Catalytic power of pyruvate decarboxylase. Rate-limiting events and microscopic rate constants from primary carbon and secondary hydrogen isotope effects

Article abstract of DOI:10.1021/ja00022a030

Isotope effects ([rate constant for light isotopic substrate]/[rate constant for heavy isotopic substrate]) for the action of the thiamin diphosphate dependent pyruvate decarboxylase of Saccharomyces carlsbergensis (EC 4.1.1.1) on pyruvate, pyruvate-1-¹³C, pyruvate-2-¹³C, and pyruvate-3-d₃ have been determined for each of the steady-state kinetic parameters k/A (second-order in pyruvate), k/B (first-order in pyruvate), and k (zero-order in pyruvate). The 1-¹³C effects are 1.008 ± 0.010 (k/A), 1.013 ± 0.024 (k/B), and 1.024 ± 0.006 (k). The 2-¹³C effects are 1.013 ± 0.009 (k/A), 0.951 ± 0.020 (k/B), and 1.039 ± 0.004 (k). The 3-d₃ effects are 0.883 ± 0.013 (k/A), 0.881 ± 0.026 (k/B), and 1.057 ± 0.005 (k). Effects with 2-oxobutanoate and 2-oxobutanoate-3-d₂ are 0.951 ± 0.012 (k/A), 0.821 ± 0.096 (k/B), and 1.057 ± 0.005 (k). Pyruvate decarboxylase was already known to be hysteretically activated by the substrate, with pyruvate binding to the regulatory site with dissociation constant 8 mM and producing unimolecular activation (0.46 s^-1) and deactivation (0.033 s^-1). The isotope effects lead to rate constants for substrate binding to the catalytic site of 8.2 × 104 M^-1 s^-1, for substrate departure from the catalytic site of 120 s^-1, for decarboxylation of 640 s^-1, and for product release of 640 s^-1. Pyruvate decarboxylase increases the rate of decarboxylation of pyruvate by thiamin alone by a factor of 3 × 10¹² at pH 6.2, 30°C. Under these conditions, conversion of activated enzyme and pyruvate to the enzymic species preceding decarboxylation is 4 × 10¹² times faster than the specific-base-catalyzed addition of thiamin to pyruvate. The enzymic species preceding decarboxylation reverts to activated enzyme and free pyruvate 6 × 10⁹ times faster than the specific-base-catalyzed reversion of the adduct of thiamin and pyruvate to thiamin and free pyruvate. Enzymic decarboxylation is 10⁷ times faster than decarboxylation of the adduct of thiamin and pyruvate.