Ornithine – intermediate in urea synthesis
Histidine Tyrosine • Citrulline – intermediate in urea synthesis
Lysine Valine • Homocysteine – intermediate in cysteine
Serine biosynthesis
Threonine • Homoserine – product of cysteine biosynthesis
❖ Selenocysteine is the 21st amino acid • Glutamate-γ-semialdehyde – serine catabolite
• Selenium atom replaces the sulfur of its elemental ❖ Potentially toxic amino acids
analog, cysteine • Homoarginine – cleaved by arginase to L-lysine
and urea; implicated in human neurolathyrism (a
profound neurological disorder characterized by
progressive and irreversible spastic paralysis of
the legs)
• β-N-Oxalyl diaminopropionic acid (β-N-
• Incorporation is specified by a large and complex ODAP) – neurotoxin implicated in human
genetic element for the unusual tRNA called neurolathyrism
tRNASec which utilizes the UGA anticodon that • β-N-Glutamylamino-propiononitrile
normally signals STOP (BAPN) – an osteolathyrogen
❖ Posttranslational modifications confer • 2,4-Diaminobutyric acid – inhibit ornithine
additional properties transcarbamylase, resulting in ammonia toxicity
• Posttranslational modifications generate novel R- • β-Methylaminoalanine – possible risk factor
groups that impart further properties for neurodegenerative diseases
• Example: –OH stabilizes collagen as protein-
bound proline and lysine residues are converted PROPERTIES OF THE FUNCTIONAL GROUPS OF
to 4-hydroxyproline and 5-hydroxylysine AMINO ACIDS
❖ Amino acids may have positive, negative, or
zero net charge
• At physiologic pH (pH 7.4), carboxyl groups exist
almost entirely as R–COO– and amino groups
predominantly as R–NH3+
❖ Amino acids are linked together by peptide
bonds
• Peptide bond – exhibits partial double-bond
character • Zwitterions – molecules that contain an equal
o O, C, N, and H atoms are coplanar (lie in the number of positively- and negatively-charged
same plane) groups and bear no net charge (neutral)
o Uncharged at any pH of physiologic interest ❖ At isoelectric pH, an amino acid bears no net
charge
• Isoelectric pH (pI) – the pH midway between
the pKa values for the ionizations on either side of
the isoelectric species
pK1 + pK2
pI =
2
❖ pKa values vary with the environment
• A nonpolar environment, which possesses less
capacity than water for stabilizing charged
species, thus raises the pKa of a carboxyl group

