Download Metabolism: Citric acid cycle

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?