Kinetics and Equilibria for the Reactions of Coenzymes with Wild Type and the Y70F Mutant of Escherichia coli Aspartate Aminotransferase

Michael D. Toney, Jack F. Kirsch

Research output: Contribution to journalArticle

28 Scopus citations

Abstract

The Y70F mutant of aspartate aminotransferase has reduced affinity for coenzymes compared to the wild type. The equilibrium dissociation constants for pyridoxamine phosphate (PMP) holoenzymes, were determined from the association and dissociation rate constants to be 1.3 nM and 30 nM for the wild type and mutant, respectively. This increase in [formula omitted] for Y70F is due to a 27-fold increase in the dissociation rate constant. Pyridoxal phosphate (PLP) association kinetics are complex, with three kinetic processes detectable for wild type and two for Y70F. A directly determined, accurate value of for wild type enzyme has been difficult to obtain because of the low value of this constant. The values of [formula omitted] for the holoenzymes were determined indirectly through the measured values for [formula omitted], glutamate–α-ketoglutarate half-reaction equilibrium constants, and the equilibrium constant for the transamination of PLP by glutamate catalyzed by Y70F. The values of [formula omitted] obtained by this procedure are 0.4 pM for wild type and 40 pM for Y70F. The increases in [formula omitted] and for Y70F correspond to ΔΔG values of 1.9 and 2.7 kcal/mol, respectively, and are directly attributed to the loss of the hydrogen bond from the phenolic hydroxyl group of Tyr70 to the coenzyme phosphate. The ΔG for association of PLP with wild type enzyme is 4.7 kcal/mol more favorable than that for PMP.

Original languageEnglish (US)
Pages (from-to)7461-7466
Number of pages6
JournalBiochemistry
Volume30
Issue number30
DOIs
StatePublished - Jul 1 1991
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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