RATE OF DECARBOXYLATION, MONITORED VIA THE KEY ENZYME-BOUND ENAMINE, OF CONJUGATED ALPHA-KETO ACIDS BY PYRUVAMIDE ACTIVATED PYRUVATE DECARBOXYLASE IS KINETICALLY COMPETENT WITH TURNOVER

The rate of formation of the covalent thiamin diphosphate-bound enamine/C2-alpha-carbanion intermediate monitored at 440 nm from conjugated mechanism-based inhibitors of the structure (E)-YC6H4CH=CHCOCOOH, where Y = p-Cl, m-NO2, m-CF3, was determined in the absence and presence of the allosteric activator pyruvamide on brewers' yeast pyruvate decarboxylase (PDC, E.C.4.1.1.1). For all three compounds the first-order rate constant for enamine formation was accelerated from 15-150-fold by conversion of the enzyme to its activated form. The rate constant for enamine formation is 10(2)-10(3) times faster than those estimated for inactivation. Comparing the k(cat) (0.44 s-1) to the rate constant for decarboxylation (0.653 s-1) for Y = p-Cl leads one to conclude that enamine formation is kinetically competent to participate in the turnover pathway. Based on the maximum absorbance developed at 440 nm, and the epsilon = 10(4) at this wavelength for a model compound (Y = H), there appear to be four active sites per tetrameric holoenzyme. The k(cat)/active site for pyruvate is estimated at ca. 40 s-1 at 20-degrees-C, and the decarboxylation rate constant for pyruvate can be estimated to be 80 s-1/active site at 20-degrees-C, assuming decarboxylation and product release are equal in kinetic significance. The rate constants for decarboxylation by activated PDC for Y = m-NO2, m-CF3 (53 and 69 s-1) are comparable to this estimated decarboxylation rate constant for pyruvate (80 s-1). The k's for Y = m-NO2, m-CF3 are also similar in magnitude to the decarboxylation rate constant (62-80 s-1 at 22-degrees-C) reported for pyruvate oxidase, an enzyme with considerable sequence homology to PDC, and one that follows the same mechanism through decarboxylation.