Bronsted analysis of aspartate aminotransferase via exogenous catalysis of reactions of an inactive mutant

M. D. Toney, J. F. Kirsch

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Abstract

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 Bronsted 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. and 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 Cα 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.

Original languageEnglish (US)
Pages (from-to)107-119
Number of pages13
JournalProtein Science
Volume1
Issue number1
StatePublished - 1992

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Aspartate Aminotransferases
Catalysis
Amines
Lysine
Protons
Proton transfer
Deuterium
Enzymes
Alkalinity
Aspartic Acid
Isotopes
Hydrolysis
Rate constants
Nitrogen
Amino Acids
Catalysts
Kinetics
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Bronsted analysis of aspartate aminotransferase via exogenous catalysis of reactions of an inactive mutant. / Toney, M. D.; Kirsch, J. F.

In: Protein Science, Vol. 1, No. 1, 1992, p. 107-119.

Research output: Contribution to journalArticle

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