Glutamine synthetase : VI. Mechanism of the dithiol-dependent inhibition by arsenite

Abstract

Dithiol-dependent inhibition by arsenite has been demonstrated in glutamine synthetase (-glutamate: ammonia ligase (ADP), EC 6.3.1.2) obtained from various sources: from Neurospora to mammalian tissues. With a partially purified enzyme preparation from rat liver, [alpha],[omega]-dimercaptoalkanes, heterocyclic and aromatic dimercapto compounds have been compared for their ability to activate the enzyme and to mediate arsenite inhibition. BAL and 1,2-dimercaptoethane proved to be the most effective dithiols in both respects.Equimolar quantities of BAL and arsenite produced the greatest inhibition of glutamine synthetase. Excess arsenite did not cause an additional inhibition, but excess BAL diminished it. Cysteine, though less effective than BAL, also reversed the inhibition.Mapharside inhibited the enzyme at low concentrations and required no dithiol. Inhibition by either mapharside or p-arsenosobenzoate could be prevented more effectively by BAL than by cysteine. Inhibition by the organic arsenicals increased progressively with the length of time of prior incubation with the enzyme; that by arsenite, however, showed little increase. In contrast, prior incubation with o-iodosobenzoate produced a spontaneous reactivation of the enzyme with time.Both Cd2+ and mercurial treatments of glutamine synthetase altered the course of arsenite inhibition. Cd2+ treatment appeared to interfere with arsenite inhibition; mercurial treatment enabled cysteine, as well as BAL to mediate the inhibition.Evidence has been presented to show that the enzyme-arsenite complex dissociates readily. Moreover, kinetic analysis gives a value for K1 as high as Km for hydroxylamine and adenosinetriphosphate. These results have invalidated the currently held assumption that dithiol-dependent arsenite inhibition results in the formation of a relatively stable ring structure with two closely juxtaposed sulfhydryl groups of the enzyme molecule. Instead, a mechanism has been proposed for the dithiol-mediated inhibition by arsenite, which entails reaction with only one sulfhydryl group of glutamine synthetase.