The effect of inorganic phosphate on the activity of the citric acid cycle can b

Question

The effect of inorganic phosphate on the activity of the citric acid cycle can be studied in mitochondrial

extracts by monitoring the rate of CO2 production. a. Why is the citric acid cycle dependent upon inorganic phosphate? b. Acetyl CoA is added to a rat liver mitochondrial extract containing excess NAD+, FAD, and GDP. The rate of CO2 production increases and then levels off very quickly; the addition of addition acetyl CoA has no effect. Explain why CO2 production begins but then quickly ceases. c. When potassium phosphate is added to the system, CO2 production resumes but eventually levels off. Explain why CO2 production resumed and why it ceased. d. The experiment in part (c) is repeated with 14C-acetyl CoA. After 2 hours, the production of CO2 has ceased. A sample of the extract (not the CO2 gas!) is analyzed by TLC and the majority of the 14C is found in a single organic molecule. Name this molecule, draw its structure, and explain why it accumulates.

Explanation / Answer

a) Citric acid cycle is dependent on inorganic phosphate, as inorganic phosphate is highly essential for the conversion of succinyl-CoA as succinate. Unless GDP is added up with inorganic phosphate, enough energy will not be produced to remove CoA from succinyl-CoA and to produce succinate. Hence, citric acid cycle is dependent on organic phosphate.

b) Feed back inhibition is the main reason for this inhibition. Initially, when the substrates are added, the cycle operates and produces enough reducing powers, along with saturating amounts of oxalo acetate.

The reducing powers must be recylced for the pathway to continue uninturrupted. The reducing powers formed initially consumes all the available oxygen. Further reducing powers generated acumulate and lead to feedback inhibition.

Another explanation is that when NAD+, FAD, and GDP are added, the cycle operates and the reducing powers generated pass through electron transport chain. ATP is synthesized and its concentration increases with time. High ATP also acts as a feedback regulator, it inhibits the enzyme citrate synthase.

c. Addition of potassium phosphate provides inorganic phosphate required for synthesis of GTP from GDP, by succinate dehydrogenase enzyme. However, accumulation of the GTP -ATP leads to inhibition of the cycle again.

d. Oxalo acetate is the molecule in which the labelled carbon is locked. With each round of citric acid cycle, two labelled Carbons enter the cycle. condensation of oxalo acetate with acetyl coA produces citrate. The newly entered carbons are at position 1 and 2 of the structure. During subsequent reactions, carbons from position 6 and and 4 ar lost. With repeated cycles, the oxaloacetate has labelled carbons over the entire structure.

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