Kevin C. Bolinget
6

making it a weaker acid, but lowers the pKa of an
amino group, making it a stronger acid
• Presence of an adjacent oppositely charged group
can stabilize, or of a similarly charged group can
destabilize, a developing charge
• The pKa values of all functional groups of an
amino acid or of a peptide dictate its net charge
at a given pH. pI, the isoelectric pH, is the pH at
which an amino acid bears no net charge, and
thus does not move in a direct current electrical
field
Chapter 4
PROTEINS: DETERMINATION OF PRIMARY
STRUCTURE
❖ Protein purification – cells contain thousands of
different proteins, each in widely varying amounts.
Thus, the highly purified protein is essential for the
detailed examination of its physical and functional
properties
1. Chromatographic techniques
• Column chromatography – consists of a
stationary phase (matrix of small beads that
can be chemically derivatized) and a mobile
phase, which percolates through a column and
collected in fractions.
Kevin C. Bolinget
BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.
• High-pressure liquid chromatography –
instead of a solvent being allowed to drip
through a column under gravity, matrix is
forced through under high pressures
a) Size-exclusion (gel filtration)
chromatography – separates proteins based
on their Stokes radius (radius of the sphere
they occupy as they tumble in solution);
employs porous beads; proteins with larger SR
are filtered first
b) Ion-exchange chromatography – proteins
are separated based on charge-charge
interactions where proteins with weaker
interactions are filtered first
c) Hydrophobic interaction
chromatography – separates proteins based
on their tendency to associate with a
stationary phase coated with hydrophobic
groups
d) Affinity chromatography – exploits high
selectivity of proteins for their ligands wherein
only the proteins that interact with the
immobilized ligands adhere
2. Selective precipitation – exploits differences
in relative solubility of individual proteins as a
function of pH (isoelectric precipitation),
polarity (precipitation with ethanol or
acetone), or salt concentration (salting out
with ammonium sulfate).
• Polyacrylamide gel electrophoresis (PAGE)
– most widely used method for determining
purity of a protein
o Used in the presence of sodium dodecyl
sulfate (SDS)
o Electrophoresis separates charged
biomolecules based on the rates at which they
migrate in an applied electrical field
o SDS binds to proteins at a ratio of one
molecule of SDS per two peptide bonds,
causing the polypeptide to unfold or denature
o Large complexes encounter greater
resistance, causing polypeptides to separate
based on their relative molecular mass (Mr)
o Individual polypeptides trapped in the
acrylamide gel after removal of the electrical
field are visualized by staining with dyes such
as Coomassie Blue
• Isoelectric focusing (IEF) – separates
different molecules by differences in
their isoelectric point (pI)
o Uses ampholytes an applied electric field to
generate a pH gradient within a
polyacrylamide matrix
o Used in conjunction with SDS-PAGE for two-
dimensional electrophoresis, which separates
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polypeptides based on pI in one dimension
and on Mr in the second
❖ Sanger sequencing – developed by Frederick
Sanger, is the first method to determine the
sequence of a polypeptide using 1-fluoro-2,4-
dinitrobenzene (Sanger reagent), which reacts with
the exposed α-amino groups of the amino-terminal
residues.
❖ Edman reaction – introduced by Pehr Edman to
selectively label the amino-terminal residue of a
peptide using phenylisothiocyanate (Edman reagent)
without disrupting the peptide bonds between other
amino acid residues
❖ Mass spectrometry – a versatile technique that
ionizes chemical species and sorts the ions based on
their mass-to-charge ratio; applicable to the
determination of primary structure, identification of
posttranslational modifications, and the detection of
metabolic abnormalities
• Time-of-flight (TOF) mass spectrometers –
employ linear flight tube; used to determine the
large masses of complete proteins
• Quadrupole mass spectrometer – used to
determine the masses of molecules of 4000 Da
or less
• Tandem mass spectrometry (MS-MS or
MS2) – involves multiple steps of mass
spectrometry selection, with some form of
fragmentation occurring in between the stages;
can be used to screen blood samples from
newborns for the presence and concentrations of
amino acids, fatty acids, and other metabolites
❖ Protein volatilization methods
1. Electrospray ionization – molecules to be
analyzed are dissolved in a volatile solvent and
introduced into the sample chamber in a minute
stream through a capillary
2. Matrix-assisted laser desorption and
ionization (MALDI) – sample is mixed with a
liquid matrix containing a light-absorbing dye and
a source of protons
3. Fast atom bombardment (FAB) – large
macromolecules dispersed in glycerol or another
protonic matrix are bombarded by a stream of
neutral atoms that have been accelerated to a
high velocity
Chapter 5
PROTEINS: HIGHER ORDERS OF
STRUCTURE
❖ Configuration – refers to the geometric
relationship between a given set of atoms
Kevin C. Bolinget
BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.
➢ Interconversion generally occurs via rotation with
retention of configuration
❖ Conformation – refers to the spatial relationship
of every atom in a molecule
➢ Interconversion requires breaking covalent bonds
FOUR ORDERS OF PROTEIN STRUCTURE
1. Primary structure – sequence of amino acids in a
polypeptide chain
2. Secondary structure – folding of short (3-30
residue), contiguous segments of polypeptide into
geometrically ordered units
• Peptide bonds restrict possible secondary
conformations
o Phi angle – angle about the Cα−N bond
o Psi angle – angle about the Co−Cα bond
o Most combinations of phi-psi angles are
disallowed due to steric hindrance, except for
glycine
• Alpha helix – a righthand-spiral conformation in
which every backbone N−H group donates a
hydrogen bond to the backbone C=O group of
the amino acid located three or four residues
earlier along the protein sequence
o polypeptide backbone of an α helix is twisted
by an equal amount about each α-carbon with
a phi angle of approximately −57° and a psi
angle of approximately −47°
o complete turn of the helix contains an average
of 3.6 aminoacyl residues, and the distance it
rises per turn (its pitch) is 0.54 nm
o only right-handed α helices are present in
proteins
o stabilized by hydrogen bonds formed between
the oxygen of the peptide bond carbonyl and
the hydrogen atom of the peptide bond
nitrogen of the fourth residue down the
polypeptide chain
o peptide bond nitrogen of proline lacks a
hydrogen atom, making it incapable of forming
a hydrogen bond with a carbonyl oxygen
o proline can only be stably accommodated
within the first turn of an α helix, when
present elsewhere, proline disrupts the
conformation of the helix, producing a bend
(kinks).
o because it possesses such a small R group,
glycine also frequently induces bends within α
helices.
• Beta sheet – consists of beta strands connected
laterally by at least two or three backbone
hydrogen bonds, forming a generally twisted,
pleated sheet
8

