Lab 5 and 6

Question 1
1.
a) FCCP - a
If FCCP makes membranes permeable to protons, there will be no proton gradient within
the mitochondria for the electron transport chain. Therefore, more oxygen will be
produced as the protein pumps won’t have to pump protons anymore. Oxygen will be
easily consumed and be produced as water.
b) Malonate - b
If malonate prevents oxidation of succinate, there will be no electrons available for the
electron transport chain and less oxygen will be produced and consumed.
c) Cyanide - b
If cytochrome oxidase is inhibited by cyanide, oxygen will not be converted to water and
so it will just be accumulated and not consumed
d) Attactylate - a
If attactylate inhibits the anti-porter, ATP won’t be transported out of the mitochondria
and ADP won’t be brought into the mitochondria and so the consumption of oxygen will be the
same
e) Oligomycin - a
If attactylate inhibits the anti-porter, ATP won’t be transported out of the mitochondria
and ADP won’t be brought into the mitochondria and so the consumption of oxygen will be the
same
f) Butylmalonate - b
If butylmalonate blocks mitochondrial uptake of succinate there will be no electrons
available for the electron transport chain and less oxygen will be produced and consumed.

2. Compound X inhibits ATP synthesis hence it uncouples ATP synthesis from electron transport chain.
If Compound X inhibits ATP synthesis, this process will be dissociated from electron transport chain.

Question 2

A(2)-A(12)
A(8)-B(5)
B(1)-B(8)

Disulfide bonds can be formed by the introduction of cysteine residues into the variable domains
producing a disulfide-stabilized Fv fragment (dsFv). The formation of a particular disulfide bond has
been said to be directly proportional to the number of residues between the linked cysteines.

Question 3

1. The spontaneous reaction is the forward because the ΔE°' is greater than zero.
2. Calculation:
ΔG° ' =−nF Δ E° '
ΔG° ' =−1× 96.5 kJ /V ×(− 0.32V +0.36 V )
ΔG ° ' =−3.86 kJ
3. This reaction is spontaneous so it can be coupled. The classmate was correct.
4. The proton produced can be used in the electron transport chain which produces ATP. The
reduction of NAD+ to NADH produces ATP.

Question 4

1. Reaction 1 - K’eq < Q substrate-limited because there is less reactant (A) than product (B)
Reaction 2 - K’eq > Q enzyme-limited as there is more reactant (B) than product (C)
Reaction 3 - K’eq >> Q enzyme-limited as there is way more reactant (C) than product (D)

2. Reaction 3 would need a bypass reaction for the reverse reaction since K’eq is way larger than Q. It
will be way harder for the reverse reaction to occur and so a bypass reaction would be needed in
order to attain it.

Question 5

1. The correct statements are: 3 and 5

Glucagon breaks down glycogen to form glucose and glucagon decreased the amount of fructose-2,6-
bisphosphate by conversion to fructose-1,6-bisphosphate.

2. The correct statements are: A, D, and F

Pyruvate + CoA-SH + NAD+ → CO2 + acetyl-CoA + NADH + H+
Pyruvate dehydrogenase is inhibited when one or more of the three following ratios are increased:
ATP/ADP, NADH/NAD+ and acetyl-CoA/CoA

Question 6

The net effect of the malate-aspartate shuttle is purely redox: NADH in the cytosol is oxidized to
NAD+, and NAD+ in the matrix is reduced to NADH. The NAD+ in the cytosol can then be reduced
again by another round of glycolysis, and the NADH in the matrix can be used to pass electrons to the
electron transport chain so ATP can be synthesized. Now here in the case there is an inhibition of this
malate-aspartate shuttle system which will eventually leads to the increment of NADH/NAD+ ratio.
This increment in NADH in the cytosol of the liver favors the reduction of Pyruvate into Lactate. Now
pyruvate is the substrate for gluconeogenesis and as we can see that increased NADH concentration is
favoring the conversion of pyruvate into Lactate. So this means that the inhibition of malate-aspartate
shuttle will increase the concentration of NADH which will favour the conversion of pyruvate into
lactate which will eventually inhibits gluconeogenesis as pyruvate is the substrate for
gluconeogenesis.

Question 7

1. The electron transport chain contains a series of electron carriers located in the inner
mitochondrial membrane. It contains four complexes. NADH donates electrons to complex-I and
drives the pumping of 10 protons from the matrix into the inter-membrane space.
The flow of electrons through ATP synthase complex results in the formation of ATP.
1 NADH = 2.5 ATP

2. FADH2 donates electrons to complex-II. It results in the pumping of only 6 electrons. So, it produces
less ATP as compared to NADH.
1 FADH2 = 1.5 ATP

Question 8

1.
0.578 g
=0.0023mol iodine
254 g/mol
0.680 g
=0.0077 mol oil
884 g/mol

Iodine:oil is 1:3 moles

Since 1 double bond react with 1 mol iodine, 3 double bonds will be needed.
2. Iodine number of the oil
0.578 g
×100 g=85 g
0.680 g

Question 9

1. Normal physiology: Insulin helps in lowering blood glucose levels. Glucagon is a catabolic hormone
and helps in sustaining plasma glucose during fasting conditions by stimulating hepatic glucose
production. In the diabetic state, there is increase in the glucagon levels after a meal, resulting in
elevated hepatic glucose production.In this scenario, when insulin is administered by the diabetic
patient,insulin is unable both to restore normal postprandial insulin concentrations and to suppress
glucagon secretion at once. This results in an abnormally high glucagon-to-insulin ratio due to which
there is production of hepatic glucose. This is how there is further elevation in the levels of blood
glucose in a diabetic patient despite the administration of insulin.

