BIOCHEMISTRY

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Introduction:
This module is a continuation of the discussion on the structures and
functions of protein.
Objective:
1. To tell the distinguish between the two models of cooperative
behavior; and
2. To know the function of proteins.
PRE-TEST

1. All of the following are considered “weak” interactions in proteins, except:

A) hydrogen bonds.
B) hydrophobic interactions.
C) ionic bonds.
D) peptide bonds.

2. The most important contribution to the stability of a protein’s

conformation appears to be the:

A) entropy increase from the decrease in ordered water molecules

forming a solvent shell around it.
B) maximum entropy increases from ionic interactions between the
ionized amino acids in a protein.
C) sum of free energies of formation of many weak interactions among
the hundreds of amino acids in a protein.
D) sum of free energies of formation of many weak interactions between
its polar amino acids and surrounding water.

3. In an aqueous solution, protein conformation is determined by two major

factors. One is the formation of the maximum number of hydrogen
bonds. The other is the:

A) formation of the maximum number of hydrophilic interactions.

B) maximization of ionic interactions.
C) minimization of entropy by the formation of a water solvent shell
around the protein.
D) placement of hydrophobic amino acid residues within the interior of
the protein.

4. Which of the following statements are NOT true?

A) The pI is the pH value at which a protein has no charges.
B) At a pH value equal to its pI, a protein will not move in the electric
field of an electrophoresis experiment.
C) An acidic protein will have a pI greater than 7.
D) A basic protein will have a pI greater than 7.
5. SDS–polyacrylamide gel electrophoresis and the isoelectric-focusing
method for the separation of proteins have which of the following
characteristics in common? Both

A) separate native proteins.

B) make use of an electrical field.
C)) require a pH gradient.
D) are carried out on supporting gel matrices

6. Which of the following statements concerning the Edman degradation

method are true?

A) Phenyl isothiocyanate is coupled to the amino-terminal residue.

B) Under mildly acidic conditions, the modified peptide is cleaved into a
cyclic derivative of the terminal amino acids and a shortened peptide
(minus the first amino acid).
C) Once the PTH amino acid is separated from the original peptide, a new
cycle of sequential degradation can begin.
D) If a protein has a blocked amino-terminal residue (as does N-formyl
methionine, for example), it cannot react with phenyl isothiocyanate.

7. Which of the following is commonly used as a protecting group during

peptide synthesis?
A) tert-butyloxycarbonyl
B) dicyclohexylcarbodiimide
C) dicyclohexylurea
D) hydrogen fluoride
8. Translation is involved in which of the following possible steps in the flow
of genetic information?

A) DNA to RNA
B) RNA to DNA
C) DNA to DNA
D) RNA to protein

9. If each of the three major classes of RNA found in a cell were hybridized
to denatured DNA from the same cell and the presence of RNA-DNA
hybrids were tested, which of the classes would be retained on the filter?
A) mRNA
B) rRNA
C) tRNA
D)qRNa

10. The following is a partial list of mRNA codons and the amino acids
they encode:
AGU = serine AGC = serine
AAU = asparagine AAC = asparagine
AUG = methionine AUA = isoleucine
Based on this list, which of the following statements are correct?

A) The genetic code is degenerate.

B) The alteration of a single nucleotide in the DNA directing the synthesis
of these codons could lead to the substitution of a serine for an
asparagine in a polypeptide.

C) The alteration of a single nucleotide in the DNA directing the synthesis

of thesecodons would necessarily lead to an amino acid substitution in
the encodedpolypeptide.

