Kinetics and equilibria for the reactions of coenzymes with wild type and the Y70F mutant of Escherichia coli aspartate aminotransferase.
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, KPMPdiss, 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 KPMPdiss 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 KPLPdiss for wild type enzyme has been difficult to obtain because of the low value of this constant. The values of KPLPdiss for the holoenzymes were determined indirectly through the measured values for KPMPdiss, glutamate-alpha-ketoglutarate half-reaction equilibrium constants, and the equilibrium constant for the transamination of PLP by glutamate catalyzed by Y70F. The values of KPLPdiss obtained by this procedure are 0.4 pM for wild type and 40 pM for Y70F. The increases in KPMPdiss and KPLPdiss for Y70F correspond to delta delta G 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 delta G for association of PLP with wild type enzyme is 4.7 kcal/mol more favorable than that for PMP.
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
M D
MD
Toney
J F
JF
Kirsch
D001051:Apoenzymes / Q000378:metabolism; D001219:Aspartate Aminotransferases / Q000235:genetics / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D006860:Hydrogen Bonding; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D011733:Pyridoxamine / Q000031:analogs & derivatives* / Q000378:metabolism
D001051:Apoenzymes; D003067:Coenzymes; D005971:Glutamates; D007656:Ketoglutaric Acids; D018698:Glutamic Acid; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D001219:Aspartate Aminotransferases; C010627:pyridoxamine phosphate