o amino acid residues of a β sheet, when viewed
edge-on, form a zigzag or pleated pattern in
which the R groups of adjacent residues
project in opposite directions
o derive much of their stability from hydrogen
bonds between the carbonyl oxygens and
amide hydrogens of peptide bonds
o interacting β sheets can be arranged either to
form a parallel β sheet, in which the adjacent
segments of the polypeptide chain proceed in
the same direction amino to carboxyl, or an
antiparallel sheet, in which they proceed in
opposite directions
o most β sheets are not perfectly flat but tend
to have a right-handed twist
• Loops, loops, & bends
o Turns and bends – refer to short segments
of amino acids that join two units of the
secondary structure, such as two adjacent
strands of an antiparallel β sheet
o β turn – involves four aminoacyl residues, in
which the first residue is hydrogen-bonded to
the fourth, resulting in a tight 180° turn;
usually exhibited by proline and glycine
o Loops – regions of irregular structure that
contain residues beyond the minimum number
necessary to connect adjacent regions of
secondary structure
o Supersecondary structures – structural
motifs that are intermediate in scale between
secondary and tertiary structures
o Helix-loop-helix motifs – provide the
oligonucleotide-binding portion of many
DNA-binding proteins such as repressors and
transcription factors
3. Tertiary structure – entire three-dimensional
conformation of a polypeptide, it is the assembly of
secondary structural units into larger functional units
such as the mature polypeptide and its component
domains
• Domain – section of the protein structure
sufficient to perform a particular chemical or
physical task such as binding of a substrate or
other ligand
• Rossman fold – N-terminal NAD(P)+-binding
domain shared by one family of dehydrogenases
• Stabilized by noncovalent interactions:
hydrophobic interactions, hydrogen bonds, ionic
interactions, and disulfide bonds
Kevin C. Bolinget
BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.
o Formation of disulfide bonds involves
oxidation of the cysteinyl sulfhydryl groups
and requires oxygen (catalyzed by protein-
suflhydryl oxidase)
o Protein disulfide isomerase facilitates the
formation of disulfide bonds that stabilize a
protein’s native conformation
o Since many eukaryotic sulfhydryl oxidases are
flavin-dependent, dietary riboflavin deficiency
often is accompanied by an increased
incidence of improper folding of disulfide-
containing proteins
• Determination of 3D structure:
(a) X-ray crystallography – protein is
precipitated to form crystals and angles and
intensities of diffracted beams of X-rays are
measured
(b) Nuclear magnetic resonance
microscopy – measures the absorbance of
radio frequency electromagnetic energy by
certain atomic nuclei
(c) Cryo-electron microscopy – extends the
resolution of EM to biologic materials by
employing cryogenic agents such as liquid
nitrogen and liquid helium to protect organic
matter from destruction
4. Quaternary structure – number and types of
polypeptide units of oligomeric proteins and their
spatial arrangement
• Types based on number of polypeptide chains
a) Monomeric – consist of a single polypeptide
chain
b) Dimeric – consist of two polypeptide chains
i. Homodimer – contain two copies of the
same polypeptide chain
ii. Heterodimer – contain different
polypeptides
PROTEIN FOLDING
❖ Native conformation of a protein is
thermodynamically favored
❖ Protein folding generally occurs via a stepwise
process
(1) As the newly synthesized polypeptide emerges
from the ribosome, short segments fold into
secondary structural units that provide local
regions of organized structure
(2) Hydrophobic regions segregate into the interior
of the protein away from solvent, forming a
“molten globule,” a partially folded polypeptide
in which the modules of the secondary structure
rearrange to give the native conformation of the
protein
❖ Auxiliary proteins are employed by cells to speed up
the process of folding
9

• Chaperone proteins – prevent aggregation,
thus providing an opportunity for the formation
of appropriate secondary structural elements and
their subsequent coalescence into a molten
globule
➢ hsp70 – binds short sequences of
hydrophobic amino acids that emerge while a
new polypeptide is being synthesized, shielding
them from solvent
➢ hsp60 (chaperonins) - provides a sheltered
environment in which a polypeptide can fold
until all hydrophobic regions are buried in its
interior, thus preempting any tendency
toward aggregation
❖ Proline-cis, trans-isomerization plays a key role in the
rate-determining steps of protein folding
• All X-Pro peptide bonds are synthesized in the
trans configuration
• cis (6%) configuration is common in β turns
• cis-trans proline isomerization is a very slow
process that can impede the progress of protein
folding by trapping one or more proline residues
crucial for folding in the non-native isomer,
especially when the native protein requires
the cis isomer
• Cyclophilins – catalyzes the isomerization of
proline from trans to cis
❖ Perturbation of protein conformation may have
pathologic consequences
• Prion diseases – transmissible spongiform
encephalopathies, are fatal neurodegenerative
diseases characterized by spongiform changes,
astrocytic gliomas, and neuronal loss resulting
from the deposition of insoluble protein
aggregates in neural cells
➢ pathogenic mechanism involves a
conformational transition of α-helix into β-
sheet structure in prion protein (PrP), a
glycoprotein encoded on the short arm of
chromosome 20
➢ Creutzfeldt-Jakob disease – cellular prion
protein (PrPC) converts into its pathogenic
isoform (PrPSc)
o Signs & symptoms: stroke-like symptoms,
dementia, startle myoclonus
o Diagnosis: EEG, MRI/CT, Spinal Tap,
postmortem biopsy
o Treatment: Supportive
o Prognosis: Almost always fatal
➢ Scrapie (in sheep)
➢ Mad cow disease (in bovine)
• Alzheimer’s disease – characterized by
refolding of misfolding of another protein
endogenous to human brain tissue, β-amyloid
Kevin C. Bolinget
BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.
➢ levels of β-amyloid become elevated, and this
protein undergoes a conformational
transformation from a soluble α helix-rich
state to a state rich in β sheet and prone to
self-aggregation
COLLAGEN
❖ Collagen illustrates the role of posttranslational
processing in protein maturation
• posttranslational modification – maturation
of proteins into their final structural state, which
often involves the cleavage or formation (or
both) of covalent bonds
❖ Collagen is the most abundant fibrous protein
• Constitutes more than 25% of the protein mass
in the human body
• One fiber contains about 1000 amino acids
• Tropocollagen – repeating unit of a mature
collagen fiber that consists of three collagen
polypeptides bundled together in a unique
conformation, the collagen triple helix
➢ Collagen triple helices are stabilized by
hydrogen bonds between residues in different
polypeptide chains, a process helped by the
hydroxyl groups of hydroxyprolyl residues
➢ Additional stability is provided by covalent
cross-links formed between modified lysyl
residues both within and between polypeptide
chains
• Collagen is initially synthesized as a larger
precursor polypeptide, procollagen
➢ Prolyl and lysyl hydroxylases – hydroxylates
numberous prolyl and lysysl residues; require
ascorbic acid
➢ Hydroxyprolyl and hydroxylysyl residues
provide additional hydrogen bonding capability
that stabilizes the mature protein
1. Prepro a-chain
2. Pro-a chain
3. Hydroxylation
4. Glycosylation
5. Triple-Helix Formation
6. Procollagen
7. Extracellular release and cleavage forming
tropocollagen
8. Tropocollagens associate forming fibrils
9. Cross-linking using Lysyl oxidase (Cu-
containing enzyme)
10

