Metabolism: Citric acid cycle

Hand-out for the CBT – version November 2011

This module is based on Chapter 17: Citric Acid Cycle of 'Biochemistry' by Berg, Tymoczko and Stryer, seventh edition (2011). The questions are taken from the self-tests in the Student Companion. The numbering of the questions is the same, except for the first question, which has been moved to the third place.

Pyruvate dehydrogenase

1. The pyruvate dehydrogenase complex is a large, highly integrated complex of several distinct enzymes. What are the potential advantages of such a multienzyme complex with respect to the isolated enzyme components?

2. The pyruvate dehydrogenase complex is a large, highly integrated complex of three distinct enzymes.

A. Give the cofactors for each enzyme. B. Give a role for each cofactor.

cofactor role

pyruvate

dehydrogenase

dihydrolipoyl

transacetylase

dihydrolipoyl

dehydrogenase

C. One of these cofactors also has the role of conveying intermediates to the different enzyme components. Which of these five cofactors has that role?

Citric acid cycle

3. Eukaryotic cells are broken open. The subcellular organelles are separated by zonal ultracentrifugation on a sucrose gradient. In which organelles would the citric acid cycle enzymes be found?

4. A. What is the cofactor required by citrate synthase?

B. Citrate synthase catalyses the reaction between oxaloacetate, acetyl CoA and water. This results in the formation of CoA and citrate. In what order does citrate synthase bind to the substrates?

C. What is the function of the histidine residues at the active site of citrate synthase?

D. What happens to the structure of the enzyme after citryl CoA is formed?

E. Where do the substrates of the enzyme bind to the enzyme?

http://www.proteopedia.org/wiki/index.php/Citrate_Synthase

5. Citrate synthase hydrolyses the thioester bond of citryl CoA. Why doesn't citrate synthase hydrolyse acetyl CoA?

6. A. What cofactors does succinate dehydrogenase contain?

B. Where in the mitochondrion is succinate dehydrogenase positioned?

C. Does succinate dehydrogenase contain an iron-sulphur cluster?

D. Does it also carry out a decarboxylation, just like citrate dehydrogenase?

7. The conversion of malate to oxaloacetate has a ∆Gº' of +29.7 kJ/mol. How is it possible that in the citric acid cycle the reaction proceeds from malate to oxaloacetate?

8. This is a figure of the pyruvate dehydrogenase reaction and the reactions of the citric acid cycle. Complete the figure. Leave room for answers of questions 14 and 17.

E. Indicate the reactions that are oxidations.

G. At which reaction does a substrate-level phosphorylation occur?

I. Which of these reactions require an FAD cofactor?

J. Indicate the reactions that are decarboxylations. 9. Aconitase reacts stereo-specifically, which means that the two carbon atoms from acetyl CoA end up at the bottom of isocitrate.

In an experiment a carbon atom of pyruvate is labelled with 14C. After how many cycles does this carbon atom first appear in released CO2, and in which step?

A. Methyl carbon

Cycle:

Step:

B. Carbonyl carbon

Cycle:

Step:

C. Carboxylate carbon

Cycle:

Step:

10. Consider the citric acid cycle steps between α-ketoglutarate and malate. NADH produced inside the mitochondria will yield about 2.5 ATP by way of oxidative phosphorylation, and FADH2 about 1.5 ATP. How many high-energy phosphate bonds (net ATP molecules) can be generated?

11. The standard free energy change when glucose is converted to 6 CO2 and 6 H2O is much larger than when glucose is converted to two lactate molecules. This has been exploited by metabolic evolution to also yield more ATP.

Net ATP yield: NADH inside mitochondria: 2.5 ATP NADH in cytoplasm: 1.5 ATP FADH2: 1.5 ATP

How many times large is the ATP yield when glucose is oxidized under aerobic conditions when compared with when it is oxidized under anaerobic conditions to lactate? Control

12. Although oxygen does not participate directly in the reactions of the citric acid cycle, the cycle operates only under aerobic conditions. Which compound causes this by having a high concentration?

13. The following questions deal with the pyruvate dehydrogenase complex.

A. If the levels of insulin drop, what will happen to the pyruvate dehydrogenase complex in liver cells?

B. If the ratio NADH/NAD+ decreases, what will happen to the pyruvate dehydrogenase complex?

C. How does pyruvate activate the complex in muscle cells during muscle contraction?

D. By what mechanism is muscle pyruvate dehydrogenase also activated?

14. A. Use the figure of question 8 to write down the answers to the following questions.

B. Indicate the enzymes that are inhibited by ATP.

C. Indicate the enzymes that are inhibited by NADH.

D. Indicate the enzymes that are feedback inhibited by succinyl CoA.

E. Indicate the enzymes that are allosterically activated by ADP.

F. Indicate the enzyme that is regulated by the availability of acetyl CoA and oxaloacetate.

15. Very important factors affecting the flux through the citric acid cycle are the ATP/ADP ratio and the availability of substrates and cycle intermediates. The NADH/NAD+ ratio is however of paramount (i.e. the highest) importance. Why is this? Biosynthetic precursors 16. Degradation of fatty acids generates high-energy phosphate bonds as well.

A. Where do these get generated?

B. Does the citric acid cycle exist in plants and bacteria?

17. The citric acid cycle also provides intermediates for biosynthesis. Use the figure in question 8 to match the biosynthetic products in mammals to their intermediates of the citric cycle (if there is one).

18. An anaplerotic reaction (of Greek origin, meaning to "fill up") is a reaction that leads to the net synthesis, or replenishment, of pathway components.

A. Do mammals need this reaction to replenish metabolites of the citric acid cycle?

B. Which anaplerotic conversion is possible in mammals?

C. Mammals lack the enzymes to convert acetyl CoA into oxaloacetate. Is the pyruvate dehydrogenase reaction operating in reverse part of this anaplerotic reaction?

19. A. Which cofactor does pyruvate carboxylase require?

B. Where does pyruvate decarboxylation take place?

C. What is the allosteric activator for pyruvate carboxylase?

20. Which enzymes have impaired activity in vitamin B1 deficiency?

Glyoxylate cycle

21. Malate synthase, an enzyme of the glyoxylate cycle, catalyses the condensation of glyoxylate with acetyl CoA.

A. Which enzyme of the citric acid cycle carries out a similar reaction?

B. How would you expect the binding of glyoxylate and acetyl CoA to malate synthase to be?

C. Why does malate synthase only bind to acetyl CoA after binding of glyoxylate?

22. All organisms require three- and four-carbon precursor molecules for biosynthesis. Yet bacteria can grow on acetate, whereas mammals cannot. Why is this?

23. The glyoxylate cycle in plants is performed in specialized peroxisomes, called glyoxisomes. The ATP equivalent for one NADH formed during the glyoxylate cycle is about 2.5. Starting with acetyl CoA, what is the approximate yield of high-energy phosphate bonds (net ATP formed) via the glyoxylate route?