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.

Answer 1

Availability of inorganic phosphate is one of the rate limiting steps for several metabolic reactions. The cells maintain a relatively low levels of Pi compared to ATP and ADP concentrations. When Pi is supplied, one of the glycolytic pathways which involve PI, the conversion of glyceraldehyde-3-phosphate to 1,3 bisphosphoglycerate, is accelerated, which results in increased production of citrate.

Likewise, the cell utilizes high energy compounds like creatine phosphate in the presence of high Pi. As a result, TCA cycle is initially accelerated, and once reaces a saturation point, the rate cannot increase further.

2. Availability of reducing agent is a rate limiting step in citric acid cycle. If more NAD+ and FAD and GDP are supplied, the rate of reactions proceed at a faster pae till the enzymes get saturated. Once the enzymes reach their maximum reaction rate, there is no increase in conversion though further acetyl-CoA is added.