Affinity labeling of succinyl-CoA synthetase from porcine heart and Escherichia coli with oxidized coenzyme A disulfide.
Incubation of oxidized coenzyme A disulfide (produced by oxidation of reduced CoA with 1 eq of sodium periodiate or of CoA disulfide with 1 eq of peracetic acid) with succinyl-CoA disulfide with 1 eq of peracetic acid) with succinyl-CoA synthetase from either porcine heart or Escherichia coli led to the formation of inactive enzyme containing 1 mol of CoA per alphabeta dimer. The bound CoA was attached through a disulfide bond to a sulfhydryl 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 desulfo-CoA, which is a competitive inhibitor of the enzyme with respect to CoA. These data are evidence that oxidized CoA disulfide is an affinity label for the CoA binding site of succinyl-CoA synthetase and are the first positive results implicating the beta subunit in the catalytic mechanism of the enzyme.
D000345:Affinity Labels; D000818:Animals; D001667:Binding, Competitive; D003065:Coenzyme A; D003066:Coenzyme A Ligases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology*; D013389:Succinate-CoA Ligases / Q000378:metabolism*; D013552:Swine
D000345:Affinity Labels; C017145:coenzyme A disulfide; D003066:Coenzyme A Ligases; D013389:Succinate-CoA Ligases; D003065:Coenzyme A