D) A tRNA with the anticodon ACU would be bound by a ribosome in the

presence of one of these codons.
Lesson 3. Protein structure and function continued.
Quaternary Structure using Hb as an example and Mb for comparison.
Many proteins are composed of more than one polypeptide chain. You have
already seen this with the collagen. Quaternary structure can provide a basis
for regulation at a distance: allosteric regulation and cooperative behavior.
We will see when studying metabolic pathways, that many of the enzymes that
control the flux through the pathway (rate-limiting step(s)) have quaternary
structures and exhibit cooperative behavior by binding small molecule
metabolites. However, there are many other proteins composed of multiple
polypeptide chains where the chains act independently.
I. Big Picture: Mb and Hb have different physiological roles. Mb is a
monomer and is found in tissues. It functions as a carrier of O2 to the
mitochondrial in a cell. O2 has limited solubility (0.1 mM) and thus a
carrier is required for rates of distribution to be sufficient for
metabolism. O2 is reduced to H20 in the respiratory chain in the
mitochondria and the energy released is used to make the energy
currency of the cell, ATP.
Hb is a tetramer composed of two types of subunits designated α and β. These
subunits are structurally homologous to each other and to Mb (Figure 2). Hb is
found in erythrocytes (red blood cells (RBCs)) and also functions as an O2
carrier. It acquires O2 from air through the lungs and delivers it through the
circulatory system to the tissues where it is picked up by Mb. The surface to
volume ratio of vertebrate cells, in contrast with bacteria, often requires
transporters for small metabolites and Mb, for O2, is an example. The RBCs
also retrieve CO2 from the tissues, the end product of oxidative metabolism,
and deliver it to the lungs where it is exhaled (Figure 1). The RBCs deliver CO2
as part of the H+/HCO3- equilibrium (think about Problem Set 1) and via Hb
itself which is able to form carbamates (-NHCO2-) via reaction of the lysines on
its surface with CO2. These carbamates are acid labile and decompose to CO2
and non-modified Hb. The O2 binding properties of Mb and Hb are distinct and
dependent on quaternary structure. The distinctive bind properties (hyperbolic
versus sigmoidal) are essential for their physiological function (see Figure 4).

Figure 1. Cartoon of O2 transport in vertebrates. Hb transports

O2 to the tissues (muscle). Metabolism in muscle, such as
glucose oxidation, results in CO 2 production and NADH
formation. NADH oxidation is coupled to reduction of O 2 in the
mitochondrial respiratory chain. The energy released from H 2O
formation, generates a proton gradient that can be used for
ATP production. Mb transports O2 to the mitochondria for this
purpose. The resulting CO2, the end product in metabolism, is
returned to the lungs via the blood where it is exhaled.
Structures: Mb and Hb (Figure 2). Note in the case of Hb that there are
multiple subunit interactions: α2β2, α1β1 and β2α1 and α2β1. The movement
between subunits occurs predominantly through the second set of subunit
interfaces.

Figure 2. Crystal structures of myoglobin and hemoglobin. Heme molecule bound is shown
in grey space-filling balls. A. Structure of the monomeric myoglobin. B. Structure of
tetrameric hemoglobin, showing similarity with myoglobin of the β1 subunit. Red and pink
monomers are alpha. Blue and grey monomers are beta.

II. Myoglobin:

A .See the quaternary structures above. Both Mb and Hb reversibly

bind to O2 via Fe2+ in protoporphyrin IX (heme, see Figure 3), a key
cofactor (see Lexicon). An octahedral complex is formed in which
the nitrogens of the pyrrole rings are equatorial ligands and the O2
and a His (in the F helix) are the axial ligands. The protein
structure prevents oxidation of Fe2+ to Fe3+ most of the time. If
oxidation does occur, there is a protein called Cytb5 (a heme
protein) that reduces Fe3+ to Fe2+.
 A, B.

Figure 3. Protoporphyrin IX. A. Protoporphyrin IX with a coordinated Fe 2+ atom. B. Deoxy Hb

(left) and oxy Hb (right) forms, with movement of the iron, porphyrin ring and His upon O 2
binding.

B. O2 binding to Mb can be measured experimentally and the

results of a typical experiment are shown in the graph below. The
rectangular hyperbola describes O2 binding to Mb. The behavior
of Mb (red line) is distinct from that described for Hb (blue and
green lines).

