13. REGULATION OF THE CITRIC ACID CYCLE
The citric acid cycle is regulated at multiple points. However, in general it is safe to say that it is inhibited by ATP and NADH. The inhibition by NADH keeps it tightly regulated by oxygen supply, since NADH is converted to NAD+ by oxidative phosphorylation. The inhibition by ATP keeps the citric acid cycle in balance with energy supply. When ATP (energy supply) is high, the citric acid cycle is inhibited and precursors to the citric acid cycle (pyruvate, acetyl CoA and amino acids) are diverted into other pathways.
Acetyl CoA, citrate, and succinyl CoA are the end products of individual steps in the citric acid cycle and their accumulation inhibits the step involved in their production. That, of course, results in inhibition of the cycle as a whole. Finally, Ca++ stimulates the citric acid cycle at several points. This is important because electrical stimulation of the muscle causes an increase in intracellular calcium levels. Thus, during exercise the citric acid cycle will be maximally stimulated in muscle. The regulation of the citric acid cycle is summarized in the very next diagram.
13.1. Citrate synthase reaction
Citrate synthase is controlled by the concentration of acetyl-CoA which is, in turn, governed by the activity of pyruvate dehydrogenase complex.
ATP is an allosteric inhibitor of citrate synthase. ATP increase the Km for acetyl-CoA. Thus reduced the affinity of enzymes towards acetyl-CoA.
High succinyl-CoA levels also decrease the affinity of citrate synthase towards acetyl-CoA.
Citrate is a competitive inhibitor for oxaloacetate on the enzyme. The effect is double-barrelled. An accumulation of citrate raises its concentration as an inhibitor, but it also lowers the concentration of oxaloacetate (OAA) as a substrate. By this double mechanism of inhibition the cycle operates with the same rate.
- Isocitrate dehydrogenase reaction. This reaction appears to be the rate-limiting step of the citric acid cycle.
Isocitrate dehydrogenase is allosterically stimulated by ADP whereas NADPH is an allosteric inhibitor.
- -ketoglutarate dehydrogenase reaction.
Succinyl-CoA and NADH inhibit the activity of a-ketoglutarate dehydrogenase. Succinyl-CoA is a competitive inhibitor for coenzyme A, which is a substrate for KDH. Here again is a double-barrelled effect. An accumulation of succinyl-CoA concentration raises its concentration as an inhibitor, but it also lowers the concentration of coenzyme A as a substrate. By this double mechanism of inhibition the cycle operates with the same rate and thus leading to still more effective inhibition.
13.2. Anaplerotic reactions
Anaplerotic means replenishing or “filling-up” reactions. Many intermediates of the citric acid cycle are used for the synthesis of other substances. Anaplerosis is defined as any reaction that can restore the concentration of a crucial but depleted intermediate. Several biosynthetic pathways utilize citric acid cycle intermediates as starting materials. The citric acid cycle is therefore amphibolic (both anabolic and catabolic).
The TCA cycle in the liver is often called an “open cycle” because there is such a high efflux of intermediates. After a high carbohydrate meal, citrate efflux and cleavage to acetyl CoA provides acetyl units for cytosolic fatty acid synthesis. During fasting, gluconeogenic precursors are converted to malate, which leaves the mitochondria for cytosolic gluconeogenesis.
Some important anaplerotic reactions of the citric acid cycle intermediate are used for the synthesis of following substrate.
Pyruvate carboxylase is a major anaplerotic enzyme. The citric acid cycle operates when oxaloacetate is available. In animal tissues especially, liver and kidney, oxaloacetate is formed by the carboxylation of pyruvate by pyruvate carboxylase in mitochondria. It is a readily reversible reaction. This enzyme is absent in mammals.
Pyruvate carboxylase is homotetramer. Each subunit of Pyruvate carboxylase contains an active site that has tightly-bound Mn+2 (or Mg+2) and covalently-bound biotin and also an allosteric site, which binds acetyl-CoA. The lysine residue of active site is covalently attached with biotin. Pyruvate carboxylase also requires a monovalent cation (K+) apart from a divalent cation (Mg+2 or Mn+2).
Pyruvate carboxylase catalyzes the addition of CO2 to pyruvate to form oxaloacetate.
The biotin of pyruvate carboxylase enzymes forms a covalent intermediate with CO2 in a reaction requiring ATP and Mg+2. The activated CO2 is then transferred to pyruvate to form the carboxyl group of Oxaloacetate.
Pyruvate carboxylase is activated by acetyl CoA and inhibited by high concentrations of many acyl CoA derivatives. As the concentration of oxaloacetate is depleted through the efflux of TCA cycle intermediates, the rate of the citrate synthase reaction decreases and acetyl CoA concentration rises. The acetyl CoA then activates pyruvate carboxylase to synthesize more oxaloacetate.