发现鸟苷5'-O-(3-硫代)三磷酸(GTPγS)是猪心脏琥珀酰辅酶A合成酶的底物,其Km和kcat值分别为3μM和0.23s-1。以GTP为底物的相应值为48μM和65s-1。通过将猪心脏琥珀酰辅酶A合成酶与[35S]GTPγS孵育来制备35S-硫代磷酸化酶。比较了该αβ(一个活性位点)酶的底物释放的硫代磷酰基与α2β2(两个活性位点)大肠杆菌酶(Wolodko,W.T.,布朗尼,E、R、O\'Connor,M.D.,还有Bridger,W.A.(1983)J.生物。Chem.258,14116-14119;西村,J.S.,还有米切尔,T.(1984)J.生物。Chem.259、9642-9645)。它被发现了,就像大肠杆菌酶一样,琥珀酰辅酶A和GTP刺激了GDP和琥珀酸酯加辅酶A的硫代磷酰基释放,分别。在1、0.1和0.01mg/ml时观察到相同的结果,贷款保证这些现象没有表现出聚集形式的猪心脏酶。虽然不排除大肠杆菌酶的交替位点催化协同作用模型,有人提出,NTP和琥珀酰辅酶A刺激的硫代磷酸基从两种酶中释放涉及“相同位点”机制,与“其他站点”机制区分开来。
Guanosine 5\'-O-(3-thio)triphosphate (GTP gamma S) was found to be a substrate of pig heart succinyl-CoA synthetase with Km and kcat values of 3 microM and 0.23 s-1, respectively. The corresponding values with GTP as substrate were 48 microM and 65 s-1. 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 (one active site) enzyme with that of the alpha 2 beta 2 (two active sites) Escherichia coli enzyme (Wolodko, W. T., Brownie, E. R., O\'Connor, M. D., and Bridger, W. A. (1983) J. Biol. Chem. 258, 14116-14119; Nishimura, J. S., and Mitchell, T. (1984) J. Biol. Chem. 259, 9642-9645). It was found, 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.