Citrate activators

Dephospho-acetyl-CoA carboxylase (Low [citrate] activates, high [fatty acyl-CoA] inhibits)... 

Lipogenesis is regulated at the acetyl-CoA carboxylase step by allosteric modifiers, phosphorylation/de-phosphorylation, and induction and repression of enzyme synthesis. Citrate activates the enzyme, and long-chain acyl-CoA inhibits its activity. Insulin activates acetyl-CoA carboxylase whereas glucagon and epinephrine have opposite actions. 

Secondary signals Citrate activates (acetyl-CoA carboxylase). 

Rate-limiting Citrate activates, insulin activates Fatty acid synthase (requires NADPH)... 

Activation of acetyl-CoA carboxylase from rat liver in vitro. Citrate activates the enzyme and converts it to a polymer. Either palmitoyl-CoA or malonyl-CoA can reverse this process. Citrate does not activate or polymerize the E. coli enzyme. 

We already learned that citrate activates the liver etyl-CoA carboxylase and converts it to a high-molecular-eight polymer (see fig. 18.11). The activation by citrate is jpropriate because excess cytosolic citrate is a good indi-ition that the energy needs of the cell are satisfied. Further-ore, citrate is the most important source of cytosolic etyl-CoA as described above and in figure 18.22. 

Bicarbonate dependence Citrate activation Acyl CoA inhibition Malonyl CoA inhibition Highest activity... 

ACC-2 produces malonyl CoA, which inhibits carnitine palmitoyl transferase I, thereby blocking fatty acid entry into the mitochondria. Muscle also contains malonyl CoA decarboxylase, which catalyzes the conversion of malonyl CoA to acetyl CoA and carbon dioxide. Thus, both the synthesis and degradation of malonyl CoA is carefully regulated in muscle cells to balance glucose and fatty acid oxidation. Both allosteric and covalent means of regulation are employed. Citrate activates ACC-2, and phosphorylation of ACC-2 by the adenosine monophosphate (AMP)-activated protein kinase inhibits ACC-2 activity. Phosphorylation of malonyl CoA decarboxylase by the AMP-activated protein kinase activates the enzyme, further enhancing fatty acid oxidation when energy levels are low. 

In 1952, Brady and Gurin reported that fatty acid synthesis from acetate by pigeon liver extracts was markedly stimulated by tricarboxylic acids 10). Subsequent work by various investigators established that acetyl-CoA carboxylase is the site of citrate activation and that acetyl-CoA carboxylase from all animal tissues requires tricarboxylic acids for activation 1, 52, 78-81, 122, 123). 

In spite of some of these uncertainties, the properties of citrate activation of acetyl-CoA carboxylase in vitro as studied extensively by Lane and co-workers leave little doubt as to citrate being a bona fide allosteric positive effector (64). However, attempts to establish... 

Kinetically, citrate activation of acetyl CoA carboxylase results in an increase in the Vmax without affecting the Km values for the substrates, acetyl-CoA, Mg +-ATP, and HCQj (see Ref. 64), Such results are in accord with the observation that citrate activates both half reactions of the carboxylase, i.e., reactions (3) and (4). Since reaction (3) requires no acetyl-CoA, while reaction (4) does not require HCOs", Mg +-ATP, Pi, or ADP, citrate s effect on these reactions could most reasonably be associated with the hiotinyl prosthetic group of the enzyme and an increase in the rate of turnover. 

Fig. 2. Effect of CoA on the sedimentation pattern of citrate-activated acetyl-CoA carboxylase. Partially purified enzyme was preincubated with or without 2 mM citrate in a medium containing 50 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride at 37°C for 30 minutes. The sample containing 2 mM citrate was divided in half after the preincubation, and CoA was added to a final concentration of 0.12 mM for further incubation (5 minutes). Samples were placed on sucrose gradients and subjected to centrifugation. Acetyl-CoA carboxylase was assayed according to the standard assay procedure. , No additions A, 2 mM citrate , 2 mM citrate, and then 0.12 mM CoA for 5 minutes. From Yeh and Kim (133).

Thrombin has been purified by chromatographic techniques on IRC 50 columns. The amino acid composition of some thrombin preparations has been studied. Thrombin prepared from citrate-activated type I prothrombin lacks tryptophan and half cystine whereas thrombin prepared from extrinsic activation of type I prothrombin contains the 19 amino acids found in prothrombin. One molecule of thrombin contains one isoleucine N-terminal and one aspartic C-terminal [9]. 

---