Guanosine 5''-O-(3-thio)triphosphate (GTP.gamma.S) was found to be a substrate of pig heart succinyl-CoA synthetase with Km and .kappa.cat [catalytic rate constant] values of 3 .mu.M and 0.23/s, respectively. The corresponding values with GTP as substrate were 48 .mu.M and 65/s. 35S-thiophosphorylated enzyme was prepared by incubation of pig heart succinyl-CoA synthetase with [35S]GTP.gamma.S. A comparison was made of thiophosphoryl group release by substrates from this .alpha..beta. (1 active sites) enzyme with that of the .alpha.2.beta.2 (2 active sites) E. coli enzyme. As in the case of the E. coli enzyme, that thiophosphoryl group release by GDP and by succinate plus CoA was stimulated by succinyl-CoA and GTP, respectively. The same result was observed at 1, 0.1 and 0.01 mg/ml, lending assurance that these phenomena were not exhibited by an aggregated form of the pig heart enzyme. While an alternating-sites catalytic cooperativity model is not ruled out for the E. coli enzyme, it is proposed that the NTP- and succinyl-CoA-stimulated release of thiophosphoryl groups from either enzyme involves a same-site mechanism, to be distinguished from an other-site mechanism.