❖ Nutritional & genetic disorders can impair collagen
maturation
• Ehlers-Danlos syndrome – can result from
deficiency of collagen-processing enzymes or
mutations in amino acid sequences in collagen
➢ Collagen types affected: I, III, V
➢ Signs and symptoms: mobile joints, increased
risk of vascular disorders, stretchy skin
• Osteogenesis imperfecta (brittle bone
syndrome/ Lobstein disease) – due to a mutation
in the gene coding for the α chains of collagen I;
replacement of glycine with amino acid containing
bulky side chains
➢ Type I: OI tarda
➢ Type II: OI congenita
• Scurvy – affects prolyl and lysyl hydroxylase,
which are involved in collagen synthesis, due to
deficiency in vitamin C
➢ Signs and symptoms: malaise, lethargy,
bleeding gums, poor wound healing, emotional
changes
Chapter 6
PROTEINS: MYOGLOBIN & HEMOGLOBIN
❖ Heme – cyclic tetrapyrrole consisting of four
molecules of pyrrole linked by methyne bridges
• Contains an oxidized iron atom, Fe2+, at the
center of the highly hydrophobic, planar,
porphyrin ring
➢ Fe2+ has six possible coordination bonds
➢ Porphyrin ring has 4 nitrogen atoms that bind
to the iron
➢ Two other coordination positions of the iron
are available for bonding to the histidine of the
protein and a divalent atom
Kevin C. Bolinget
BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.
➢ Other proteins with metal-containing
tetrapyrrole prosthetic groups include the
cytochromes (Fe and Cu) and chlorophyll
(Mg)
• Synthesis occurs partly (begins) in the
mitochondria and partly in the cytoplasm
➢ Pathway is initiated by the synthesis of 5-
aminolevulinic acid (5-ALA) from the amino
acid glycine and succinyl-CoA from the citric
acid cycle (Krebs cycle)
➢ Rate-limiting enzyme responsible for this
reaction, ALA synthase, is negatively regulated
by glucose and heme concentration
❖ Myoglobin – oxygen reservoir in red muscle, which
releases oxygen during deprivation (e.g. severe
exercise) for use in muscle mitochondria for aerobic
synthesis of ATP
• Monomeric
• Compactly folded 153-aminoacyl residue
polypeptide
• About 75% of residues are present in eight right-
handed α helices (A-H)
• Oxygenation of myoglobin is accompanied by the
motion of the iron, of His F8 (proximal histidine),
and of residues linked to His F9 (distal histidine)
• Apomyoglobin provides a hindered environment
for the heme iron
➢ Isolated heme binds carbon monoxide (CO)
25,000 times more strongly than oxygen
• Partial pressure of oxygen needed to achieve half-
saturation of the binding sites (P50) is 1 mm Hg
❖ Hemoglobin – iron-containing oxygen-transport
metalloprotein in red blood cells
• Tetramer of two different polypeptide subunits
➢ α globin chain – chromosome 16
➢ β globin chain – chromosome 11
• Hemoglobin variants are a part of the normal
embryonic and fetal development and can also be
pathologic mutants
➢ α2β2 (HbA) – normal adult hemoglobin
➢ α2γ2 (HbF) – fetal hemoglobin
11

➢α2βS2 (HbS) – sickle cell hemoglobin
➢α2δ2 (HbA2) – a minor adult hemoglobin
➢ζ2ε2 (Hb Gower 1) – embryonic hemoglobin
➢α2β2-gluc (lysyl) (HbA1C) – glycated
hemoglobin
➢ γ4 (Hb Barts) – produced in the
disease alpha-thalassemia
➢ α2β2-CO (HbCO) – carboxyhemoglobin
• Allosteric properties of hemoglobins result from
their quaternary structures
• Oxygenation of hemoglobin triggers
conformational changes in the apoprotein
➢ Hb binds four molecules of O2 per tetramer,
one per heme
➢ A molecule of O2 binds to a hemoglobin
tetramer more readily if other O2 molecules
are already bound (cooperative binding)
• Partial pressure of oxygen needed to achieve half-
saturation of the binding sites (P50) is 26 mm Hg
➢ Oxygen-dissociation curve for myoglobin is
hyperbolic
➢ Oxygen-dissociation curve for hemoglobin is
sigmoidal
• Subunit composition of hemoglobin tetramers
undergoes complex changes during development
➢ Human fetus initially synthesizes ξ2ε2
➢ By the end of the first trimester, ξ and ε
subunits have been replaced by α and γ
subunits, forming HbF (α2γ2), the hemoglobin
of late fetal life
➢ While synthesis of β subunits begins in the
third trimester, the replacement of γ subunits
by β subunits to yield adult HbA (α2β2) does
Kevin C. Bolinget
BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.
not reach completion until some weeks
postpartum
• Oxygenation of hemoglobin is accompanied by
large conformational changes
➢ T (Taut) state – low-affinity, predominant in
the peripheries
➢ R (Relaxed) state – high-affinity of
unoxygenated hemes to O2; predominant in
the lungs
• After releasing O2 at the tissues, hemoglobin
transports CO2 & protons to the lungs
➢ Carries CO2 as carbamates (15% of CO2)
formed with the amino terminal nitrogens of
the polypeptide chain
➢ Carbamate formation changes the charge on
amino terminals from positive to negative,
favoring salt bridge formation between α and
β chains.
➢ Remaining CO2 is carried as bicarbonate,
which is formed in erythrocytes by the
hydration of CO2 to carbonic acid (H2CO3)
➢ Deoxyhemoglobin binds one proton for every
two O2 molecules released, contributing
significantly to the buffering capacity of the
blood
➢ Bohr Effect – reciprocal coupling of proton
and O2 binding
12

