Thiophosphorylation as a probe for subunit interactions in Escherichia coli succinyl coenzyme A synthetase. Further evidence for catalytic cooperativity and substrate synergism.
Succinyl-CoA synthetase has an (alpha beta)2 subunit structure and shows half-of-the-sites reactivity with respect to the formation of the phosphohistidyl residues that acts as a catalytic intermediate. Adenosine 5'-O-(3-thio)triphosphate has been found to be a substrate, but the overall maximum velocity is 3 orders of magnitude lower than that seen with ATP. Moreover, steps of the reaction involving thiophosphoryl transfer are much slower than the corresponding phosphoryl transfers. These properties of adenosine 5'-O-(3-thio)triphosphate as a substrate have been exploited to test the concept of alternating sites catalytic cooperativity proposed earlier as a rationale for the subunit structure of succinyl-CoA synthetase. As predicted by this model for catalysis, the rate of discharge of thiophosphate from the enzyme in the presence of succinate and CoA is stimulated by ATP. Neither of two nonhydrolyzable analogs of ATP has an equivalent effect. The results indicate that the transfer of the thiophosphoryl group from the enzyme to succinate at one active site is not favored until the neighboring active site is phosphorylated by ATP, with accompanying reciprocal changes in the conformations of the two halves of the enzyme molecule.
D000214:Acyl Coenzyme A / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008956:Models, Chemical; D013389:Succinate-CoA Ligases / Q000378:metabolism*; D013873:Thionucleotides / Q000378:metabolism
D000214:Acyl Coenzyme A; D046911:Macromolecular Substances; D013873:Thionucleotides; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; C012046:succinyl-coenzyme A; D003066:Coenzyme A Ligases; D013389:Succinate-CoA Ligases