Transition-state responses to amino acid perturbations in yeast pyruvate decarboxylase: a carbon kinetic isotope effect study

Carbon kinetic isotope effects (C-12/C-13 at the carboxyl carbon of pyruvic acid) and their pH dependence were measured for the decarboxylation reaction catalyzed by the substrateregulated yeast pyruvate decarboxylase (PDC) and several of its site-specific variants. The active-site variants studied were E477Q, D28A and E51D; the regulatory-site variants, each with two amino acid substitutions, were C221A/C222A, C221E/C222A and C221D/C222A. The isotope effects for the regulatory-site variants were not significantly different from the values for the wild-type enzyme (1.0046 +/- 0.0003 at pH 6.0, 25 degreesC). For the active-site variants, the isotope effects were 1.0018 +/- 0.0009 (E477Q), 1.0398 +/- 0.0021 (D28A) and 1.0143 +/- 0.0011 (E51D) at pH 6.0 and 25 C. The results were interpreted in terms of shifts in the rate-limiting steps and uniform binding changes in the free-energy profiles for decarboxylation phase of the PDC reaction. Copyright (C) 2004 John Wiley Sons, Ltd.