2. In contrast to the toxicity of 2-fluoro-citrate, L-(2-fluoro-)succinate is not an inhibitor of succinate dehydrogenase and the product 2-fluoro-fumarate is converted by fumarate hydratase to L-(2-fluoro-2- hydroxy)malate that breaks down to oxaloacetate. Does the oxidation of 2-fluoro-succinate yield the same number of reducing equivalents to (a) the FAD coenzyme of succinate dehydrogenase as the normal substrate? Write the reaction with structural formulas to illustrate your answer. 2-fluoro-fumarate is a substrate of fumarate hydratase forming only L-(2-fluoro-2-hy- (b) droxy)malate, which breaks down spontaneously to oxaloacetate. Write the equation with structural formulas. Oxaloacetate has been shown to be a very po- (c) tent inhibitor of succinate dehydrogenase. For the enzyme isolated from cardiac muscle the Kj value is ~4 x 10-6M, as shown in the L-B plot on the right. Yet, this inhibition is never discussed in biochemistry textbooks. From your reading in the textbook, suggest two reasons why this in- hibition may not be metabolically consequential in the mi- tochondrion. 0.5 1.0 1.5 2.0 Buccinate (mM) Inhibition of succinate dehydrogenase by oxalo- acetate. O, no oxaloacetate; A, 5.4 µM ox- aloacetate; ●, 10.8 µM oxaloacetate. 1(umole"!min.mg protein)

6. The AG of the citric acid cycle reaction catalyzed by succinyl-CoA synthetase is approximately 0 kJ/mol. Which of the following statements explains why we would expect this reaction to be highly exergonic, but why the overall AG = 0 in reality. A) The reaction cleaves a thioether linkage, and the released free energy is used to generate a new thioester linkage in a product molecule. B) The reaction cleaves a thioester linkage, and the released free energy is used to generate GTP. C) The reaction cleaves a thioester linkage, and the released free energy is used to generate a new thioester linkage in a product molecule. D) None of the above

25. The AG" values for the two reactions are given. 1. 2. oxaloacetate + acetyl-CoA + H₂O → citrate + COASH oxaloacetate + acetate → citrate Enzymes for reactions 1 and 2 are citrate synthase and citrate lyase, respectively. Determine the AG" for the hydrolysis of acetyl-CoA acetyl-CoA + H₂O acetate + COASH + H+ plane een isoimorfbold bns (94) opg Vp1903 9911 AG¹⁰ ? AGO = -32.2 kJ/mol AG"=-1.9 kJ/mol

3. Acetylcholinesterase is a serine hydrolase enzyme im- portant in nerve signal transmission, hydrolyzing acetylcho- line, an ester molecule with a positively charged quaternary nitrogen group. The structure of the physiologically relevant substrate of this enzyme is shown on the right. The quater- H₂C nary nitrogen group serves to anchor the molecule in the active site Gly121 Oxy- anion hole Gly122 Ala204 Substrate ACh His447 Catalytic triad Ser203 Glu202 Glu334 Ser229 CH3 N+ CH3 CH3 The a Scale document down rine protease family, consisting of a catalytic triad Ser203- His447-Glu334 with Ser203 supplying the nucleophilic hydroxyl group and an oxyanion hole com- prised of peptide NH groups of Gly121, Gly122, and Ala204, illustrated in the diagram above, for which carbon (green), nitrogen (blue), and oxygen (red) atoms are shown while hydrogen atoms are white. The enzyme catalyzed reaction can be represented by the following scheme: (a)( k1 K2 E + S = ES K-1 K3 EYE + P where ES…

7. Complete the scheme for isocitrate dehydrogenase. Name the reactant and product. Draw and name the intermediate. COOH HIC H H-C -COOH HO-C-H COOH NAD(P)* NAD(P) H +H+ CO₂ بر COOH HIC-H HIC-H C=O COOH

19. Which of the following statements is true for the shown reactions? i C-S-COA CH₂ H₂C-C-OH CH₂ coo W Coo CH₂ C=0 I Acetyl-CoA CH3 x 214 Coo- 1 CH ₂ I CHOH CH3 Y A. Deficiency of oxaloacetate stimulates the formation of X and y B. Insulin signaling stimulates the formation of X and Y C. Both A and B D. Neither A nor B

3. (а) (coenzyme A), NAD+ (oxidized nicotinamide adenine dinucleotide), FAD (oxidized flavin adenine dinu- cleotide), and a five-membered ring for oxidized lipoamide, and using structural formulas outline the reactions for the oxidative decarboxylation of a-ketoglutarate catalyzed by the aKG dehydrogenase complex. Name the 3 enzymes catalyzing the reactions underlying the conversion of a-ketoglutarate. Using the following abbreviations for cofactors: TPP (thiamine pyrophosphate), COASH

11. Write the mechanism of all three enzymatic steps of the a-ketoglutarate dehydrogenase complex, indicating which steps occur in which enzyme (E1, E2, E3) active sites.

3. The enzyme enolase catalyzes the removal of one water molecule ("dehydration") of 2- Phosphoglycerate (2-PGA). The numbering scheme of the enolase carbons is labeled HC-0-P-O + H20 on 2-PGA. || CH2 HO-CH2 2-Phosphoglycerate Phosphoenolpyruvate Part of the catalytic mechanism is as follows: Mg2+ PO?- Mg?+ PO? 'o. Ó H Mg?+ Mg²+; PO? :-C-H C=C-C-H OH OH Нон Enolase но но H-N*-H Lys345 Lys345 Glu211 Glu211 2-Phosphoglycerate bound to enzyme Enolate intermediate Phosphoenolpyruvate а. Draw the mechanism of the reaction by adding arrows to show how electrons move. Mg2+ PO?- Mg2+ po PO H H Mg2+ С -с-с-н Mg2+ C=c-C-H H OH OH Enolase H. H-N-H но но .. H-N-H Lys345 Lys345 Glu211 Glu211 2-Phosphoglycerate bound to enzyme Enolate intermediate Mg?+ PO O. он Mg2+: PO? c=C-C-H OH HOH H но C=C H-N*-H Lys345 Glu211 Enolate intermediate Phosphoenolpyruvate

3. Trace the course of the radioactive label in 2-[14C] glucose through glycolysis and the citric acid cycle. At what points(s) in the cycle will the radioactivity be released as 14CO2? How many turns of the cycle will be required for complete conversion of the radioactivity to CO2? 4. Repeat problem 3 for pyruvate that is 14C-labeled at its methyl group.