Biochemistry 1

• Biochemistry of Amino Acids

• Biochemistry of Proteins
• Portrait of Allosteric Protein
• Enzymes
 Protein Classification
Simple – composed only of amino acid residues

Conjugated – contain prosthetic groups

(metal ions, co-factors, lipids, carbohydrates)
Example: Hemoglobin – Heme
 Protein Classification
One polypeptide chain - monomeric protein
 More than one - multimeric protein
 Homomultimer - one kind of chain
 Heteromultimer - two or more different
chains

(e.g. Hemoglobin is a heterotetramer. It has

two alpha chains and two beta chains.)
 Protein Classification
Globular –
1) polypeptide chains folded into spherical or
globular form
2) water soluble
3) contain several types of secondary structure
4) diverse functions (enzymes, regulatory
proteins)
Fibrous
5) polypeptides arranged in long strands or
sheets
6) water insoluble (lots of hydrophobic AA’s)
7) strong but flexible
8) Structural (keratin, collagen)
 Protein Function
• Catalysis – enzymes
• Structural – keratin
• Transport – hemoglobin
• Trans-membrane transport – Na+/K+ ATPases
• Toxins – rattle snake venom, ricin
• Contractile function – actin, myosin
• Hormones – insulin
• Storage Proteins – seeds and eggs
• Defensive proteins – antibodies
 Globular Proteins
Myoglobin/Hemoglobin
Hemeproteins (Mb & Hb): group of specialized proteins that
contain heme group as a tightly bound prosthetic group.

prosthetic group: is a non-protein compound that is permanently

associated with protein.

The role of heme group is dependent on the environment

created by the three-dimensional structure of the protein.

e.g. heme in cytochrome  electron carrier,

enzyme catalase  active site
Myoglobin and hemoglobin  Oxygen carrier
 Myoglobin/Hemoglobin
 First protein structures
determined
 Oxygen carriers
 Hemoglobin transport O2 from
lungs to tissues
 Myoglobin O2 storage protein
 Structure and function of Hemoglobin
Mb and Hb subunits structurally similar
8 alpha-helices
Contain heme group
Mb monomeric protein
Hb heterotetramer (α22)

- Hb found exclusively in red

blood cells
-transport O2 from lungs to Heme
capillaries of tissues and
transfer CO2 from tissues
to lungs
-composed of 4 polypeptides
held together by non-
covalent interaction
-each subunit is similar to
myoglobin and contains a
heme group myoglobin hemoglobin
 Myoglobin is O2 binding protein found in almost all mammals mainly in
muscles and heart
 Its main function is to store O2 for periods where energy demands is
high, it also increases the rate of transport of oxygen within the muscle
cells.
 Compact structure, 80% of its polypeptide chain is α-helix that labeled
A to h that terminated by proline or by -bends
 The interior of the
myoglobin is composed of
NON-polar a.a. they
packed together stabilized
by hydrophobic
interaction.
 Charged a.a are located at
the surface.
Structure of Heme
Heme is a complex of protoporphyrine IX (4 pyrrole rings linked by
methene bridges) and ferrous iron (Fe+2)
Ferrous ion has 6 coordination bonds: 4 with the N of pyrrole rings
and 2 are perpendicular
one with N of histidine and the other is with O2

protoporphyrine IX

porphyrine
 Heme group Distal histidine: stabilizes the
binding of O2 to heme
 Heme = Fe++ bound to
tertapyrrole ring
(protoporphyrin IX complex)
 Heme non-covalently bound to
globin proteins through His
residue
 O2 binds non-covalently to
heme Fe++, stabilized 
through H-bonding with another
His residue
 Heme group in hydrophobic
crevice of globin protein

Proximal histidine
 Heme group
 Heme = Fe++ bound to
tertapyrrole ring
(protoporphyrin IX complex)
 Heme non-covalently bound
to globin proteins through
His residue
 O2 binds non-covalently to
heme Fe++, stabilized
through H-bonding with
another His residue
 Heme group in hydrophobic
crevice of globin protein
Oxygen binding to
Heme group Distal histidine: stabilizes
the binding of O2 to heme

O2 binds non-covalently to
heme Fe++

Heme
Oxygen

Ferrous ion Proximal histidine

Hb tetramer can be described as two identical dimers, (α)1 and (α)2
The interaction between α and  subunits is strong (hydrophobic, ionic and
hydrogen interactions )
The interaction (α)1 and (α)2 is weak interaction (primary hydrophobic).
The two dimers can move with respect to each other two conformations
according to the presence or absence of O2
 T-form (Taut or tense): the deoxy form of Hb. The two α dimers interact
through a network of ionic bonds and hydrogen bonds that constrain the
movement of the dimer. This form is the low O2 affinity form of Hb
 R-form (relaxed form): the binding of O2 causes rupture of some ionic
bonds and H-bonds  the polypeptide chains have more freedom of
movement. The R-form is the high O2 affinity form of Hb.
Oxygen binding to Myoglobin
• Myglobin has one heme group bind only one oxygen molecule.
• Hemoglobin has 4 heme groups  bind to 4 oxygen molecules
• O2 dissociation curve has hyperbolic shape
• Myglobin has higher
O2 affinity than of

