Activation of acetate by Methanosarcina thermophila. Purification and characterization of phosphotransacetylase.

Phosphotransacetylase (EC 2.3.1.8) was purified 83-fold to a specific activity of 2.5 mmol of acetyl-CoA synthesized per min/mg of protein from Methanosarcina thermophila cultivated on acetate. This rate was 10-fold greater than the rate of acetyl phosphate synthesis. The native enzyme (Mr 42,000-52,000) was a monomer and was not integral to the membrane. Activity was optimum at pH 7.0, and 35-45 degrees C. The enzyme was stable to air and to temperatures up to 70 degrees C, but was inactivated at higher temperatures. Phosphate and sulfate partially protected against heat inactivation. Potassium or ammonium ion concentrations above 10 mM were required for maximum activity of the purified enzyme; the intracellular potassium concentration of M. thermophila approximated 175 mM. Sodium, phosphate, sulfate, and arsenate ions were inhibitory to enzyme activity. Western blots of cell extracts showed that phosphotransacetylase was synthesized in higher quantity in acetate-grown cells than in methanol-grown cells.

Enhanced activity of 3'-pyrophospho coenzyme A.

3'-Super(pyro)phospho CoA synthesized by enzymic transfer of 5'-beta, alpha-pyrophosphoryl group from dATP to dephospho CoA at adenosine 3'-OH site catalysed by Streptomyces morookaensis ATP nucleotide pyrophosphokinase (EC 2.7.6.4). CoApp was found to be about twice more active than normal CoA as a cofactor of phospho-transacetylases from C. kluyveri, L. mesenteroides and E. coli. Acetyl CoApp was found to be about six times more active than normal acetyl CoA as an allosteric effector with E. coli phosphoenolpyruvate carboxylase. Significance of these findings was discussed.

Irreversible inhibition of phosphotransacetylase by S-dimethylarsino-CoA.

S-Dimethylarsino-CoA was synthesized by acylation of CoA with dimethylchloroarsine. The new analogue of acetyl-CoA was tested as an active-site-directed irreversible inhibitor of phosphotransacetylase (EC 2.3.1.8), carnitine acetyltransferase (EC 2.3.1.7) and citrate synthase (EC 4.1.3.7). Irreversible inhibition was observed only with phosphotransacetylase, which was derivatized via a simple bimolecular process (k2 = 197 +/- 15 min-1 . M-1). Acetyl-CoA provided complete substrate protection against the inactivation, while phosphate (a substrate) and desulfo-CoA (a competitive inhibitor) provided a partial protection. The inactivation was not reversed by dithiothreitol. The new reagent was a linear competitive inhibitor versus acetyl-CoA with both carnitine acetyltransferase (Ki = 41 microM) and citrate synthase (Ki = 20 microM). Chemical studies showed that S-dimethylarsino-CoA reacts with the thiol of N alpha-acetylcysteine but not with the side-chain functional groups of histidine and lysine. The nature of the chemical modification of cysteine was determined by investigating a model system. Thus the chemical reaction between the thioarsenite linkage of S-dimethylarsinobenzylmercaptan and the thiol of cysteine was shown to involve transesterification of the dimethylarsino group.

S-acetonyl-CoA. A nonreactive analog of acetyl-CoA.

We have synthesized S-acetonyl-CoA from CoASH and 1-bromoacetone. This thioether-containing structural analogue of acetyl-CoA is a potent competitive inhibitor, with respect to acetyl-CoA, of citrate synthase, phosphotransacetylase, and carnitine acetyltransferase. This analog will not activate Escherichia coli phosphoenolpyruvate carboxylase or rat liver pyruvate carboxylase, two enzymes which require acetyl-CoA as an obligate activator. Furthermore, acetonyl-CoA will not compete with acetyl-CoA for binding to these enzymes showing the apparent absolute requirement of these two enzymes for a thioester group on the activating ligand. S-Acetonyl-CoA should be a useful reagent in the investigation of acetyl-CoA-requiring processes.