Set 3 - Plant Sciences

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PROBLEM SET 3
TCA cycle
1.
To date this quarter you have encountered reactions involving the cofactors listed below:
biotin
lipoic acid
thiamin pyrophosphate
flavin adenine dinucleotide (FAD)
co-enzyme A
NAD
Indicate the type of reaction that each cofactor is typically involved in.
2.
Suggest a likely or possible enzyme catalyzed reaction sequence whereby α−ketoadipic
acid (an α-keto, six carbon dicarboxylic acid) may be synthesized from acetyl CoA and
α−ketoglutarate. Use structures and show all cofactors (they may be abbreviated)
required. Please base your answer on analogous reactions discussed this quarter.
COOH
|
C=O
|
(CH2)3
|
COOH
3. Malonate inhibits one enzyme of the TCA cycle. If yeast grown aerobically are incubated in
malonate, succinate accumulates. What reaction does malonate inhbit? Write the name of the
enzyme or write the reaction.
4. Suppose you had a patient with a much-elevated level of pyruvate in blood and urine. One
possible explanation is a genetic defect in the enzyme pyruvate dehydrogenase; but another
plausible explanation is a specific vitamin deficiency. Explain first what vitamin might be
deficient in the diet, and why that would account for high urinary excretion of pyruvate. How
would you determine which explanation is correct?
5. In a few words, what is the relationship of the pyruvate dehydrogenase complex reaction to
glycolysis and the citric acid cycle?
6. Briefly describe the possible metabolic fates of pyruvate produced by glycolysis in humans
and explain the circumstances that favor each.
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7. At what point in the citric acid cycle do the methyl carbon from acetyl-CoA and the carbonyl
carbon from oxaloacetate become chemically equivalent?
8. The citric acid cycle is frequently described as the major pathway of aerobic catabolism,
which means that it is an oxygen-dependent degradative process. However, none of the reactions
of the cycle directly involves oxygen as a reactant. Why is the pathway oxygen-dependent?
9. There are a number of human diseases in which one of the enzymes necessary for the
synthesis or breakdown of glycogen is defective. There are few, if any, humans with defects in
the enzymes of the citric acid cycle. Explain this observation in terms of the role of the citric
acid cycle.
10. In 1935, Szent-Gyorgi showed that the production of CO2 in an extract of muscle increased
when succinate was added. For every mole of succinate added, many extra moles of CO2 were
produced. Explain this effect in terms of the known catabolic pathways.
11. You are in charge of designing a new bacterium that will derive all of its ATP from sunlight
by photosynthesis. Will you put the enzymes of the citric acid cycle in this organism? Briefly
explain why or why not?
Suggested problems from the texts:
G&G:
Chapter 20: 5, 6, 7
V,V,&P;
Chapter 16: 1, 3, 6, 7, 8, 9
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Answer Key
1.
Biotin is used in ATP-dependent carboxylation reactions.
Lipoic acid participates in oxidation of hydroxy groups to carbonyls and forms a covalent
adduct with the substrate undergoing oxidation.
thiamin pyrophosphate- (1) decarboxylation of α−keto acids and (2) formation and cleavage
of α−hydroxy ketones
flavin adenine dinucleotide (FAD)- two electron donor and acceptor – 2 hydrogen donor
and acceptor- oxidation/reduction reactions
co-enzyme A- carrier of acyl units, formation of thioester linked acyl units
NAD- hydride donor and acceptor-participates in oxidation reduction reactions.
2.
Use enzymes from TCA cycle:
O
C
O-
C
O
H 2O
CH 3
O
+
CoA
S CoA
(C H 2)2
1
CO O -
acetyl CoA
α -ketoglutarate
O
-O O C
H 2C
C
C
OOH
Homocitric acid
(C H 2)2
CO O -
1. Citrate synthase
2
O
C
O-
C
O
NADH + H
NAD
+
O
-O O C
CH 2
(C H 2)2
+
C O2
HC
2. Aconitase
C
O-
C
H
3. Isocitrate dehydrogenase
O H(C H 2)2
3
CO O -
CO O -
α -ketoadipic acid
3. Since succinate accumulates, this means that the conversion of succinate to fumarate is
blocked. Therefore the enzyme inhibited is succinate dehydrogenase.
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4. The most likely explanation is that the patient has a deficiency of thiamin, without which the
cell cannot make thiamine pyrophosphate, the cofactor for pyruvate dehydrogenase. The
inability to oxidize pyruvate produced by glycolysis to acetyl-CoA would lead to accumulation
of pyruvate in blood and urine. The most direct test for this deficiency is to feed a diet
supplemented with thiamin and determine whether urinary pyruvate levels fall.
5. The pyruvate dehydrogenase complex converts pyruvate, the product of glycolysis, into
acetyl-CoA, the starting material for the citric acid cycle.
6. Pyruvate may be oxidized to acetyl-CoA, which enters the citric acid cycle; this occurs when
the tissue has enough oxygen to recycle the NADH produced by the citric acid cycle. Under
anaerobic conditions, pyruvate is reduced to lactate, recycling NADH to NAD. When an excess
of glucose is obtained in the diet, pyruvate may be oxidized to acetyl-CoA, to be used in the
synthesis of lipids for storage.
7. With succinate.
8. The citric acid cycle produces NADH, which normally is recycled by passage of electrons
from NADH to oxygen via the respiratory chain. With no oxygen to accept electrons from
NADH, the accumulation of NADH effectively stops the citric acid cycle.
9. The citric acid cycle is central to all aerobic energy-yielding metabolism, and also plays a
critical role in biosynthetic reactions by providing precursors. Mutations in the enzymes of the
citric acid cycle are likely to be lethal during fetal development.
10. Succinate acts "catalytically" as an intermediate in the citric acid cycle; it is not consumed
but is regenerated by the operation of the cycle. Its addition to an extract depleted in citric acid
cycle intermediates allows the cycle to resume operating, oxidizing acetyl-CoA to CO2.
11. Even though the citric acid cycle is not needed for catabolic reactions in this organism, the
enzymes of the cycle are still essential. These enzymes produce precursors of amino acids (such
as α−ketoglutarate and oxaloacetate), of heme (succinyl-CoA), and of a variety of other essential
products.
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