Catalysis of proton transfer to thiamin diphosphate (TDP) and 2-(1-hydroxyethyl)thiamin diphosphate (HETDP) by pyruvate decarboxylase isozymes (PDC; EC 188.8.131.52) from Saccharomyces carlsbergensis was investigated by determining the solvent discrimination tritium isotope effect, (kH/kT)disc, on the reaction of pyruvate to form acetaldehyde in the presence of the nonsubstrate allosteric effector pyruvamide. The fractionation factors for TDP C(2)-L (φC(2) = 0.98 ± 0.06) and HETDP C(α)-L (φC(α) = 1.01 ± 0.07) (L = H or D) do not contribute significantly to observed enzymic isotopic discrimination. The value of (kH/kT)disc = 1.0 for reprotonation of TDP C(2)-L under single-turnover conditions ([E] > [S]) is consistent with C(2)-hydron transfer via a catalytic group (φ = 1) equilibrated with solvent. [1-L]Acetaldehyde formation under transient steady-state ([E] < [S]) conditions shows solvent discrimination tritium isotope effects that increase over the range (kH/kT)disc = 0.39 (single turnover) to 0.86 (ten turnovers). The 2-fold increase in the value of (kH/kT)disc for the [1-L]acetaldehyde product under steady-state compared to single-turnover conditions is attributed to a fractionation factor of φ1 = 0.88 ± 0.06 for the residue(s) involved in C(α)-hydron transfer to form HETDP. This provides evidence that catalysis of acetaldehyde formation by PDC involves specific protonation of both HETDP C(α)-L and TDP C(2)-L (φ2 = 1.0 ± 0.1) and requires at least two catalytic groups. Values of φ ≤ 1 for protonation of TDP C(2)-L and HETDP C(α)-L provide no evidence that the exocyclic 4′-amino or -imino group (φ ≥ 1.2) provides significant intramolecular catalysis in the enzyme-bound coenzyme.
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