Degree of saturation
Hemoglobin
• P50 of Mb is about 1 Oxygen dissociation curve

mmHg
• P50 for Hb is 26
• P50 is O2 Partial
pressure needed to
half saturation of
the Mb or of Hb
Concentration of Oxygen (Partial pressure)
Oxygen
transport
proteins

Efficient O2
transport protein
should bind to O2 at
high partial pressure
(loading in lung) and
release it (low
affinity) at low
Partial pressure of
(unloading in the
tissue)
 Oxygen Binding Curves

tissues
Mb has hyperbolic O2
binding curve lungs
Mb binds O2 tightly.
Strong-binding
Releases at very low
pO2

Hb has sigmoidal O2 Transition from weak

to strong binding
binding curve
Hb high affinity for
O2 at high pO2 (lungs)
Hb low affinity for O2
at low pO2 (tissues) Weak-binding
 O2 Binding to Hb shows positive cooperativity
• O2 Binding to Hb shows sigmoidal shape low
binding affinity at low con of Oxygen and high Hb
affinity at higher con
• cooperative binding by the four subunit of Hb
•  The binding of one O2 molecule at one heme
group increases the oxygen affinity of the
remaining heme groups in the same hemoglobin
molecule. The affinity of hemoglobin for the last
O2 bound is 300 times greater than its affinity
for the first O2
• O2 affinity increases as each O2 molecule binds
• Increased affinity due to conformation change
• Deoxygenated form = T (tense) form = low affinity Increasing
• Oxygenated form = R (relaxed) form = high affinity for
O2
affinity
• Hemoglobin is efficient in delivering the O2 to the
tissues from lung, myglobin which has hyperbolic
• O2-dissociation curve is unable to do that
Cooperative O2 Binding to Hb
Myoglobin-Oxygen binding
 Allosteric Interactions
• Allosteric interaction occur when specific
molecules bind a protein and modulates activity
• Allosteric modulators or allosteric effectors bind
reversibly to site separate from functional binding
or active site
• Modulation of activity occurs through change in
protein conformation
• 2,3 bisphosphoglycerate (BPG), CO2 and protons
are allosteric effectors of Hb binding of O2
 How is CO2 Exported?
CO2 + H2O  H2CO3  HCO3 + H+
Most of the CO2 produced in metabolism is hydrated and transported as
bicarbonate ion. the hydration of CO 2 by the zinc-dependent enzyme
carbonic anhydrase.
 Binding of Hemoglobin to CO2
Some CO2 is carried as carbamate bound to the uncharged α-amino group
Carbon dioxide is transported in the form of a carbamate on the amino
terminal residues of each of the polypeptide subunits.
Direct binding of CO2 to Hb stabilizes the T- form (deoxy) of Hb 
resulting in a decrease in its affinity for oxygen
The formation of a carbamate also results in release of a proton into
solution  indirectly induces the Bohr effect

O H+

O H H
O
H
H2N C C P rotein C N C C P rotein
O
R O R O

Amino Terminus Carbamate on Amino Terminus

 Bohr Effect
 Increased CO2 leads to
decreased pH
CO2 + H2O <-> HCO3- + H+
 At decreased pH several key
a.a’s protonated, causes Hb to
be converted to T-
conformation (low affinity)
HbO2 + H HbH + O2.
 Deoxy form of Hb has higher
affinity of H than O2
 Protonation of some amino acids
stabilizes the deoxyhemeglobin
(T-form)
 HCO3- combines with N-
terminal α-amino group to form
carbamate group.
 Carbamation stabilizes T-
conformation
 Bisphosphoglycerate (BPG)