2. A diabetic person naturally has low levels of insulin and hence low levels of glucose in his body.
When the body does not have enough glucose for energy, there is reduction in the glycolysis and the
body burns stored fats instead of glucose.This leads to the formation of ketone bodies (ketones). An
increase in the production of ketone bodies only means that the glucose store is almost depleted and
the body is relying on lysis of fat for energy.Hence, the body is in starvation mode.

Question 10

dADP, dGDP, dCTP, and dUDP are formed under the experiment. The inhibitor of thymidylate
synthase block the activity of this enzyme which would otherwise convert dUDP to dTDP. As an
inhibitor of thymidylate synthase is used, so there will be no dTDP synthesis occurs. All others will be
formed.

Hence by blocking this enzyme lower down the thymidine diphosphate nucleotide concentration
which interrupt the DNA replication. The ribonucleotide reductase is responsible for the reduction of
respective nucleotides into their deoxyribonucleic forms by then which can be considered as
precursors for DNA synthesis. deoxy Uridine diphosphare is also a product of this enzyme which is
further modified by thymidylate synthase to convert into deoxy thymedine diphosphate. But this
convertion is blocked in the above experiment leads to one nucleotide scarce for DNA replication.

Question 11

Part I
1. The Vmax of the reaction is 80 nmol/min because it plateaus from that point on.
2. The Km is the concentration of half the Vmax which is 50 μM.
3.
( 80 nM /min ) ( 43,000 nM )
V 0= =36.99 nM /min
(50,000+ 43,000 nM )

Part II
1. No effect as enzymes do not change the reaction equilibrium, they only accelerate the rate of the
reaction.

It should be taken into consideration that when the reactant is added, or when the substrate is
present which acts as the reaction, the reaction will lead to the formation of products only when it is
stable thermodynamically. From the above reaction, it is clear that the enzyme is acting as a catalyst
without changing the substrate thereby enhancing the rate at which the products are being formed
which depends upon another factor called as the activation energy. Lets consider a particular
substrate mediated reaction needs more energy to proceed to form the produce. This is the activation
energy and the enzymes reduce this activation energy such that less time will be needed to complete
the work of forming the products. In short, the enzyme does not alter the equilibrium state or the
way in which the substrates are involved or the products are formed but decreases the activation
energy thereby enhancing the rate of conversion rather than acting on the reactants

2.
a) Both Km and VMax will remain the same and the ΔG°' will also not change. In case where the
concentration of the substrate is doubled, there will be no increase in the rate of reaction
taking into consideration that the enzyme has already been saturated with the substrate
and hence the Vmax value will not show any change.

The Km value indicates the substrate concentration at the point where the rate of reaction
has reached half of the Vmax value. This value never changes and it depends solely on the
enzyme specifically used for a substrate. Low Km value indicates that there would be more
efficient binding to form the ES complex as compared to high Km value. But once
introduced and the reaction proceeds, the Km value always remain constant.

For the Standard Gibbs free energy state, we should note that the enzymes are not
associated with changing the equilibrium of the reaction which in simple terms means that
it cannot increase the amount of product which is formed after the reaction is complete but
will lead to the formation of the same amount of product but the time required for the
formation will be reduced due to reduction in the activation energy. In case where the
substrate concentration increases, there wont be a further increase in the V max value
although the rate at which the enzyme catalyzes the reaction should also be considered but
in general, there wont be further decrease in the activation energy or the reaction will
remain stable at equilibrium.

b) In case where the concentration of enzyme is doubled, there would be presence of more
active sites for the substrate to bind to and hence the reaction will now proceed in the
forward direction. This indicates that there will be an increase in the rate of reaction
thereby increasing the Vmax value or the maximum rate achieved as the enzyme
concentration corresponds to the rate of reaction.

There would be no effect on the Km value taking into consideration that change in the
enzyme concentration have changes associated with only the rate of reaction. This
indicates that the Km value will remain constant.

In this case the enzyme concentration increases and thus there will be an increase in the
rate of reaction taking into consideration the Km value of the enzyme. But in terms of free
energy change of reaction, this would remain constant and hence there would be no
change. So, Standard Gibbs free energy change would not change taking into consideration
only the activation energy is affected. A negative value although indicates that the products
will be formed and in the above mentioned case this value would be relatively negligible
taking into consideration enzyme catalyzed reaction is occurring. This needs further
mention regarding the points one is considering.

3. For the inhibitor A, the V max value is 2.4 uM/sec and the 1/2 V max value is 1.2 uM/sec which
corresponds to the Km value of 4mM. In this case, it can be seen that the Km value remains the same
although the Vmax value is reduced indicating that the inhibition is a type of non-competitive inhibition.

For inhibitor B, the Vmax value is 3.8 uM/sec and the 1/2 V max value is 1.9 uM/sec and this corresponds
to the Km value of around 14.4 mM. In this case, it can be seen that the V max value remains almost
same but the Km value increases with respect to the Km value when no inhibitor is added and this
implies that the type of inhibition is competitive inhibition.