DISTRIBUTION OF THE THIAMIN DIPHOSPHATE C(2)-PROTON DURING CATALYSIS OF ACETALDEHYDE FORMATION BY BREWERS-YEAST PYRUVATE DECARBOXYLASE

The distribution of tritium derived from enzyme-bound [thiazole-2-T]th iamin diphosphate (TDP) during the reaction of pyruvate to form acetal dehyde catalyzed by pyruvate decarboxylase isozymes (PDC; EC 4.1.1.1) from Saccharomyces carlsbergensis was determined under single-turnover conditions ([E] > [S] > in the presence of the nonsubstrate allosteri c effector pyruvamide. The specific radioactivity of the [1-L]acetalde hyde product and solvent ([L]H2O) was 43 +/- 4% and 54 +/- 2%, respect ively, of the initial specific radioactivity of PDC-bound [thiazole-2- T]TDP and was independent of the extent of the single-turnover reactio n. There is little (less than or equal to 3%) or no return of the abst racted C(2)-hydron to the C(2) position of PDC-bound TDP. This provide s evidence that the abstracted C(2)-hydron is involved in the specific protonation of the C(cr) position of the PDC-bound intermediate 2-(1- hydroxyethyl)thiamin diphosphate (HETDP), which is cleaved to form [1- L]acetaldehyde and PDC-bound [thiazole-2-H]TDP. The partial exchange o f C(2)-derived tritium into solvent requires that (1) hydron transfer from C(2) occurs to a catalytic base in which the conjugate catalytic acid is partially shielded from hydron exchange with the solvent, (2) the conjugate catalytic acid transfers the C(2)-derived hydron to the C(alpha) position of HETDP, and (3) hydron transfer to C(2) to regener ate the coenzyme occurs either from solvent directly or from a second catalytic acid of the enzyme that undergoes rapid hydron exchange with the solvent. The observed rate constant k(obsd) = 1.4 min(-1) for C(2 )-hydron exchange in PDC-bound TDP in the absence of substrate corresp onds to a pK(a) value for C(2)-H at the active site of 17.5.