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.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000345:Affinity Labels; D001426:Bacterial Proteins / Q000302:isolation & purification; D055598:Chemical Phenomena; D002621:Chemistry; D003066:Coenzyme A Ligases / Q000302:isolation & purification*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D013389:Succinate-CoA Ligases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*
D000345:Affinity Labels; D001426:Bacterial Proteins; D000244:Adenosine Diphosphate; C018808:adenosine 5'-diphosphate 2',3'-dialdehyde; D003066:Coenzyme A Ligases; D013389:Succinate-CoA Ligases