STUDIES ON THE MECHANISM OF ACTION OF ADENOSINE 5′-TRIPHOSPHATE-CREATINE PHOSPHOTRANSFERASE. INHIBITION BY MANGANESE IONS AND BY P-NITROPHENYL ACETATE

Abstract

Creatine kinase (adenosine 5[image]-triphosphate-creatine phosphotransferase, EC 2.7.3.2) is irreversibly inhibited by incubation with Mn2+ ions. The rate of inhibition varies with the enzyme preparation and the protein concentration, and is stopped but not reversed by the addition of cysteine. Inhibition is markedly enhanced by ATP, ADP and phosphocreatine, but not by creatine or pyrophosphate. A progressive diminution in the rate of the subsequent reaction with iodoacetate indicates that inhibition by Mn2+ ions causes loss of the reactive sulphydryl groups. The results are interpreted to indicate a Mn3+ ion-catalysed oxidation of the sulphydryl groups and to provide further evidence that the sulphydryl groups are in close proximity to the substrate-binding sites. The reaction of p-nitrophenyl acetate with creatine kinase is first-order with respect to both enzyme and inhibitor over the range investigated. The progress curve for the reaction at pH 7[center dot]0 can be divided into an initial reaction, causing loss of activity, and a steady-state catalysis. The inhibition at pH 7[center dot]0 can be attributed mainly to the modification of the reactive sulphydryl groups. Above pH 7[center dot]0 the ionization of new more-rapidly-reacting groups, with pK values of about 9[center dot]0, is revealed whose modification also causes loss of activity. An increased rate of reaction of the sulphydryl groups is excluded and the reacting groups are tentatively identified as [epsilon] -amino groups of lysine. The presence of the complete substrate reaction mixture gives strong protection against inactivation by p-nitrophenyl acetate at pH 9[center dot]0. Single substrates alone or plus Mg2+ ions are without effect. The pH profile of the inhibition process in the presence of a substrate equilibrium mixture reveals that protection is achieved by raising the pK of the reacting groups by 0[center dot]85 pH unit. The results are interpreted to indicate that a conformational change occurs in creatine kinase during the trans-phosphorylation reaction and that the integrity of the groups reacting above pH 7[center dot]0 are essential for the maintenance of conformation.