The distribution of tritium derived from enzyme-bound [thiazole-2-T]thiamin diphosphate (TDP) during the reaction of pyruvate to form acetaldehyde catalyzed by pyruvate decarboxylase isozymes (PDC; EC 220.127.116.11) from Saccharomyces carlsbergensis was determined under single-turnover conditions ([E] > [S]) in the presence of the nonsubstrate allosteric effector pyruvamide. The specific radioactivity of the [1-L]acetaldehyde product and solvent ([L]H2O) was 43 ± 4% and 54 ± 2%, respectively, of the initial specific radioactivity of PDC-bound [thiazole-2-T]TDP and was independent of the extent of the single-turnover reaction. There is little (≤3%) or no return of the abstracted C(2)-hydron to the C(2) position of PDC-bound TDP. This provides evidence that the abstracted C(2)-hydron is involved in the specific protonation of the C(α) 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 of 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(α) position of HETDP, and (3) hydron transfer to C(2) to regenerate 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 kobsd = 1.4 min-1 for C(2)-hydron exchange in PDC-bound TDP in the absence of substrate corresponds to a pKa value for C(2)-H at the active site of 17.5.
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