395G Exam 3
11 Dec 2002
100 points total
1. One-carbon transfers are mediated by the coenzyme tetrahydrofolate (H4folate) in most organisms. However, some Archaebacteria use the structurally related coenzyme,
tetrahydromethanopterin (H4MPT) for the same transfers. An important reaction involving these cofactors is the reduction of the bound one-carbon unit from the CH2 (methylene) oxidation state to the CH3 (methyl) oxidation state, catalyzed by a specific reductase (see reactions A and B). In the mammalian enzyme (reaction A), NADPH is the electron donor for the reduction; in the
Archaebacteria enzyme (reaction B), a different cofactor, F420H2 serves as electron donor. A: CH2-H4folate + NADPH + H+ <==> CH3-H4folate + NADP+ ∆G°' = -39.8 kJ/mol B: CH2-H4MPT + F420H2 <==> CH3-H4MPT + F420 ∆G°' = -5.5 kJ/mol E°' (V) NADP+ + 2H+ + 2 e- <==> NADPH + H+ -0.320 F420 + 2H+ + 2 e- <==> F420H2 -0.350 F = 96494 J.volt-1.mol-1 R = 8.314 J.K-1.mol-1
10 pts. a. Using the values given, calculate the standard redox potential (E°') for the CH2/CH3 couple for each system (i.e. CH2-H4folate/CH3-H4folate vs. CH2-H4MPT/CH3-H4MPT).
First calculate ∆E°' for each reaction: ∆G°' = -n
F
∆E°'
‡
∆E°' = ∆G°'/-n
F
A: ∆E°' = -39.8 kJ/mol/(-2)(96.494 kJ/V/Mol) = 0.206 volts
B: ∆E°' = -5.5 kJ/mol/(-2)(96.494 kJ/V/Mol) = 0.028 volts
Now calculate E°' from ∆E°' = E°'
acceptor- E°'
donorA: CH
2-H
4folate = acceptor; NADPH = donor
0.206 V = E°'
acceptor- (-0.320 V)
E°'
acceptor= 0.206 - 0.320 = - 0.114 V
B: CH
2-H
4MPT = acceptor; F
420H
2= donor
0.028 V = E°'
acceptor- (-0.350 V)
4 pts. b. Why do you think the Archaebacteria evolved to use F420H2 instead of NADPH as the electron donor for the reduction of CH2-H4MPT?
Because the CH
2-H
4MPT/CH
3-H
4MPT redox couple is so much more negative (by ~
200 mV) than the CH
2-H
4folate/CH
3-H
4folate redox couple, NADPH is not a
strong enough reductant to ensure the reduction goes significantly in the direction
of CH
3-H
4MPT - need a stronger reductant = F
420H
22. You have been sent samples of two new metabolic inhibitors for analysis. Using a preparation of isolated liver mitochondria incubated with pyruvate, O2, ADP and Pi you find that addition of
inhibitor A blocks both electron transport and oxidative phosphorylation. When you add
inhibitor B in addition to A, you find to your surprise that electron transport is restored, but not oxidative phosphorylation.
5 pts. a. Classify these inhibitors in regard to their mode of action in electron transport and oxidative phosphorylation and briefly describe where they might be acting.
Inhibitor A = phosphorylation inhibitor Inhibitor B = uncoupler
When A is added alone, both electron transport and oxidative phosphorylation are blocked. As long as the mitochondria are tightly coupled, inhibition of either process will inhibit the other, so it is not possible to distinguish the site of inhibition. Addition of B restores electron transport, but not phosphorylation, suggesting that B is an uncoupler (dissapates the proton gradient). Compound A must be an inhibitor of phosphorylation, at the ATP synthase.
2 pts. b. Name a pair of known inhibitors that would give the same result. A = oligomycin or dicyclohexylcarbodiimide (DCCD)
B = dinitrophenol (DNP)
5 pts. 3. Nigericin is an ionophore that exchanges K+ for H+ across membranes. Predict the effect of
treatment of functioning mitochondria with nigericin on electron transport and ATP synthesis (oxidative phosphorylation). Explain your predictions. What effects would valinomycin, an ionophore that transports K+, but not H+, have on functioning mitochondria?
