Active site directed irreversible inactivation of brewers' yeast pyruvate decarboxylase by the conjugated substrate analog (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid: development of a suicide substrate

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Abstract
(E)-4-(4-Chlorophenyl)-2-oxo-3-butenoic acid (CPB) irreversibly inactivated brewers'' yeast pyruvate decarboxylase (PDC) in a biphasic, sigmoidal manner, as is found for the kinetic behavior of substrate. An expression was derived for 2-site irreversible inhibition of allosteric enzymes, and the kinetic behavior of CPB fit the expression for 2-site binding. The calculated Ki of 0.7 mM and 0.3 mM for CPB were assigned to the catalytic site and the regulatory site, respectively. The presence of pyruvic acid at high concentrations protected PDC from inactivation, whereas low concentrations of pyruvic acid accelerated inactivation by CPB. Pyruvamide, a known allosteric activator of PDC, enhanced inactivation by CPB. The results can be explained if pyruvamide binds only to a regulatory site, but CPB and pyruvic acid compete for both the regulatory and the catalytic centers. [1-14C]CPB lost 14CO2 concurrently with the inactivation of the enzyme. CPB was being turned over by PDC, in addition to inactivating it. CPB can be labeled a suicide-type inactivator for PDC.

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