Positional oxygen isotope exchange as a probe for the mechanism of catalysis by Escherichia coli succinyl coenzyme A synthetase.
Succinyl-CoA synthetase of Escherichia coli has an alpha 2 beta 2 subunit structure. The enzyme shows strict half-sites reactivity with respect to the phosphorylation of a histidine residue in the alpha subunit that represents a step in catalysis. Several lines of evidence indicate that this behavior may result from cooperative interactions between alternatingly functional active sites, so that subsequent steps in catalysis at one site may be promoted by phosphoryl transfer to the site on the neighboring half of the molecule. This study is directed toward learning more about the nature of these cooperative interactions. Here we have used positional isotope exchange (i.e., exchange of 18O between the beta, gamma bridge and the beta nonbridge position of ATP) as a test for transient bisphosphorylation. Succinyl-CoA synthetase was ATP) as a test for transient bisphosphorylation. Succinyl-CoA synthetase was prepared in which one of the two active sites was thiophosphorylated; this species thus has one of its two active-site histidine residues occupied and unavailable for further reaction with ATP. Treatment of this monothiophosphorylated enzyme with [beta, gamma-18O]ATP resulted in no significant scrambling of isotope into the nonbridge position, clearly indicating that the enzyme does not undergo even transient bisphosphorylation. We interpret the results in terms of a model of catalysis in which phosphoryl transfer to the second site occurs in concerted fashion with transfer from the first.
Williams
S P
SP
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Bridger
W A
WA
D000255:Adenosine Triphosphate / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D046911:Macromolecular Substances; D010103:Oxygen Isotopes; D010766:Phosphorylation; D013389:Succinate-CoA Ligases / Q000378:metabolism*
D046911:Macromolecular Substances; D010103:Oxygen Isotopes; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; D013389:Succinate-CoA Ligases