Evidence for Succinyl Phosphate as an Enzyme-bound Intermediate in the Reaction Catalyzed by Succinyl Coenzyme A Synthetase

When purified succinyl coenzyme A synthetase from Escherichia coli was incubated with [I4C]succinyl phosphate and coenzyme A, [14C]succinyl coenzyme A was formed. Incubation of the enzyme with [32P]succinyl phosphate and adenosine 5'-diphosphate led to adenosine 5 '-triphosphate (ATP) synthesis. When the enzyme was incubated with [y-32P]ATP and succinate evidence was obtained for synthesis of succinyl phosphate, which was separated and identified by paper electrophoresis. Evidence that the enzyme is phosphorylated by succinyl phosphate was also obtained. The data support the conclusion that enzyme-bound succinyl phosphate is an intermediate in the reaction.