Reaction of substrates with 35S-thiophosphorylated succinyl-CoA synthetase of pig heart. Similarities to the case of the Escherichia coli enzyme.
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.
D000214:Acyl Coenzyme A / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D003066:Coenzyme A Ligases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D046911:Macromolecular Substances; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D013329:Structure-Activity Relationship; D013389:Succinate-CoA Ligases / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D013552:Swine; D013873:Thionucleotides / Q000378:metabolism*
D000214:Acyl Coenzyme A; D046911:Macromolecular Substances; D013462:Sulfur Radioisotopes; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; C012046:succinyl-coenzyme A; D003066:Coenzyme A Ligases; D013389:Succinate-CoA Ligases