o Dependent upon cooperative interactions
between the hemes of the hemoglobin
tetramer
o CO2 generated in peripheral tissues
combines with water to form carbonic
acid, which dissociates into protons and
bicarbonate ions
o Deoxyhemoglobin acts as a buffer by
binding protons and delivering them to the
lungs
o In the lungs, the uptake of oxygen by
hemoglobin releases protons that combine
with bicarbonate ion, forming carbonic
acid, which when dehydrated by carbonic
anhydrase becomes carbon dioxide, which
then is exhaled.
❖ Numerous mutations affecting human hemoglobins
• 2,3-BPG stabilizes the T structure of hemoglobin
➢ 2,3-bisphosphoglycerate – synthesized by
erythrocytes in response to low Po2
o Hemoglobin tetramer binds one molecule
of BPG in the central cavity formed by its
four subunits
o Space between the H helices of the β
chains lining the cavity is sufficiently wide
to accommodate BPG only when
hemoglobin is in the T state
o BPG stabilizes deoxygenated (T-state)
hemoglobin by forming additional salt
bridges that must be broken prior to
conversion to the R state
➢ 2,3-bisphosphogylcerate synthase/2-
phosphatase (BPGM) – catalyzes the
synthesis of BPG from the glycolytic
intermediate 1,3-bisphosphoglycerate
o BPG is hydrolyzed to 3-phosphoglycerate
by the 2-phosphatase activity of BPGM and
to 2-phosphoglycerate by a second
Kevin C. Bolinget
BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.
enzyme, multiple inositol
polyphosphate phosphatase (MIPP)
➢ Physiologic changes that accompany prolonged
exposure to high altitude include increases in
the number of erythrocytes, the
concentration of hemoglobin within them, and
the synthesis of BPG
o Elevated BPG lowers the affinity of HbA
for O2 (increases P50), which enhances the
release of O2 at peripheral tissues
have been identified
• Hemoglobinopathy – mutations in the genes
that encode the α or β subunits of hemoglobin
that compromise its biologic function
➢ More than 7% of the world’s population are
carriers for hemoglobin disorders
• Methemoglobinemia – heme iron is ferric
(Fe3+) rather than ferrous (Fe2+)
➢ Can arise by oxidation of Fe2+ to Fe3+ as a side
effect of agents such as sulfonamides, from
hereditary hemoglobin M (histidine F8 (His
F8) has been replaced by tyrosine), or
consequent to reduced activity of the enzyme
methemoglobin reductase
➢ Congenital methemoglobinemia – due to a
deficiency of NADH-cytochrome b5
reductase
➢ Signs & symptoms: chocolate cyanosis
➢ Treatment: ascorbic acid, methylene blue
• Sickle cell disease – at least one of the beta-
globin subunits in hemoglobin is replaced with
hemoglobin S
➢ Hb S – gene defect is a point mutation in the β
globin gene, which results in glutamate being
substituted by valine at position 6
o nonpolar amino acid valine has replaced
the polar surface residue Glu6 of the β
subunit, generating a hydrophobic “sticky
patch” on the surface of the β subunit of
both oxyHbS and deoxyHbS
13

