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Phosphotransacetylase from Bacillus subtilis: Purification and physiological studies

1973; Elsevier BV; Volume: 321; Issue: 1 Linguagem: Inglês

10.1016/0005-2744(73)90065-x

1878-1454

Thomas A. Rado, James A. Hoch,

Enzyme Catalysis and Immobilization

Phosphotransacetylase (acetyl CoA:orthophosphate acetyl transferase, EC 2.3.1.8) was purified over 600-fold from crude extracts of Bacillus Subtilis. The purified enzyme was activated by K1 and NH4+ and inhibited by Mn2+ and Ca2+. Apparent Michaelis constants for the substrates were determined. The equilibrium constant of the reaction was found to favor the formation of acetyl-CoA. The enzyme was capable of cleaving the short chain fatty acid esters propionyl-CoA and butyryl-CoA but the V and affinity for these substrates were less than that observed for acetyl-CoA. The enzyme was unable to cleave succinyl-CoA or palmityl-CoA but palmityl-CoA was a competitive inhibitor, with respect to CoA-SH, of the reaction. The adenine nucleotides, ATP, ADP, and AMP, were inhibitory at 1.0 mM concentration. No inhibition by pyruvate or the reduced pyridine nucleotides was observed. A molecular weight of 90 000 was calculated for the enzyme from chromatography on Sephadex G-150 columns. Physiological studies revealed that maximal enzyme activity was attained during the exponentail phase of growth. Studies with citric acid cycle mutants were consistent with the conclusion that phosphotransacetylase is not regulated with the citric acid cycle.