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.
1H-NMR was used to follow the aspartate aminotransferase-catalysed exchange of the alpha-protons of aspartate and glutamate. The effect of the concentrations of both the amino acids and the cognate keto acids on exchange rates was determined for wild-type and the R386A and R292V mutant forms of aspartate aminotransferase. The wild-type enzyme is found to be highly stereospecific for the exchange of the alpha-protons of L-aspartate and L-glutamate. The R386A mutation which removes the interaction of Arg-386 with the alpha-carboxylate group of aspartate causes an approximately 10,000-fold decrease in the first order exchange rate of the alpha-proton of L-aspartate. The R292V mutation which removes the interaction of Arg-292 with the beta-carboxylate group of L-aspartate and the gamma-carboxylate group of L-glutamate causes 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 respectively. Apparently both Arg-386 and Arg-292 must be present for optimal catalysis of the exchange of the alpha-protons of L-aspartate and L-glutamate, perhaps because the interaction of both these residues with the substrate is essential for inducing the closed conformation of the active site.
Mahon
M M
MM
Department of Biochemistry, University College Dublin, Belfield, Dublin, Ireland.
Graber
R
R
Christen
P
P
Malthouse
J P
JP
D001219:Aspartate Aminotransferases / Q000737:chemistry* / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry*; D003903:Deuterium; D018698:Glutamic Acid / Q000737:chemistry*; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D011522:Protons; D013237:Stereoisomerism; D014367:Tryptophan Synthase / Q000737:chemistry
D011522:Protons; D001224:Aspartic Acid; D018698:Glutamic Acid; D003903:Deuterium; D012696:Glycine Hydroxymethyltransferase; D001219:Aspartate Aminotransferases; D014367:Tryptophan Synthase