Solvent-derived protons in catalysis by brewers' yeast pyruvate decarboxylase

Area of Science:

• Biochemistry
• Enzymology
• Chemical Kinetics

Background:

• Pyruvate decarboxylase isozymes (PDC) are crucial enzymes in carbohydrate metabolism.
• Thiamin diphosphate (TDP) and its hydroxyethyl derivative (HETDP) are key coenzymes in PDC catalysis.
• Understanding the catalytic mechanism of PDC, particularly proton transfer, is essential.

Purpose of the Study:

• To investigate the role of proton transfer in the catalysis of pyruvate to acetaldehyde by Saccharomyces carlsbergensis PDC.
• To determine the contribution of coenzyme protonation to the overall enzymic reaction using solvent discrimination tritium isotope effects.
• To elucidate the number and nature of catalytic groups involved in proton transfer during PDC-catalyzed reactions.

Main Methods:

• Measurement of solvent discrimination tritium isotope effects ((kH/kT)disc) on pyruvate conversion to acetaldehyde.
• Investigation under both single-turnover and transient steady-state conditions.
• Analysis of fractionation factors (phi) for proton transfer to thiamin diphosphate (TDP) and 2-(1-hydroxyethyl)thiamin diphosphate (HETDP).

Main Results:

• Fractionation factors for TDP C(2)-L and HETDP C(alpha)-L did not significantly contribute to isotopic discrimination.
• Single-turnover reprotonation of TDP C(2)-L showed no significant isotopic discrimination, consistent with solvent-equilibrated C(2)-hydron transfer.
• Steady-state acetaldehyde formation exhibited increasing solvent discrimination tritium isotope effects, indicating a fractionation factor of phi = 0.88 +/- 0.06 for C(alpha)-hydron transfer to form HETDP.

Conclusions:

• PDC-catalyzed acetaldehyde formation involves specific protonation of both HETDP C(alpha)-L and TDP C(2)-L.
• The reaction mechanism requires at least two catalytic groups for efficient proton transfer.
• No significant intramolecular catalysis by the coenzyme's exocyclic 4'-amino or -imino group was observed.