Choline Acetyltransferase: INHIBITION BY THIOL REAGENTS

Abstract

Abstract Choline acetyltransferase (EC 2.3.1.6) catalyzes the reversible transfer of the acetyl group from acetyl coenzyme A to choline. Previous studies are consistent with the idea that an active site sulfhydryl group reacts with acetyl coenzyme A to form an acetyl-thioenzyme intermediate and coenzyme A (Roskoski, R., Jr. (1973) Biochemistry 12, 3709). Choline then reacts with the acetyl-enzyme to form acetylcholine and the regenerated enzyme. It has been found that the enzyme is inactivated by N-ethylmaleimide with a second order rate constant of 32.5 m-1 s-1 at pH 7.4 at 37°. The enzyme is completely protected against N-ethylmaleimide inactivation by acetyl coenzyme A and is substantially protected by acetylcholine. Choline does not appreciably protect against thiol reagent inactivation. The postulated acetyl-enzyme intermediate, isolated by Sephadex gel filtration, is also resistant to thiol reagent inactivation, but becomes susceptible to inactivation as the acetyl group is transfered from enzyme to substrate. At saturating concentrations of acetyl coenzyme A and choline (during turnover), the enzyme is resistant to inactivation. Decreasing the acetyl coenzyme A concentration during turnover renders the enzyme susceptible to N-ethylmaleimide inactivation. These studies provide further evidence for an active site—SH and for a kinetically significant acetyl-thioenzyme intermediate. The results also suggest that the reaction of choline with the acetylenzyme (deacylation) is rate-limiting during turnover.