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Broønsted analysis of aspartate aminotransferase via exogenous catalysis of reactions of an inactive mutant
Author(s) -
Toney Michael D.,
Kirsch Jack F.
Publication year - 1992
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560010111
Subject(s) - chemistry , transamination , kinetic isotope effect , amine gas treating , catalysis , lysine , cysteine , medicinal chemistry , stereochemistry , enzyme catalysis , brønsted–lowry acid–base theory , enzyme , amino acid , deuterium , organic chemistry , biochemistry , physics , quantum mechanics
Primary amines functionally replace lysine 258 by catalyzing both the 1,3‐prototropic shift and external aldimine hydrolysis reactions with the inactive aspartate aminotransferase mutant K258A. This finding allows classical Brønsted analyses of proton transfer reactions to be applied to enzyme‐catalyzed reactions. An earlier study of the reaction of K258A with cysteine sulfinate (Toney, M.D. & Kirsch, J.F., 1989, Science 243 , 1485) provided a β value of 0.4 for the 1,3‐prototropic shift. The β value reported here for the transamination of oxalacetate to aspartate is 0.6. The catalytic efficacy of primary amines is largely determined by basicity and molecular volume. The dependence of the rate constants for the reactions of K258A and K258M on amine molecular volume is nearly identical. This observation argues that the alkyl groups of the added amines do not occupy the position of the lysine 258 side chain in the wild type enzyme. Large primary C α and insignificant solvent deuterium kinetic isotope effects with amino acid substrates demonstrate that the amine nitrogen of the exogenous catalysts directly abstracts the labile proton in the rate‐determining step.