The reaction of organophosphorus compounds with hydrolytic enzymeS—II.: The inhibition of citrus acetylesterase

Abstract

A purification method, using acetone precipitation, and a simple colonmetric assay method for citrus acetylesterase, using 2,6-dichlorobenzenonejndophenylacetate as a substrate are described. Reaction rate constants have been determined of the inhibition of citrus acetylesterase at pH 7.5 and 25° by a number of organophosphorus compounds of the general formula R′R″. P(O)X. There is a correlation between the rate of enzyme inhibition and the strength of the P—X bond as expressed by alkaline hydrolysis and p K a values of HX. The influence of the structure of the groups R′ and R″ on the rate of inhibition is parallel to that of the reactivity measured as the rate of alkaline hydrolysis but a number of exceptions occur. In a series of alkyl p-nitrophenyl methylphosphonates the rate of enzyine inhibition increases when the alkyi chain is lengthened whereas the rate of the alkaline hydrolysis decreases. In one case the influence of the temperature on the reaction rate has been investigated. It seems that the rapid reaction of the compound with the enzyme, compared With alkaline hydrolysis is caused by a lowering of the activation enthalpy rather than by an increase of the activation entropy. In a number of cases the influence of the pH on the inhibition rate was studied. This influence was found to be rather small.