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 αβ or, extrapolated, 2 mol/mol inactive α₂β₂ 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 ↔ succinyl‐CoA exchange reaction, in comparison to the CoA ↔ 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.