Nishimura J S, Mitchell T
J Biol Chem. 1985 Feb 25;260(4):2077-9.
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.
发现鸟苷5'-O-(3-硫代)三磷酸(GTPγS)是猪心琥珀酰辅酶A合成酶的底物，其Km和kcat值分别为3μM和0.23 s-1。以GTP为底物时的相应值分别为48μM和65 s-1。通过将猪心琥珀酰辅酶A合成酶与[35S]GTPγS孵育制备了35S-硫代磷酸化酶。比较了该αβ(一个活性位点)酶的底物释放硫代磷酸基团的情况与α2β2(两个活性位点)大肠杆菌酶的情况(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)。结果发现，与大肠杆菌酶的情况一样，GDP以及琥珀酸加辅酶A释放硫代磷酸基团分别受到琥珀酰辅酶A和GTP的刺激。在1、0.1和0.01 mg/ml时观察到相同的结果，这表明猪心酶的聚集形式未表现出这些现象。虽然不排除大肠杆菌酶存在交替位点催化协同模型，但有人提出，两种酶中NTP和琥珀酰辅酶A刺激的硫代磷酸基团释放都涉及“同一位点”机制，以区别于“其他位点”机制。