 2,3-Bisphosphoglycerate is an
important allosteric effecter of
hemoglobin Binding of BPG to Hb
causes low O2 affinity
 One molecule binds at the
interface of all four subunits,
and makes contacts with the β
-subunits. BPG binds in the
cavity between β-Hb subunits
 Its binding stabilizes the
deoxyhemoglobin state so
stabilizes T-conformation. This
promotes oxygen dissociation
from oxyhemoglobin.
 2,3-Bisphosphoglycerate
 2,3-BPG concentration increases in response to chronic hypoxia
as in pulmonary obstruction or to high altitude or chronic
anemia.
 2,3-BPG is present in erythrocytes at about 5 mM (at sea
level), At high altitudes it is present at 8 mM. Shifts the curve
to the right  increase the O2 delivery to the tissue.
 At high altitudes, wherein the partial pressure of oxygen is
low, one would want hemoglobin to give up more of its bound
oxygen to the tissues
 Fetal Hb (α22) has low affinity for BPG, allows fetus to
compete for O2 with mother’s Hb (α2 β2) in placenta
 Role of 2,3-BPG in transfused blood
Carbon monoxide binding to Hb
 CO binds tightly to one or more of heme iron forming carbon
monoxyhemoglobin (HbCO) and hemoglobin is shifted to R-form 
causing the remaining heme with high O2 affinity  shifts the O2-
binding curve to the hyperbolic (left)  inability of affected
hemoglobin to deliver O2 to the tissue
Formation of methemoglobin:
 oxidation of the heme component of Mb and Hb into ferric (Fe+3)
state form metmyglobin and methmoglobin
 The oxidized heme can't bind the O2
 This oxidation can result from drugs or toxins or from inherited
defects
 Occasional oxidation of heme is corrected by the enzyme NADH-
cytochrome b5 reductase that found in the red blood cell.
 Methemoglobin binds strongly to CN (poison that inhibits the
cytochromal electron transport), so in the case of the cyanide
poisoning amyle nitrite is taken which able to oxidize the heme group
 sequestering the CN
Types of Hemoglobins
There are 4 different types of hemoglobins known:
The most common is Hb A that form 90% of total Hb and consists of α2 β2
Hb F (α22) less than 2%
Hb A2 (α22) 2-5%
Hb A1C (α2 β2-glucose) 3-9 %
Fetal hemoglobin (Hb F): tetramer α22
 Hb F is major Hb in the fetus and newborn. During the last month of
pregnancy , it accounts for 60 % of the total Hb.
 In the first few weeks of pregnancy embryonic Hb is synthesized Hb Gower1
(ζ2ε2) after that the liver starts HbF synthesis. After the development of
the bone marrow the Hb A is synthesized at about the eighth month of the
pregnancy and gradually replaces the Hb F
Binding of the 2,3-BPG to HbF (α22)
 HbF has higher affinity for O2 than dose HbA, bec it has lower binding
affinity to 2,3-BPG and this facilitates the transfer of O2 from maternal
circulation across the placenta to the red blood cells of the fetus
 2 globin chains (HbF) lack some positively charged amino acids found that
found in the β globin (Hb A) reduce the 2,3BPG binding  higher affinity to
O2
Hemoglobin A2 (Hb A2 (α22))
 Hb A2 is a minor component of normal
adult hemoglobin, appear firstly about 12
week after the birth and can form about
2% of the total Hb
Hemoglobin A1c
 Under physiologic conditions HbA is slowly
and non-enzymatically glycosylated
 The extent of glycosylation is dependent
on the plasma level of particular hexoses
 The most abundant glycosylated Hb is
HbA1c which has glucose unit that
covalently linked to amino group of N-
terminal valines of the beta chain
 In the case of Diabetes mellitus, the
amount of HbA1c will increase
Hemoglobinopathies
Defined as a family of disorders caused either by production of
structurally abnormal hemoglobin molecule, synthesis of insufficient
quantities of normal hemoglobin or rarely both
 Sickle- cell anemia (HbS)
 Hemoglobin C disease (HbC)
 Thalassemia
Sickle- cell anemia (Hemoglobin S disease “HbS”)
 a glutamate residue is replaced by valine residue in the β-chains. This
results in two fewer negative charges for the tetrameric structure.
 The substitution of a hydrophobic amino acid for a hydrophilic one makes
the resulting molecule “sticky.” This is because a hydrophobic patch has
been created, which causes molecules to stick together at this point. This
causes aggregation to occur in deoxyhemoglobin.
 Subsequent to strand formation, several strands can assemble to form an
insoluble fiber, which is what gives sickled cells there shape.
 People with sickle cell anemia suffer from repeated crises brought on by
physical exertion.
Hemoglobinopathies
ickle- cell anemia (Hemoglobin S disease “HbS”)
Hemoglobin C disease
 HbC is a hemoglobin variant having a single substitution in the sixth
position of the β-globin chain. In this case lysine is substituted.
 Patients have a relatively mild chronic hemolytic anemia and they don't
suffer from infractive crises
Hemoglobin SC disease
in this disease some β-globin chains have sickle-cell mutation and other β-
globin chains carry mutation found in HbC
Thalassemias
 Thalassemia is a hereditary hemolytic disease in which an imbalance in the
synthesis of globin chains occurs
 Normally the synthesis of α-chains and β-chains are coordinated so that
each α-globin has its β-globin
 in the thalassemia the synthesis of either α- or β-globin chain is
defective
α-thalassemia: defect in the synthesis of the α-globin and there are 4
different levels of this type
β-thalassemia: β-globin is decreased or absent, there are 2 different level
of this type
 Protein Classification
Globular
1) polypeptide chains folded into spherical or
globular form
2) water soluble
3) contain several types of secondary structure
4) diverse functions (enzymes, regulatory
proteins)
Fibrous
5) polypeptides arranged in long strands or
sheets
6) water insoluble (lots of hydrophobic AA’s)
7) strong but flexible
8) Structural (keratin, collagen)
Fibrous proteins

