Inhibition of Escherichia coli acetyl coenzyme A carboxylase by acyl-acyl carrier protein

Escherichia coli acetyl coenzyme A carboxylase (ACC), the first enzyme of the fatty acid biosynthetic pathway, is inhibited by acylated derivatives of acyl carrier protein (ACP). ACP lacking an acyl moiety does not inhibit ACC. Acylated derivatives of ACP having chain lengths of 6 to 20 carbon atoms were similarly inhibitory at physiologically relevant concentrations. The observed feedback inhibition was specific to the protein moiety, as shown by the inability of the palmitoyl thioester of spinach ACP I to inhibit ACC.     

Rat liver pyruvate carboxylase. Inhibition by chromium nucleotide complexes.

The effect of inert coordination complexes of chromium (III) with various nucleotides on the catalytic activity of rat liver pyruvate carboxylase was determined. The chromium nucleotides are effective initial inhibitors of pyruvate carboxylase and the inhibition becomes more severe with time. The initial rate decreases for several minutes, reaching a new slower rate that is then maintained until considerable net reaction occurs. Incubation of the enzyme with chromium nucleotides in the presence of Mg2+ and HCO3- causes maximal inhibition of the reaction and linear initial rates are then observed. This effect is similar to that found with yeast hexokinase (Dannenberg, K.D., and Cleland, W.W. (1975) Biochemistry 14, 28-39). The specificity of the carboxylase toward the nucleotide complexes suggests that the alpha and beta nucleotide phosphates are as important as the gamma phosphate in binding to the enzyme. A stable pyruvate carboxylase chromium nucleotide complex was not observed. These results are quite different from those found with yeast hexokinase where a stable complex between CrATP, sugar, and enzyme is found and hexokinase appears to be specific toward the beta, gamma phosphates of its nucleotide substrates.     

Decarboxylation of oxalacetate by pyruvate carboxylase

The decarboxylation of oxalacetate by pyruvate carboxylase in the absence of ADP and Pi is stimulated 400-fold by the presence of oxamate, which is an inhibitory analogue of pyruvate. The observation of substrate inhibition when either oxamate or oxalacetate is varied at a fixed concentration of the other indicates that both molecules bind at the same site on the enzyme. The pH profiles for this reaction show no evidence of the involvement of an enzymic acid-base catalyst, suggesting that the proton and CO2 units may be exchanged directly between the reactants (although CO2 sequestered in the active site may be an intermediate in the process). The pH profiles of the full reverse reaction of pyruvate carboxylase in which oxalacetate decarboxylation is coupled to ATP formation and where Pi is the variable substrate do, however, indicate that such an acid-base catalyst is involved in the other partial reaction of the enzyme in proton transfer to and from biotin. The enzyme also displays two oxamate-independent oxalacetate decarboxylating activities, one of which is biotin-dependent and the other is independent of biotin.     

Proteolysis of liver acetyl coenzyme A carboxylase by cathepsin B

Proteolysis of acetyl-CoA carboxylase was examined with cathepsin B. When chicken liver acetyl-CoA carboxylase was incubated with cathepsin B at pH 6.3, the native 220-kDa polypeptide was primarily cleaved into two polypeptides of 125 and 115 kDa, and further degraded to polypeptides of 100-50 kDa.     

A multisubunit acetyl coenzyme A carboxylase from soybean

A multisubunit form of acetyl coenzyme A (CoA) carboxylase (ACCase) from soybean (Glycine max) was characterized. The enzyme catalyzes the formation of malonyl CoA from acetyl CoA, a rate-limiting step in fatty acid biosynthesis. The four known components that constitute plastid ACCase are biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and the alpha- and beta-subunits of carboxyltransferase (alpha- and beta-CT). At least three different cDNAs were isolated from germinating soybean seeds that encode BC, two that encode BCCP, and four that encode alpha-CT. Whereas BC, BCCP, and alpha-CT are products of nuclear genes, the DNA that encodes soybean beta-CT is located in chloroplasts. Translation products from cDNAs for BC, BCCP, and alpha-CT were imported into isolated pea (Pisum sativum) chloroplasts and became integrated into ACCase. Edman microsequence analysis of the subunits after import permitted the identification of the amino-terminal sequence of the mature protein after removal of the transit sequences. Antibodies specific for each of the chloroplast ACCase subunits were generated against products from the cDNAs expressed in bacteria. The antibodies permitted components of ACCase to be followed during fractionation of the chloroplast stroma. Even in the presence of 0.5 M KCl, a complex that contained BC plus BCCP emerged from Sephacryl 400 with an apparent molecular mass greater than about 800 kD. A second complex, which contained alpha- and beta-CT, was also recovered from the column, and it had an apparent molecular mass of greater than about 600 kD. By mixing the two complexes together at appropriate ratios, ACCase enzymatic activity was restored. Even higher ACCase activities were recovered by mixing complexes from pea and soybean. The results demonstrate that the active form of ACCase can be reassembled and that it could form a high-molecular-mass complex.     

Regulation of hepatic acetyl coenzyme A carboxylase by phosphorylation and dephosphorylation

Acetyl CoA carboxylase, in a partially purified preparation, was inactivated by ATP in a time- and temperature-dependent reaction. Adenosine 3′,5′-monophosphate did not affect the inactivation. Further purification separated the carboxylase from a protein fraction which could greatly enhance the inactivation of the enzyme.Inactivation of the enzyme with [γ-³²P]ATP resulted in the incorporation of ³²P which copurified with the enzyme. No label was incorporated when [U-¹⁴C]ATP was used. When carboxylase inactivated by exposure to [γ-³²P]ATP was precipitated with antibody, isotope incorporation into the precipitate paralleled enzyme inactivation. The phosphate was bound to serine and threonine residues by an ester linkage.Sodium fluoride completely inhibited the activation of partially purified enzyme by magnesium ions. Activation by magnesium, accompanied by the release of protein-bound ³²P, was antagonistic to inactivation of the enzyme by ATP.The data presented in this communication are consistent with a mechanism for controlling acetyl CoA carboxylase activity by interconversion between phosphorylated and dephosphorylated forms. Phosphorylation of the enzyme by a portein kinase decreases enzyme activity, whereas dephosphorylation by a protein phosphatase reactivates the enzyme.     

Requirement of acetyl-coenzyme A carboxylase kinase for coenzyme A

Phosphorylation and inactivation of acetyl-coenzyme A (CoA) carboxylase by acetyl-CoA carboxylase kinase in the presence of ATP and Mg2+ requires coenzyme A. Coenzyme A did not enhance the phosphorylation of alternative substrates of the carboxylase kinase such as protamine or histones. Analogs of coenzyme A were also effective in stimulating the inactivation of carboxylase. The KA of CoA for stimulated carboxylase inactivation was 25 microM. The presence of coenzyme A did not alter the Km of the carboxylase kinase for its substrates, ATP and acetyl-CoA carboxylase. Fluorescence binding studies showed that CoA binds to carboxylase but not to the kinase. The KD of CoA binding to carboxylase is 27 microM. These results indicate that coenzyme A, acting on acetyl-CoA carboxylase, may play an important role in the regulation of the covalent modification mechanism for acetyl-CoA carboxylase.