Active Site Directed Irreversible Inactivation of Brewers' Yeast Pyruvate Decarboxylase by the Conjugated Substrate Analogue (E)-4-(4-Chlorophenyl)-2-oxo-3-butenoic Acid: Development of a Suicide Substrate

(E)-4-(4-Chlorophenyl)-2-oxo-3-butenoic acid (CPB) was found to irreversibly inactivate brewers' yeast pyruvate decarboxylase (PDC., EC 4.1.1.1) in a biphasic, sigmoidal manner, as is found for the kinetic behavior of substrate. An expression was derived for two-site irreversible inhibition of allosteric enzymes, and the kinetic behavior of CPB fit the expression for two-site binding. The calculated K_i's 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., was found to enhance 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 was found to lose ¹⁴CO₂ concurrently with the inactivation of the enzyme. Therefore, CPB was being turned over by PDC., in addition to inactivating it. CPB can be labeled a suicide-type inactivator for PDC.