o Sticky patch is exposed only in deoxyHbs
o At low Po2, deoxyHbs can polymerize to
form long, insoluble fibers that distort the
erythrocyte into a sickle shape
➢ Diagnosis: Hb electrophoresis
➢ Treatment: supportive, transfusion,
hydroxyurea
• Thalassemia - results from the partial or total
absence of one or more α or β chains of
hemoglobin
➢ α Thalassemia – connected to the deletion
of the 16p chromosome
o Result in decreased α-globin production
o Hemoglobin Bart hydrops fetalis
syndrome – characterized by excess fluid
builds up in the body before birth, results
in stillbirth
o HbH disease – milder form (silent type)
➢ β Thalassemia – due to point mutations in
the beta globin (HBB) gene
o β thalassemia major (βo/βo genotype) –
no functional β chains are produced, and
thus no hemoglobin A can be assembled;
most severe form of β-thalassemia
o β thalassemia minor (β/βo or β/β+
genotype) – only one of the two β globin
alleles contains a mutation, so β chain
production is not terribly compromised
and patients may be relatively
asymptomatic
Chapter 7
ENZYMES: MECHANISMS OF ACTION
❖ Enzymes (Gk. enzymos, “leavened”) – substances
that catalyze the conversion of one or more
compounds (substrates) into one or more different
compounds (products)
• Mostly proteins (ribozymes – RNA enzyme)
• Enhance the rates of the corresponding
noncatalyzed reaction by factors of 106 or more
• Neither consumed nor permanently altered as a
consequence of their participation in a reaction
• Aside from being highly efficient, enzymes are
also extremely selective
Kevin C. Bolinget
BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.
• Specific not simply for the type of reaction
catalyzed, but also for a single substrate or a
small set of closely related substrates
➢ Enzymes are stereospecific catalysts that
typically catalyze reactions of only one
stereoisomer of a given compound (e.g. D-
but not L-sugars)
• Can produce chiral products from nonchiral
substrates since they bind to substrates to at
least “three points of attachment”
❖ Enzymes are classified by reaction type
• Traditional names still persist
➢ E.g. pepsin (Gk: pepsis, “digestion”), trypsin
(Gk.: tyrein, “to wear down”)
• International Union of Biochemistry (IUB)
developed an unambiguous system of enzyme
nomenclature in which each enzyme has a unique
name and code number that identify the type of
reaction catalyzed and the substrates involved
1. Oxidoreductases – enzymes that catalyze
oxidations and reductions (transfer of
electrons)
2. Transferases – enzymes that catalyze group
transfer of electrons (moieties)
3. Hydrolases – enzymes that catalyze
hydrolytic cleavage of C–C, C–O, C–N, and
other covalent bonds (transfer of functional
groups to water)
4. Lyases – enzymes that catalyze cleavage of
C–C, C–O, C–N, and other covalent bonds
by atom elimination, generating double bonds
5. Isomerases – enzymes that catalyze
geometric or structural changes within a
molecule
6. Ligases – enzymes that catalyze the joining
together (ligation) of two molecules in
reactions coupled to the hydrolysis of ATP
➢ Example: Hexokinase (IUB name: ATP:D-
hexose 6-phosphotransferase E.C. 2.7.1.1)
o This name identifies hexokinase as a
member of class 2 (transferases), subclass 7
(transfer of a phosphoryl group), sub-
subclass 1 (alcohol is the phosphoryl
acceptor), and “hexose-6” indicates that
the alcohol phosphorylated is on carbon
six of a hexose
❖ Enzymes contain small molecules or metal ions that
participate directly in substrate binding or in catalysis
• Prosthetic groups – usually metal ions, are
tightly and stably incorporated into a protein’s
structure by covalent or noncovalent forces
➢ Metalloenzymes – enzymes that contain
tightly bound Fe, Co, Cu, Mg, Mn, and Zn
14

• Cofactors – commonly metal ions, can associate
either directly with the enzyme or in the form of
a cofactor-substrate complex
➢ Unlike prosthetic groups, cofactors must be
present in the medium surrounding the
enzyme for catalysis to occur
➢ Metal-activated enzymes – enzymes that
require a metal cofactor
• Coenzymes – organic molecules that serve as
recyclable shuttles that transport many substrates
from one point within the cell to another
➢ First stabilize species that are too reactive
➢ Second, serve as an adaptor that facilitates the
recognition and binding of small chemical
groups by their target enzymes
• Holoenzymes – active forms of enzymes that
represent the apoenzyme (inactive form) bound
to its cofactor, coenzyme, or prosthetic groups
• Many coenzymes, cofactors, & prosthetic groups
are derivatives of B vitamins
❖ Enzymes catalysis occurs at the active site
(recognition site for binding substrates)
• Lock and Key model – the lock is the enzyme
and the key is the substrate, that only the
correctly sized key (substrate) fits into the key
hole (active site) of the lock (enzyme), as
postulated by Emil Fischer (1894)
• Induced Fit Theory – assumes that the
substrate plays a role in determining the final
shape of the enzyme and that the enzyme is
partially flexible (proposed by Daniel Koshland)
❖ Catalysts do not affect reaction equilibrium
• Catalyst speed up the forward and back reaction
to the same extent
Kevin C. Bolinget
BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.
• Enzymes enhance reaction rates by lowering
activation energies (the lower the activation
energy for a reaction, the faster the rate)
❖ Enzymes employ multiple mechanisms to facilitate
catalysis
• Catalysis by proximity – for molecules to
interact, they must be in bond-forming distance
of one another
➢ The higher their concentration, the more
frequently they will encounter one another,
and the greater will be the rate of their
reaction
• Acid-base catalysis
➢ Specific acid or base catalysis – reactions
for which the only participating acid or base
are protons or hydroxide ions
➢ General acid catalysis or general base
catalysis – reactions whose rates are
responsive to all the acids or bases present;
mediated by weak acids or bases
➢ Exhibited by HIV protease, an enzyme of the
aspartic protease family
o ➀ Aspartate X acts as a base to activate a
water molecule by abstracting a proton
o ➁ The activated water molecule attacks
the peptide bond, forming a transient
tetrahedral intermediate
o ➂ Aspartate Y acts as an acid to facilitate
breakdown of the tetrahedral intermediate
and release of the split products by
donating a proton to the newly formed
amino group. Subsequent shuttling of the
proton on Asp X to Asp Y restores the
protease to its initial state.
• Covalent catalysis – involves the formation of
a covalent bond between the enzyme and one or
more substrates
15
 BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.

