The aspartate aminotransferase-catalysed exchange of the alpha-protons of aspartate and glutamate: the effects of the R386A and R292V mutations on this exchange reaction

H-1-NMR was used to follow the aspartate aminotransferase-catalysed exchang e of the alpha-protons of aspartate and glutamate. The effect of the concen trations of both the amino acids and the cognate keto acids on exchange rat es was determined for wild-type and the R386A and R292V mutant forms of asp artate aminotransferase, The wild-type enzyme is found to be highly stereos pecific for the exchange of the alpha-protons of L-aspartate and L-glutamat e. The R386A mutation which removes the interaction of Arg-386 with the alp ha-carboxylate group of aspartate causes an similar to 10 000-fold decrease in the first order exchange rate of the alpha-proton of L-aspartate. The R 292V mutation which removes the interaction of Arg-292 with the beta-carbox ylate group of L-aspartate and the gamma-carboxylate group of L-glutamate c auses even larger decreases of 25 000- and 100 000-fold in the first order exchange rate of the alpha-proton of L-aspartate and L-glutamate respective ly. Apparently both Arg-386 and Arg-292 must be present for optimal catalys is of the exchange of the alpha-protons of L-aspartate and L-glutamate, per haps because the interaction of both these residues with the substrate is e ssential for inducing the closed conformation of the active site. (C) 1999 Elsevier Science B.V. All rights reserved.