Problem set-2.3 & 2.4

Question 1

globins (myoglobin & hemoglobin) heme group function

Answer 1

-heme group stores and transports oxygen

Question 2

cytochrome heme group function

Answer 2

-carries electrons

Question 3

Why do we need oxygen carrier proteins?

Answer 3

-O2 molecule is nonpolar–>can’t go through aqueous solutions
-can’t diffuse/go long distances

Question 4

Why do oxygen carrier proteins require a heme group to function?

Answer 4

-iron can bind to oxygen well and carry it
-iron is reactive to the body
-heme makes iron less reactive

Question 5

Myoglobin function

Answer 5

-found in tissues
-helps with oxygen diffusion and storage
-found in neurons protects brain from hypoxia and ischemia

Question 6

Hemoglobin function

Answer 6

assists with diffusion of oxygen from the lungs to the rest of
the body.

Question 7

Which has a higher affinity for carbon monoxide, free heme or the heme group in myoglobin?

Answer 7

Free heme has a higher affinity for carbon monoxide, binding with an affinity 20,000x greater than for
O2.

Question 8

How does the residue His64 of myoglobin affect affinity of the protein for carbon monoxide?

Answer 8

His64 sterically hinders CO binding to the heme.

Question 9

What are the functions of His64 and His93 in myoglobin?

Answer 9

-His64/His E7 forms a hydrogen bond with the oxygen molecule in an O2 dimer that is not
interacting with the iron of the heme group.
-His93 (also known as His F8) forms a bond with the iron atom to coordinate the heme group

Question 10

Describe the differences between T and R states of hemoglobin.

Answer 10

T state is the tense state of hemoglobin and occurs in deoxyhemoglobin because the T state is more
stable in the absence of oxygen. It also has a relatively low affinity for oxygen. The R state is relaxed;
this conformation is stabilized by oxygen and therefore occurs in oxygenated hemoglobin. The R state
has a higher affinity for oxygen relative to the T state.

Question 11

How does binding of oxygen to the heme group of T state hemoglobin trigger conformational
changes to convert to hemoglobin to the R state?

Answer 11

In T state hemoglobin the heme group is slightly warped out of planar conformation so that the iron
interacts with a histidine in the F helix. Binding of oxygen pulls the heme back into a more planar
conformation. This shifts the position of the histidine and the rest of the F helix, the movement of
which trigger changes in the ion pairs at the interface between the α1 and β2 subunits that promote
conversion to R state.

Question 12

How does the environment of respiring tissues promote reversion of hemoglobin from the R to T
state?

Answer 12

Respiring tissues have higher CO2 concentrations and lower pH than the lungs, both of which interact
with hemoglobin to stimulate reversion of the high affinity R state hemoglobin back to the T state,
lowering the affinity of hemoglobin for CO2. This is known as the Bohr effect.
Low pH stabilizes the T state because it increases the probability of His residue protonation. Protonated
His residues form ionic interactions that stabilize the T state. CO2 binds to terminal amino groups on
hemoglobin, forming a negatively charged carbamate group. These carbamate groups form ionic
interactions with positively charged amino groups and side chains at the interface between the αβ
dimers to stabilize the T-state.

Question 13

What is an allosteric effect? Briefly give an example of homotropic versus heterotropic allosteric
effects. How do cooperative binding and allosteric effects relate to one another?

Answer 13

An allosteric effect is when binding of a ligand on one site of a protein complex affects binding
properties of another part of a protein complex to either increase or decrease affinity for a second
ligand. If the ligand at both sites is the same, like the two GroES ligands that bind GroEL, it is
homotropic allosteric effect. If the ligand at one site is different from the other site, such as tryptophan
affecting binding to DNA in the Trp repressor, it is a heterotropic allosteric effect. Cooperative binding
occurs when an allosteric effect increases affinity for the second ligand.

Question 14

Why does hemoglobin have a sigmoidal oxygen binding curve?

Answer 14

Hemoglobin initially has a low affinity for oxygen, but transitions to increased affinity as oxygen
concentrations increase due to sequential cooperative binding of oxygen to the individual subunits. This
results in a second higher affinity state. This change in affinity with increased binding due to increased
oxygen concentrations causes the sigmoidal oxygen binding curve for hemoglobin.

Question 15

Why does mutation of Glu6 in the hemoglobin beta subunit cause sickle cell anemia?

Answer 15

Mutation of Glu6 to Val in the hemoglobin beta subunit causes a hydrophobic knob that can fit into
hydrophobic clefts on other subunits to promote aggregation into rigid linear chains of hemoglobin
molecules. These rigid chains deform erythrocytes, which can cause the cells to rupture or become
stuck and clog capillaries.

