Affinity labeling of succinyl-CoA synthetase from Escherichia coli by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate.

The 2'3'-dialdehyde of adenosine 5'-diphosphate, oADP, exhibited the properties of an affinity label with Escherichia coli succinyl-CoA synthetase. Inactivation of this synthetase by oADP followed pseudo-first-order kinetics and was competitively blocked by ADP. The stoichiometry of labeling of the synthetase was 1 mol/mol alpha beta or, extrapolated, 2 mol/mol inactive alpha 2 beta 2 molecule. oADP also exhibited the properties of a substrate, bringing about rapid dephosphorylation of the enzyme. Further specificity of oADP was demonstrated in partially inactivated succinyl-CoA synthetase by selective inhibition of the succinate in equilibrium succinyl-CoA exchange reaction, in comparison to the CoA in equilibrium succinyl-CoA exchange reaction. Modification of the synthetase by oADP resulted in cross-linking of the enzyme, casting uncertainty over the subunit binding site for ADP. Modification of the synthetase by ADP-2'-semialdehyde occurred at a faster rate than that by oADP but exhibited biphasic inhibitor concentration dependence and did not exhibit saturability.