Ionophores are not pumps, but rather carriers or channels that allow selected ions to flow down their concentration gradients. Nigericin exchanges K+ for H+ ions, thereby discharging the proton
gradient across the inner mitochondrial membrane (allows protons to flow back into
mitochondria in exchange for K+ flowing out). Since the proton gradient is generated by electron
transport and discharged by the ATP synthase-mediated formation of ATP, nigericin uncouples these two processes. I.e., electron transport will continue (or even increase) but ATP synthesis will cease . Valinomycin cannot transport protons, so it will have little effect on either electron
6 pts 4. a. Briefly summarize the mechanism by which light energy is converted to chemical energy in the light reactions of photosynthesis. (Just outline the fundamental processes involved.)
Light absorption by PSI and PSII causes excitation of an electron to its singlet state. Electron is ejected and it enters electron transport chain . Protons are pumped during electron transport through the cytochrome bf complex to produce a proton gradient across the thylakoid membrane, This protonmotive force provides energy for ATP synthesis via ATP synthase. The electrons eventually flow to reduce NADP + to NADPH .
2 pts b. The chemical energy produced by the light reactions resides in two compounds needed to carry out the dark reactions. Name them.
NADPH, ATP
2 pts c. List the ultimate electron donor and final electron acceptor in non-cyclic electron transport of the light reactions.
H2O = ultimate electron donor
NADP = ultimate electron acceptor
5 pts. d. Ordinary sunlight is very “dilute”. Briefly explain how the photosynthetic apparatus deals with this problem.
Each photosynthetic reaction center chlorophyll is associated with a large number of antenna chlorophylls which absorb photons, and pass the energy eventually to the reaction center chlorophyll by exciton or resonance transfer . Thus, the antenna chlorophylls capture photons from "dilute" sunlight and funnel their energy to the reaction center.
5 pts e. Which is the rate-limiting step in the dark reactions? Briefly discuss the manner by which this step is regulated by light.
ribulose-1,5-bisphosphate + CO2 + H20 ‡ 2 3-phosphoglycerate (RUBISCO reaction)
Regulation is indirectly by light:
Enzyme is allosteric and is stimulated by 3 different changes:
1) Increase in pH. When chloroplasts are illuminated, protons transported from stroma to thylakoid space resulting in increase in stroma pH which stimulates enzyme since located on stromal face of thylakoid membrane.
2) Mg+2 stimulates activity - enters stroma as protons leave during illumination 3) NADPH stimulates, which is generated by PSI during illumination.
5 pts. 5. a. Which is the rate-limiting step in the b-oxidation of fatty acids and how is it regulated?
Transport of the fatty acyl CoA substrates into mitochondria via the carnitine
acyltransferase I (CATI) - this enzyme is inhibited by malonyl CoA, an
intermediate of fatty acid synthesis.
5 pts. b. Which is the rate-limiting step in the synthesis of fatty acids and how is it regulated?
Synthesis of malonyl CoA by acetyl CoA carboxylase - this enzyme is activated by
citrate or insulin, and inhibited by palmitoyl CoA or glucagon.
2 pts. 6. Acetate provides all of the carbon atoms for cholesterol, however, it is not the direct precursor. Name the activated compound that serves as the direct precursor.
Acetyl-CoA or mevalonate
2 pts. 7. a. Increased ketogenesis results from a decrease/increase in carbohydrate metabolism coupled to a decrease/increase in fatty acid oxidation (circle the correct word).
3 pts. b. Which reaction, missing in liver, allows peripheral tissues such as brain to utilize acetoacetate as an energy source?
b-oxoacid CoA transferase: acetoacetate + succinyl CoA ‡ acetoacetyl CoA + succinate
4 pts 8. b-oxidation of fatty acids involves the removal of two pairs of electrons in each round. What
electron carriers are used, and at what point(s) do these electrons enter the mitochondrial electron transport chain?
The two oxidation steps are catalyzed by acyl-CoA dehydrogenase, which uses FAD , and 3-hydroxyacyl-CoA dehydrogenase, which uses NAD +. The electrons on the acyl-CoA
dehydrogenase-bound FADH 2 enter the mitochondrial electron transport chain at coenzyme Q
(ubiquinone) via electron transfer flavoprotein (ETF ). The electrons on NADH enter the mitochondrial electron transport chain at complex I (NADH-CoQ reductase ).
