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Transition‐state responses to amino acid perturbations in yeast pyruvate decarboxylase: a carbon kinetic isotope effect study
Author(s) -
Chen Lan,
Yuan Yang,
Huskey W. Phillip
Publication year - 2004
Publication title -
journal of physical organic chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.325
H-Index - 66
eISSN - 1099-1395
pISSN - 0894-3230
DOI - 10.1002/poc.774
Subject(s) - decarboxylation , chemistry , kinetic isotope effect , pyruvate decarboxylase , yeast , pyruvic acid , active site , stereochemistry , amino acid , catalysis , enzyme , biochemistry , deuterium , physics , alcohol dehydrogenase , quantum mechanics
Carbon kinetic isotope effects ( 12 C/ 13 C 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°C). 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 © 2004 John Wiley & Sons, Ltd.