Collagen, elastin and keratin

Functions:
• Structural functions in the body:
• Collagen and elastin are found as component of skin, connective
tissues, sclera and cornea of the eye and blood vessel walls.
• Keratin is found in skin and hair.
• Each fibrous protein has its special mechanical properties resulting from
its unique but relatively simple structure.
• Fibrous proteins consist of specific amino acids arranged into regular
secondary structural elements. This is in contrast to globular proteins
whose structure resulted from a complex interaction of secondary,
tertiary, and sometimes quaternary structural elements.
Collagen
• Collagen is the most abundant protein in the human body
Collagen
• Functions of collagen:
• Collagen found as gel that serves to strengthen the
structures as in the extracellular matrix or vitreous
humor of the eye.
• In other tissues collagen may be bundled in tight
parallel fibers that provide great strength as
tendons.
• In the cornea of the eye, collagen stacked so
transmit light with minimum of scattering.
• Collagen in the bone occurs as fibers arranged at an
angle to each other so as to resist mechanical
shear from any direction.
Structure of collagen

Collagen is formed from three

polypeptides called α-chains which
wrap around each other in a triple
helix forming a rope-like structure.

The three polypeptide chains are

held together by H-bonds.

Variations in amino acids sequence of

the α-chains result in structural
components that are in the same size
(around 1000 amino acids) but
slightly with different properties.

The α-chains are combined to form

the various types of collagen found d
in tissue.
Amino acid sequence:

The primary structure of collagen is unusual in that glycine is found in every third position of
the polypeptide chain, the glycine residue is a part of a repeating sequence –Gly-X-Y where X
is frequently is proline and Y often hydroxyproline or hydroxylysine

Triple-helical structure
• Collagen doesn't fold into a compact structure
• It has an elongated triple-helical structure, the amino acid side chains are placed outside
of the molecule.
• This allow the interaction between triple-helical molecules that lead to aggregation of
collagen monomers into long fiber
Hydroxyproline and hydroxylysine:

These amino acids are rarely found in other proteins and found extensively in collagen

They resulted from hydroxylation of Proline and Lysine after their incorporation into
polypeptide chains. (Posttranslation modification)

Hydroxproline is important in stabilizing the triple-helical structure

Glycosylation

The hydroxyl group of the hydroxylysine residues of collagen maybe glycosylated. Most
commonly glucose and galactose.
 Elastin
Elastin is a connective tissue protein with a rubber like properties. Elastin
fibers like are found at lungs, wall of blood vessels and elastic ligaments.
Elastin can be stretched to several times their normal length but recoil to
their original shape when the stretching force is relaxed.
 Elastin
Structure of elastin
Amino acid composition
 Elastin is composed primarily of small, non-polar amino acid residues as
glycine, alanine and valine.
 Elastin also rich in lysine and proline but little hydroxyproline and no
hydroxylysine.
Interchain cross-link
 Elastin fiber are formed as three dimensional network of cross-linked
polypeptide that have an irregular conformation.
 The cross-link involve lysine. 4 lysine residue from 4 separate chains can be
covalently joined to produce a desmosine cross link  results in inter-
connected, rubbery network that can stretch and bend in any direction when
stressed giving connective tissue its elasticity.
 α-Keratins
• The α-Keratins are proteins that form tough
fibers. They are found in hair, nails and outer
epidermal layer of mammals.
• α-Keratins are also constituents of
intermediate filaments of the cytoskeleton in
certain cells.
• α-Keratins are rich in cysteine  covalent
disulfide cross-links between adjacent
polypeptide chains thus producing fibers that
insoluble and resistant to stretching.
 α-Keratins
• The α-Keratins of hair is an example of a protein constructed
almost of α-helices.
• Hair is composed of dead cells. Each cell is packed with keratin
macrofibrils
• Macrofibriles are formed of microfibril embedded into a protein
matrix. Each microfibril is formed from protofibrils.
• Protofibrils are formed from α-helix protein
 Protein Classification
• Globular:
–Hemoglobin
• Fibrous
–Collagen
The End