PROBLEM SET 10. ENZYMES OF THE CITRIC ACID CYCLE

 

In Problem set 9, we have predicted the intermediates in three possible “Citric Acid Cycles”. Only one of these produces CO2 and H2O so we concluded that this is the cycle we are looking for. However, we would like to know if the alternative cycles occur. Now that we have the intermediates, we can look in the databases to see if the necessary enzymes have been described. This will also tell us which isomers are formed and which co-enzymes are required. From these data we can draw some further conclusions.

1. The conversion of acetyl-CoA to citric acid is catalyzed by citrate si-synthase (EC 2.3.3.1). The standard free energy for the reaction at pH 7 and 25º is – 7.7 kcal/mol. What can you conclude about the initial condensation reaction?

2. Citrate (pro 3S) lyase (EC 4.1.3.6) catalyzes the reaction:

citrate = acetate + oxaloacetate

The reaction is freely reversible under physiological conditions. (ΔGº’ = -493 cal/mol at 38º C). This provides a way to use free acetate instead of acetyl-CoA.

a. What mechanistic problem does the condensation reaction create?

b. Citrate (proS) lyase has been resolved into two enzymes. Write two reactions that would solve the mechanistic problem of synthesizing citrate from acetate and oxaloacetate.

c. Write a mechanism for second reaction.

3. Look up the enzyme, isocitrate dehydrogenase (NAD+) (EC 1.1.1.41).

a. What does the stereochemistry tell you about the aconitase reaction?

b. Write two different mechanisms for conversion of isocitrate to 2-oxoglutarate and CO2.

4. Look up the enzyme that converts 2-oxoglutatate to succinyl-CoA in mammalian cells..

a. Write a detailed mechanism for the reaction

b. How many different enzymatic activities are required. List  them.

5. Which enzyme catalyzes the conversion of  succinyl-CoA to succinate in mammalian cells?

a. Write a mechanism for the conversion

b. Look up the definition of a “ligase” in the IUBM database. Does this agree with your mechanism?

6. Look up the enzymes for conversion of succinate to fumarate, fumarate to malate and malate to OAA. Which co-enzymes are involved?

7. Based on your predictions and the information you obtained by looking up the enzymes, construct the accepted Citric Acid Cycle. Include all the reactants, the co-enzymes, the names of the enzymes and their EC numbers. Then, write a net reaction for oxidation of acetyl-CoA to CO2 and H2O that agrees with the cycle.

8. How many ATPs or ATP equivalents are formed by the oxidation of acetyl-CoA to CO2 and H2O?

9. How much energy is produced by oxidation of stearic acid to CO2 and H2O?

10. In the last problem set, we predicted three “Citric Acid Cycles”. We have just found all the enzymes for the cycle that explains the oxidation of acetyl-CoA to CO2 and H2O. Do the enzymes for the alternative cycles exist?

Leave a Comment