➢ Introduces a new reaction pathway whose Chapter 8

activation energy is lower—and the reaction ENZYME KINETICS
therefore is faster—than the reaction pathway
in homogeneous solution ❖ Enzyme kinetics – quantitative measurement of
➢ Particularly common among enzymes that the rates of enzyme-catalyzed reactions and the
catalyze group transfer reactions systematic study of factors that affect these rates.
➢ Follows a “ping-pong” mechanism—one in • Constitutes a central tool for the analysis,
which the first substrate is bound and its diagnosis, and treatment of the enzymic
product released prior to the binding of the imbalances that underlie numerous human
second substrate diseases
❖ Changes in free energy determine the direction and
equilibrium state of chemical reactions
• Gibbs free energy change (△G) – describes
quantitatively both the direction in which a
chemical reaction will tend to proceed and the
• Catalysis by strain – strain raises the energy of
concentrations of reactants and products that will
molecules, which often decreases the activation
be present at equilibrium
energy for chemical reactions, facilitating catalysis
o ΔG for a chemical reaction equals the sum of
the free energies of formation of the reaction
products ΔGP minus the sum of the free
energies of formation of the substrates ΔGS
o ΔG0 denotes the change in free energy that
accompanies transition from the standard
state, one-molar concentrations of substrates
and products, to equilibrium
❖ Analysis of certain enzymes aids diagnosis o ΔG0′, which defines ΔG0 at a standard state of
Serum Enzyme Major Diagnostic Use 10−7 M protons, pH 7.0
Aminotransferases o If the free energy of formation of the products
Aspartate aminotransferase is lower than that of the substrates, the signs
Myocardial infarction
(AST, or SGOT) of ΔG0 and ΔG0′ will be negative, indicating
Alanine aminotransferase (ALT, that the reaction as written is favored in the
Viral hepatitis
or SGPT)
direction left to right (spontaneous
Amylase Acute pancreatitis
reaction)
Hepatolenticular
Ceruloplasmin degeneration (Wilson ΔG0 = –RT ln Keq
disease) o ΔG is independent of the mechanism of the
0

Muscle disorders and reaction, and provides no information

Creatine kinase
myocardial infarction concerning rates of reactions
γ-Glutamyl transferase Various liver diseases • Reaction equilibria are linked to the standard
Lactate dehydrogenase isozyme free-energy change for the reaction
Liver diseases
5 ➢ A large negative value for ΔG0′ reflects a
Lipase Acute pancreatitis favorable reaction equilibrium
β-Glucoscerebrosidase Gaucher disease ➢ ↑ positivity of ΔG0′ favors substrate
Various bone disorders,
Phosphatase, alkaline (isozymes) stabilization
obstructive liver diseases
❖ Numerous factors affect reaction rate
Serum Enzyme Major Diagnostic Use
Aminotransferases • Kinetic theory (collision theory) – states that
Aspartate aminotransferase for two molecules to react they (1) must
Myocardial infarction approach within bond-forming distance of one
(AST, or SGOT)
Alanine aminotransferase (ALT, another, or “collide,” and (2) must possess
Viral hepatitis
or SGPT) sufficient kinetic energy to overcome the energy
Amylase Acute pancreatitis barrier for reaching the transition state
1. Temperature – raising the ambient
temperature increases the kinetic energy of
molecules (which increases their rapidity of

Kevin C. Bolinget
16

motion, and therefore the frequency with which
they collide)
2. Reactant concentration – frequency with
which molecules collide is directly proportionate
to their concentrations
o ΔG for a chemical reaction equals the sum of
the free energies of formation of the reaction
products ΔGP minus the sum of the free
energies of formation of the substrates ΔGS
3. pH – rate of all enzyme-catalyzed reactions
exhibits a significant dependence on hydrogen ion
concentration
o Most intracellular enzymes exhibit optimal
activity at pH values between 5 to 9
o For acid-base catalysis enzymes, the residues
involved must be in the appropriate state of
protonation for the reaction to proceed
❖ Factors that contribute to the activation energy
1. Entropy – lowering the entropy raises the
activation energy
2. Solvation shell of hydrogen-bonded water
3. Need for substrate distortion and proper
functional group alignment
❖ Catalytic power and specificity of enzymes
• Covalent interactions between enzymes and
substrates lower the activation energy
• Binding energy is a major source of free energy
used by enzymes to lower the activation energies
of reactions
o Derived from the free energy released in
forming many weak bonds
o Weak interactions are optimized in the
reaction transition state
o Binding energy contributes to reaction
specificity and catalysis
• Weak binding interactions between the enzyme
and the substrate provide a substantial driving
force for enzymatic catalysis
Kevin C. Bolinget
BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.
❖ Michaelis-Menten & Hill equations model the effects
of substrate concentration
• Michaelis-Menten equation – illustrates the
relationship between initial velocity (vi)
and substrate concentration [S]
o Michaelis constant (Km) – the
substrate concentration at which vi is half the
maximal velocity (Vmax/2) attainable at a
particular concentration of the enzyme
• Assumptions based on the Michaelis-Menten
equation
o If [S] < Km, the initial reaction velocity is
directly proportional to [S]
o If [S] > Km, reaction velocity is maximum and
unaffected by further increases in[S]
o If [S] = Km, initial velocity is half-maximal
• Lineweaver-Burk equation (Double
Reciprocal Plot) – plot of 1/vi as y as a function
of 1/[S] as x therefore gives a straight line whose
y intercept is 1/Vmax and
whose slope is Km/Vmax
o Useful in distinguishing between certain types
of enzymatic reaction mechanisms
o Useful in analyzing enzyme inhibition
o Increase in Km results to a decreased affinity
• Cooperative binding – exclusive property of
multimeric enzymes that bind substrate at
multiple sites
o Hill equation – describes the behavior of
enzymes that exhibit cooperative binding
17