Figure 4. O2 dissociation curves for Mb and Hb in whole blood and their differences. Mb’s O 2
dissociation curve is hyperbolic: Its p50 (the value of pO2 when YO2 = 0.5) is 2.8 torr where 1
torr = 1 mm Hg at 0ºC; 760 torr = 1 atm). Hb exhibits a sigmoidal binding curve and the
amount of O2 bound at 100 torr (arterial pressure) is substantially different from that at venous
pressure (20-30 torr) where the O2 needs to be unloaded.
 C. Mb binding to O2 is described by a standard binding analysis:


Mb + O2 Mb•O2 and the

Kd = [Mb][O2]/[Mb•O2] and YO2 = Mb•O2/[Mb•O2+ Mb]

The binding is described as the fraction of Mb saturated by O 2

that is,

(YO2) = [O2]/ (Kd + [O2]).

Since O2 is a gas, its concentration is expressed as partial pressure of O 2

(pO2). p50 is the partial pressure of O2 when 50% is bound.

III. Hemoglobin

Hemoglobin has four subunits and the difference in its ability to bind O 2
relative to Mb is based on the communications between the subunits.

Historical Digression

Max Perutz (1914 – 2002) began his work on Hb in 1937, and Hb was the first
protein crystallized (1949). It was nearly fifteen years later when the x-ray
structure was solved (1962) and it required Perutz to invent the method of
isomorphous replacement to solve the phase problem. Hb was a first in many
categories, and its rich history is fascinating. Vernon Ingram (MIT Biology)
established for the first time that a “single” mutation of an amino acid (E to V)
is responsible for a genetic disease – sickle cell anaemia. It was Linus Pauling
(again!) who proposed this theory first. Hb was the first system, and still today
is the best studied system, in which cooperativity of ligand binding was
established.End historical digression

Binding of one ligand effects the binding of additional ligands. Based on the
observation of cooperativity of binding, a number of theories
(Monod/Wyman/Changeux; Koshland – covered more below) and a
phenomenological description were put forth to explain the experimental
observations. These theories describe a major mechanism of regulatory
control for key (rate-limiting) enzymes in metabolic pathways.

We will focus on two issues (expanded on below):

1. the basis for the sigmoidal O2 binding curve of Hb (Figure 4).

+
2. the mechanism of allosteric regulation by H (the Bohr
effect) or 2, 3-bisphosphoglyceric acid (BPG – see
structure below).

A. Cooperativity in O2 binding to Hb is displayed in the graph above. One

can observe that in the lungs where the pO 2 is 100 torr, that Hb is
>90% saturated with O2. In the tissue however, where pO2 is 20 to 30
torr, the Hb binds O2 at 50% saturation. Under the same conditions,
the graph also shows that O 2 is tightly bound to Mb. Thus Mb is able
to bind the O2 released from the Hb.

New vocabulary used in the description of cooperative binding of ligands.

 Allostery = binding of a ligand to a specific site, affects binding of the
same ligand or a different ligand to an additional site.

Homotropic = both ligands are the same. In the case of Hb, the ligand is
O2. Heterotropic = the ligands are not the same. O 2 binds and the
allosteric effectors can be

+ -
H (Bohr effect), Cl , CO2, bis-phosphoglyceric acid, BPG.

There are a number of models that describe cooperative behavior: In the case
of hemoglobin, at issue is how the binding of O 2 of one subunit can increase
the binding of O2 to additional subunits. The distances between the hemes in
the subunits are 35 and 27Å!

Monod/Changeux/Wyman model (1) and Koshland/Nemethy/Filmore

model (2) to explain cooperativity.

While these models are mathematically distinct, both intuitively describe the
cooperative behavior and in most cases, it is experimentally difficult to
distinguish between them.

