Adenosine 5'-O-(3-thio)triphosphate, a substrate and potent inhibitor of Escherichia coli succinyl-CoA synthetase. Additional evidence for a cooperative alternating-sites mechanism.

Abstract

Adenosine 5'-O-(3-thio)triphosphate (ATP gamma S) has been shown to be a potent inhibitor of Escherichia coli succinyl-CoA synthetase. This inhibition was competitive with respect to ATP and GTP (Ki values of 0.8 and 0.7 microM, respectively) and mixed with respect to CoA and succinate. ATP gamma S previously had been shown to be a weak substrate of the enzyme, probably because of the relatively sluggish reactivity of the thiophosphoryl enzyme intermediate (Wolodko, W. T., Brownie, E. R., O'Connor, M. D., and Bridger, W. A. (1983) J. Biol. Chem. 258, 14116-14119). In our work, reaction of thiophosphoryl enzyme with ADP was greatly stimulated by succinyl-CoA, an observation that is consistent with the concept of alternating-sites cooperativity. Thiophosphoryl group release did not appear to be accompanied by "other-site" phosphorylation, in contrast to ATP stimulation of thiophosphoryl group release in the presence of succinate and CoA (Wolodko et al., see above). In addition, ADP did not appear to be required in the latter reaction.