Kinetic studies with phosphotransacetylase: II. The acetylation of arsenate by acetyl coenzyme A

Abstract

1. 1. Phosphotransacetylase (acetyl-CoA:orthophosphate acetyltransferase, EC 2.3.1.8) catalyses the reaction Arsenate + acetyl-CoA → acetyl arsenate + CoA We have studied the kinetics of this process, using a range of Tris buffers, at 25 °C. 2. 2. The kinetic form of the acylation shows that both substrates are adsorbed by the enzyme, probably under Michaelis-Menten conditions. Arsenate is found to be taken up much more strongly than previously believed. Kinetic analysis shows the outline mechanism to be random bimolecular in type, and leads to the values of the various Michaelis parameters as a function of pH. 3. 3. The arsenate dianion (HAsO 4 2−) is bound by an acidic group(s) in the enzyme, p K a > 9. This group engages at least two of the arsenate oxygen atoms since the monoanion (H 2AsO 4 −) is not taken up by the enzyme. 4. 4. Binding of acetyl-CoA involves a group, pK a ⋍ 8.5 in the free enzyme, active in its protonated form. The group, of pK a ⋍ 7.2 in the enzyme-substrate complex, which assists CoA binding in the forward process of the phosphotransacetylase catalysed equilibrium CoA + acetyl phospate ⇌ acetyl-CoA + phospate is not detected in the present arsenate reaction. This suggests that the enzyme-CoA and enzyme-acetyl-CoA adducts have different conformations. 5. 5. The rate of unimolecular transformation, k, of the enzyme-substrate ternary complex is affected by at least two acid-base groups, both of which probably originate in the enzyme. One, active when protonated, has pK a ⋍ 9.0 , and another, active in its basic form, has pK a ⋍ 7.0 . These groups are probably identical with those of similar p K a identified for the forward step of the acetyl phosphate equilibrium above. 6. 6. The acetylation of arsenate appears likely to follow a detailed mechanism which is essentially the microscopic reverse of that of the CoA-acetyl phosphate reaction previously studied. It follows that the acetylation of phosphate should display all the features here revealed for arsenate.