1. In the Monod model described in Figure 5A, T is the tight, or taut,

state (by convention) that weakly binds O2, while R is the relaxed state
that tightly binds O2.
A B
 Figure 5. Monod/Changeux/Wyman model.

Assumptions of the Monod model:

a. The protein must exist in at least two conformations that are in

equilibrium: the R and T states. T is the state where substrate has low
affinity for Hb, while R has high affinity.
b. The states must have multiple equivalent binding sites,
quaternary structure. There are 4 in the case of Hb.
c. The protein is either in the all R or all T states. There are NO mixed
states.

I. Koshland/Nemethy/Filmore KNF sequential model (Figure 6).

Figure 6. Koshland/Nemethy/Filmore model.

Assumptions of the Koshland model (2):

a. The binding affinity to one site influences the binding affinity to a

neighboring site.

b. This model is able to account for negative cooperativity (binding at

one site, decreases the affinity at the second site) that is seen in a

number of enzymatic reactions.

Neither model in its pure form accounts for the experimentally

observed behavior of Hb. A combined model is required.
We have 100s of structures from different species of Hb in the
oxygenated and deoxygenated states. The molecular basis for
interconversion of the R and T states is understood based on
structures and decades of study. The conformational change is
triggered by binding of O 2 to one heme of Hb (Figure 7). Blue is
2+
deoxyHb and red is oxyHb. In the deoxyHb, Fe is too large to fit into
2+
the opening in the porphyrin ring. Upon binding of O 2 to Fe , the size
2+
is reduced and Fe fits into the hole, dragging down the His (Figure
3B) axial ligand (F8) and the F helix along with it as it comes into the
plane of the porphyrin ring.

Figure 7. Conformational changes occur in both the F helix of hemoglobin and the heme upon
oxygen binding. When oxygen is bound, the protein transitions from the deoxygenated
structure (blue) to the oxygenated structure (pink) as the F helix moves down, pulled by its
heme-coordinating His F8.

+
B. Regulation by H or 2, 3-bisphosphoglyceric acid (BPG). We will
focus briefly on the Bohr effect. The diagram below shows the
effect of pH on O2 binding to Hb. The experimental observation
is that at pH 7.2 and 20 torr, more O2 is unloaded than at pH
7.6 (Figure 8). Can we provide an explanation for this
observation based on structures?
 Figure 8. Binding of oxygen to
the heme of hemoglobin is pH
dependent.

To think about the basis of this pH effect you need to recall PS 1 and the
-
CO2/HCO3 equilibria. You also need to know that deoxyHb is a stronger base
than O2•Hb. Finally, in RBCs, there is an enzyme carbonic anhydrase, which
catalyzes the hydration of CO2 by water.

Using these equations, you can think about how to maximize unloading of O 2
in the tissues and unloading of CO2 in the lungs.

First example: In muscle during exercise, glucose is converted to CO 2. The

+
CO2 then diffuses to the capillaries and RBCs and equilibrates to H and
- +
HCO3 . Some of the side chains of Hb act as a buffer. The H s bind to deoxyHb
and thus shift the equilibrium (above graph, right) to the right, releasing more
O2 .

Second example: The deoxyHb is transported through the circulatory system

back to the lungs where it picks up O 2. O2 shifts the equilibrium to the left
+ -
generating H . The protons then react with HCO 3 to generate CO2 and H2O.
+
The CO2 is exhaled. In addition, the H (transient reduction in pH) can react
with the carbamylated-Hb to also release CO2.