o Linear form of the Hill equation – used to
evaluate S50, the substrate concentration that
produces half-maximal velocity, and the
degree of cooperativity n (slope of the line)
▪ If n=1, binding sites follow the
Michaelis-Menten kinetics
▪ If n>1, positive cooperativity
❖ Kinetic analysis distinguishes competitive from
noncompetitive inhibition
• Competitive inhibitors – inhibitor binds to the
substrate-binding portion of the active site
❖ Regulation of metabolite flow can be active or
thereby blocking access by the substrate
o Acts by decreasing the number of free enzyme
molecules available to bind substrate, that is,
to form ES, and thus eventually to form
product
❖ Controlling an enzyme that catalyzes a rate-limiting
• Noncompetitive inhibitors – binding of the
inhibitor does not affect binding of the substrate
Kevin C. Bolinget
BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.
• Dixon plot – alternative to the Lineweaver-Burk
plot for determining inhibition constants.
o Initial velocity (vi) is measured at several
concentrations of inhibitor, but at a fixed
concentration of the substrate (S).
Chapter 9
REGULATION OF ENZYME ACTIVITIES
passive
• The substrates for most enzymes are usually
present at a concentration close to their KM. This
facilitates passive control of the rates of product
formation in response to changes in levels of
metabolic intermediates.
• Active control of metabolite flux involves changes
in the concentration, catalytic
activity, or both of an
enzyme that catalyzes a
committed, rate-limiting
reaction
• Metabolite flow tends to be
unidirectional
reaction regulates an entire metabolic pathway
• Rate-limiting reaction – slowest step in a
metabolic pathway or series of chemical
reactions, which determines the overall rate of
the other reactions in the pathway
o Has high activation energy
o Inhibition would lead to reduce metabolite
influx in the entire pathway
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❖ Regulation of enzyme quantity
• Proteins are continuously synthesized and
degraded
o Protein turnover – balance between protein
synthesis and protein turnover
• Control of enzyme synthesis
o Inducers – stimulate the transcription of the
gene that encodes them
▪ Examples of inducible enzymes:
▪ Tryptophan pyrrolase
▪ Threonine dehydratase
▪ Tyrosine-α-ketoglutarate
aminotransferase
▪ Urea cycle enzymes
▪ HMG-CoA reductase
▪ δ-aminolevulinate synthase
▪ cytochrome P450
o Repressors – inhibits the expression of genes
o Transcription factors – stimulate synthesis
of enzymes; activity is controlled by the
interaction of hormones and other
extracellular signals with specific cell-surface
receptors
• Control of enzyme degradation
o Ubiquitin-proteosome pathway –
principal mechanism of protein degradation in
animals
▪ 26S proteasome – site of degradation
▪ Ubiquitination – covalent attachment of
multiple ubiquitin molecules; targets
proteins to the interior of the proteosome
Kevin C. Bolinget
BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.
• Regulation of enzyme activity
a) Covalent modification – principal
mechanism of protein degradation in animals;
binding of dissociable ligands
b) Allosteric modification – binding an
effector molecule at a site other than
the enzyme's active site
c) Association with membranes,
oligonucleotides, or other proteins
❖ Allosteric effectors regulate certain enzymes
• Feedback inhibition – process by which the
end product of a multistep biosynthetic pathway
binds to and inhibits an enzyme catalyzing one of
the early steps in that pathway
o Inhibit the enzyme that catalyzes the first
committed step in a particular biosynthetic
sequence
o Typically bear little or no structural similarity
to the substrates of the enzymes they inhibit
o Negative allosteric effector binds at an
allosteric site, one spatially distinct from the
catalytic site of the target enzyme
o Allosteric enzymes - those for which
catalysis at the active site may be modulated
by the presence of effectors at an allosteric
site
• Feedback regulation – can both be stimulatory or
inhibitory
❖ Many hormones act via second messengers
• Second messengers – specialized allosteric
effectors that elicit the rate of enzyme-catalyzed
reactions within target cells
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 BIOCHEMISTRY (MD-1)
Harper’s Illustrated Biochemistry, 30th ed.

❖ Regulatory covalent modifications can be reversible ❖ Individual regulatory events combine to form
or irreversible sophisticated control networks
• Reversible – refers to the fact that the modified • One well-studied example of such a network is
protein can be restored to its original, the eukaryotic cell cycle that controls cell division
modification-free state, not the mechanism by o Upon emergence from the G0 or quiescent
which restoration takes place. E.g. acetylation, state, the extremely complex process of cell
ADP-ribosylation, methylation, and division proceeds through a series of specific
phosphorylation phases designated G1, S, G2, and M
• Irreversible – activation of proproteins (inactive
precursor proteins) or zymogens (proprotein
form of enzymes)
• Histone code – represents a classic example of
epigenetics, the hereditary transmission of
information by a means other than the sequence
of nucleotides that comprise the genome
❖ Protein phosphorylation is extremely versatile
• Protein phosphorylation-
dephosphorylation – transfer of the terminal
phosphoryl group of ATPs to the hydroxyl
groups of seryl, threonyl, or tyrosyl residues,
forming O-phosphoseryl, O-phosphothreonyl, or
O-phosphotyrosyl residues (catalyzed by protein
kinases)
o Phosphorylation can increase an enzyme’s
catalytic efficiency, converting it to its active
form in one protein, while phosphorylation of
another protein converts it to an intrinsically
inefficient, or inactive form
• Protein phosphatases – hydrolytic removal of
phosphoryl groups (dephosphorylation)

Kevin C. Bolinget
20