Journal of the American Chemical Society p. 8402 - 8409 (1991)
Update date:2022-08-11
Topics:
Alvarez, Francisco J.
Ermer, Joachim
Hübner, Gerhard
Schellenberger, Alfred
Schowen, Richard L.
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.
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