Question 16

Why do bruises change color as they heal?

Answer 16

The change of color in bruises as they heal is due to breakdown of heme as the leaked red blood cells
are disposed of. Intact heme bond to iron is dark red in color, but is converted by heme oxygenase to
biliverdin, which is green. Biliverdin is reduced to form bilirubin which is yellow.

Question 17

Why is free heme dangerous in vivo?

Answer 17

Free heme is reactive and causes accumulation of reactive oxygen species (ROS). ROS cause oxidative
damage that lipids, proteins and DNA that lead to inflammation and tissue injury that can result in
vascular dysfunction.

Question 18

Free heme is reactive and causes accumulation of reactive oxygen species (ROS). ROS cause oxidative
damage that lipids, proteins and DNA that lead to inflammation and tissue injury that can result in
vascular dysfunction.

Answer 18

Hemozoin are large crystals of heme formed by the malaria-causing parasite Plasmodium falciparum.
Heme groups are linked together as dimers through bonding between the central iron and the
carboxylate side chain of a second heme. These dimers are incorporated into a larger crystal structure
that can reach 100-200 nm in length and contain 80,000 individual heme molecules.
P. falciparum makes hemozoin because during part of its life cycle it proliferates in red blood cells and
damages them, releasing free heme. To protect itself from the toxic effects of the free heme, P.
falciparum sequesters it into the hemozoin crystals.

Question 19

What is the mechanism of action for the anti-malarial drug chloroquinone?

Answer 19

Chloroquinone binds to heme dimers to prevent their incorporation into larger crystals. The
chloroquinone-heme complex prevents heme detoxification, so is highly toxic to P. falciparum cells and
disrupts the cellular membrane resulting in lysis and autodigestion.

Question 20

Describe the effect of enzymes on (a) the activation energy required to form products, (b) the rate
of product formation, and (c) the equilibrium between substrate and product.

Answer 20

a. Enzymes lower the activation energy required to form product
b. Enzymes increase the rate of product formation
c. Enzymes have NO effect on equilibrium between product and substrate

Question 21

What are the three ways that enzymes reduce activation energy for product formation?

Answer 21

Enzymes decrease the activation energy through: (a) providing catalytically active groups for specific
reactions, (b) reduction of entropy by binding substrates in an orientation that facilitates reaction
catalysis, (c) using the differential binding energy of the substrate in its transition state compared to the
normal state

Question 22

What is the difference in affinity of enzymes for substrate versus transition states? Explain why
there is a difference in affinity and why this is important to catalytic activity.

Answer 22

Enzymes (E) have higher affinity for the substrate transition state (TS) than for the substrate (S). This is
because there are more stabilizing weak intermolecular interactions in the E-TS interaction than in the E-
S interaction. This makes conversion of S to TS energetically favorable in the active site pocket, because
formation of each weak intermolecular interaction releases energy, and this one of the primary ways
enzymes decrease activation energy.

Question 23

What are the four essential components of the chymotrypsin active site? What are their
functions?

Answer 23

a) The catalytic triad: site of catalytic activity, contains a His, Ser and Asp. The His activates the Ser by
accepting a proton, leaving an alkoxide ion acts as a nucleophile to form a covalent tetrahedral
intermediate with the substrate. The Asp stabilizes the positive charge of the His residue.
b) The oxyanion hole: forms a hydrogen bond with the negatively charged oxygen atom connected to
the substrate C1 to stabilize the tetrahedral transition state intermediate
c) Substrate binding pocket: forms hydrogen bonds with the substrate peptide backbone in a short
antiparallel beta sheet conformation to stabilize the transition state chain in the binding pocket.
d) Specificity pocket: binds to the substrate residue just prior to the scissile bond and determines
where on the peptide chain the protease cleaves.

Question 24

What are the functions of each residue in the chymotrypsin catalytic triad?

Answer 24

Ser: forms the nucleophilic alkoxide ion which attacks the C1 carbonyl carbon to form the covalent bond
linking the tetrahedral intermediate to the enzyme; hydrogen bonds with the negatively charged oxygen
of the tetrahedral intermediate to stabilize it.
His: (a) acts as a base to accept a proton from Ser, forming the strongly nucleophilic alkoxide ion, (b)
donates a proton to the NH3 leaving group of the first peptide release, (c) deprotonates the water to
form the hydroxide ion that is the nucleophile for the second reaction to break the acyl-enzyme covalent
bond that releases the second peptide.
Asp: stabilizes the positively charged form of His through hydrogen bonding.