The kinetics of inactivation of adenylosuccinate lyase: evidence for a substrate-induced conformational change

Abstract

The kinetics of inactivation of adenylosuccinate lyase by the alkylating agents N-ethyl maleimide and iodoacetamide and by photooxidation have been investigated. The inactivation by the alkylating agents indicates that there are several groups on the enzyme whose reaction affects activity. The presence of AMP, a product of the enzymic reaction, decreases the rate of inactivation by both N-ethyl maleimide and iodoacetamide, while the other product, fumarate, has no effect on the rate. In the case of photoinactivation, the rate is accelerated by the presence of AMP. Fumarate, which again has no effect by itself, causes an overall protection of the enzyme from photoinactivation when added in the presence of AMP. The results, suggesting that fumarate is unable to combine with the free enzyme but does combine with the enzyme–AMP complex, are consistent with the strongly preferred sequence of product release determined by a previous initial-rate kinetic study. Both the kinetics of inactivation and the initial-rate kinetics may be interpreted in terms of a mechanism involving the operation of an enzymic conformational change which is allowed only when either AMP or adenylosuccinate is bound to the enzyme.