AFFINITY LABELING OF SUCCINYL-COA SYNTHETASE FROM PORCINE HEART AND ESCHERICHIA-COLI WITH OXIDIZED COENZYME-A DISULFIDE

Abstract

Incubation of oxidized coenzyme A disulfide (produced by oxidation of reduced CoA with 1 eq of sodium periodate or of CoA disulfide with 1 eq of peracetic acid) with succinyl-CoA synthetase [EC 6.2.1.4, EC 6.2.1.5] from either porcine heart or E. coli led to the formation of inactive enzyme containing 1 mol of CoA/.alpha..beta. dimer. The bound CoA was attached through a disulfide bond to a SH group of the .beta. subunit. Release of CoA and restoration of activity was achieved by incubation of the modified enzyme with thiols, such as dithiothreitol. Interaction of oxidized CoA disulfide with enzyme was inhibited competitively by disulfo-CoA, which is a competitive inhibitor of the enzyme with respect to CoA. Oxidized CoA disulfide is apparently an affinity label for the CoA binding site of succinyl-CoA synthetase and are the 1st positive results implicating the .beta. subunit in the catalytic mechanism of the enzyme.