9. Answer the following questions using the diagram below.
4 pts a. What intermediate in purine biosynthesis will accumulate in mutant bacteria unable to synthesize the following:
•10-formyltetrahydrofolate glycinamide ribonucleotide (GAR)
•glycine b-5-phosphoribosylamine (PRA)
•aspartate carboxyaminoimidazole ribotide (CAIR)
•glutamine phosphoribosyl pyrophosphate (PRPP)
2 pts b. Which step requires biotin as a coenzyme? none
2 pts c. Which step(s) can be classified as amidotransferases? 1, 4
2 pts d. One step releases a TCA cycle intermediate. Which step is it, and what is the intermediate? Step 8 - fumarate O OH OH O CH2 P O P O-O O O- O-O -2O3P O OH OH NH2 C O CH NH C O O CH H N CH2 NH H N CH2 C O NH2 CH2 O C O C N CH N C C CH N C O C N CH N C C H2N C H O N HN HC N C -OOC N CH N C C HN HC COO-H2N CH2 COO-O C N CH N C C H2N H2N CH2 O -2O3P 2 9 8 7 6 5 4 3 2 1
Formylglycinamide ribotide (FGAR) Phosphoribosyl pyrophosphate (PRPP)
Glycinamide ribotide (GAR)
5-Aminoimidazole-4-(N-succinylocarboxamide) ribotide (SAICAR)
5-Aminoimidazole-4-carboxamide ribotide (AICAR) 5-Formaminoimidazole-4-carboxamide ribotide
Inosine Monophosphate (IMP)
Ribose-5-phosphate Ribose-5-phosphate Ribose-5-phosphate Ribose-5-phosphate Ribose-5-phosphate Ribose-5-phosphate 1 3 4 7 8 9 10 b-5-phosphoribosylamine
2 pts. 10. Which of the following statements about E. coli ribonucleotide reductase is true?
a. electrons are transferred directly from NADPH to the active site thiols b. the reaction mechanism involves the formation of a thiyl free radical
c. an active site histidine free radical and a dinuclear iron form the functional center of the R2 subunit
d. the enzyme contains just two types of nucleotide binding sites: a catalytic site and an allosteric regulatory site (two of each type per a2b2 tetramer)
2 pts 11. The enzymes that catalyze the oxidation of dihydroorotate to orotate differ in prokaryotes and eukaryotes. Name the electron acceptor and the cellular location of the enzyme in mammalian cells.
FAD or Coenzyme Q (CoQ); mitochondria
2 pts 12. Which of the following statements about glutamine amidotransferases is false?
a. ATP serves to force the equilibrium in the amination direction
b. the nucleophilic ammonia produced at the active site attacks a phosphorylated intermediate c. these enzymes can use ammonia directly, but with much higher Km than for glutamine
d. the nucleophilic ammonia is produced from the a-amino group of glutamine
13. 5-Fluorouracil is a widely used chemotherapeutic agent. 2 pts a. Which metabolic reaction is the target of this drug?
Thymidylate synthase: dUMP + CH2-tetrahydrofolate ‡ dTMP + DHF
3 pts b. Why is this reaction required in dividing cells?
dTMP is a required precursor for DNA synthesis (after its phosphorylation to dTTP). Rapidly dividing cells are replicating their DNA, thus requiring continuous production of dNTPs, including dTTP.
6 pts. 14. List three types of metabolic compartmentation, giving an example of each.
1.
Compartmentation within intracellular organelles, e.g. fatty acid synthesis in
cytoplasm vs. fatty acid oxidation in mitochondria.
2.
Distribution of metabolites between free and protein-bound states; e.g.
intracellular folates are virtually all protein-bound - no free folates.
3.
Substrate channeling - local flux of metabolic intermediates through sequential
enzyme reactions; e.g. carbamoyl phosphate synthesis via CPS.
6 pts. 15. A common variant of human methylenetetrahydrofolate reductase (MTHFR) can lead to deficiency in reductase activity, and elevated plasma homocysteine if the patient is homozygous for this polymorphism, even though the mutation (A ‡ V) is quite far from the active site of the enzyme. However, if the patient has adequate nutritional intake of folic acid, plasma homocysteine levels return to normal. Explain these observations.
MTHFR catalyzes the reduction of CH2-THF to CH3-THF, which is the required methyl donor for the
remethylation of homocysteine in the methionine biosynthesis pathway. It has been shown that the A ‡ V variant destabilizes the enzyme, by shifting the equilibrium between active dimer and inactive monomer to the monomer state. However, if the folate substrate is bound to the enzyme, it stabilizes the dimer form, even in the A ‡ V variant. Thus, homozygotes for the variant will have decreased MTHFR activity and elevated homosyteine. However, if they have adequate folic acid in their diets, their MTHFR will remain saturated with substrate, stabilizing the enzyme, and protecting against loss of activity.