Medical Digression - Mutant Hemoglobins

Many mutations in Hb are associated with disease – often anaemia – a lower

number of red blood cells and not enough Hb. The most common disease is
sickle cell anaemia. In the US, there are 70 000 people affected by sickle cell
anaemia, though about 2 million are heterologous for the sickle cell trait. Its
occurrence is roughly 1 in 36 000 Hispanic Americans and 1 in 500 African
Americans. The disease is recessive, requiring two copies of the sickle cell gene
containing the single mutation and is characterized by the C shaped
erythrocytes that tend to get stuck in blood vessels, form clots causing damage
to potentially many organs (bone pain, necrosis, skin ulcers), and are very
fragile compared to wild type (normal) erythrocytes. The lifetime of sickle cell
erythrocytes is about a tenth the length of the wild type cells (10-20 days
instead of ~120 days). It is this sickle cell trait that resistance to malaria
parasites persists. For more information on sickle cell anaemia, see the
webpage on Sickle Cell Diseases at the National Heart, Lung, and Blood
Institute from the National Institute of Health.

End medical digression

Problem Set 3

1. Proteins Are Built from a Repertoire of 20 Amino Acids 1. (a)

Examine the four amino acids given below:

Indicate which of these amino acids are associated with the

following properties:
(a) aliphatic side chain
(b) basic side chain
(c) three ionizable groups
(d) charge of ;1 at pH 7.0
(e) pK ~10 in proteins
(f) secondary amino group
(g) designated by the symbol K
(h) in the same class as phenylalanine
(i) most hydrophobic of the four
(j) side chain capable of forming hydrogen bonds
(b) Name the four amino acids.
(c) Name the other amino acids of the same class as D.

2. Draw the structure of cysteine at pH 1.

3. Match the amino acids in the left column with the appropriate
side chain types in the right column.

(a) Lys (1) nonpolar aliphatic

(b) Glu (2) nonpolar aromatic

(c) Leu (3) basic
(d) Cys (4) acidic
(e) Trp (5) sulfur-containing
(f) Ser (6) hydroxyl-containing

4. How many different dipeptides can be made from the 20 L amino

acids? What are the minimum and the maximum number of pK
values for any dipeptide?
5. For the pentapeptide Glu-Met-Arg-Thr-Gly,
(a) name the carboxyl-terminal residue.
(b) give the number of charged groups at pH 7.
(c) give the net charge at pH 1.
(d) write the sequence using one-letter symbols.
(e) draw the peptide bond between the Thr and Gly residues,
including both side chains.

6. If a polypeptide has 400 amino acid residues, what is its

approximate mass?
(a) 11,000 daltons (c) 44,000 daltons
(b) 22,000 daltons (d) 88,000 daltons

7. Which of the following statements about the peptide bond are

true?
 (a) The peptide bond is planar because of the partial double-
bond character of the bond between the carbonyl carbon and the
nitrogen.
(b) There is relative freedom of rotation of the bond between the
carbonyl carbon and the nitrogen.
(c) The hydrogen that is bonded to the nitrogen atom is trans to
the oxygen of the carbonyl group.
(d) There is no freedom of rotation around the bond between the
a carbon and the carbonyl carbon.

8. Which of the following properties are common to a-helical and b

pleated sheet structures in proteins?
(a) rod shape
(b) hydrogen bonds between main-chain CO and NH groups
(c) axial distance between adjacent amino acids of 3.5 Å
(d) variable numbers of participating amino acid residues

9. Match the levels of protein structures in the left column with the
appropriate descriptions in the right column.
(a) primary (1) association of protein subunits
(b) secondary (2) overall folding of a single
can
chain,
(c) tertiary include a-helical
and b sheet structures
(d) quaternary (3) linear amino acid sequence
(4) repetitive arrangement of
amino acids that are near each
other in the linear sequence
10. Which of the following statements are true?
(a) Ribonuclease (RNase) can be treated with urea and reducing
agents to produce a random coil.
(b) If one oxidizes random-coil RNase in urea, it quickly regains
its enzymatic activity.
(c) If one removes the urea and oxidizes RNase slowly, it will
renature and regain its enzymatic activity.
(d) Although renatured RNase has enzymatic activity, it can be
readily distinguished from native RNase.
Republic of the Philippines
Mountain Province State Polytechnic College
Bontoc, Mountain Province
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