Abstract
Two refined crystal structures of aspartate aminotransferase from E.coli are reported. The wild type enzyme is in the pyridoxal phosphate (PLP) form and its structure has been determined to 2.4 Å resolution, refined to an R-factor of 23.2%. The structure of the Arg292Asp mutant has been determined at 2.8 Å resolution, refined to an R–factor of 20.3%. The wild type and mutant crystals are isomorphous and the two structures are very similar, with only minor changes in positions of important active site residues. As residue Arg292 is primarily responsible for the substrate charge specificity in the wild type enzyme, the mutant containing a charge reversal at this position might be expected to catalyze transamination of arginine as efficiently as the wild type enzyme effects transamination of aspartate [Cronin,C.N. and Kirsch,J.F. (1988) Biochemistry, 27, 4572–4579]. This mutant does in fact prefer arginine over aspartate as a substrate, however, the rate of catalysis is much slower than that of the wild type enzyme with its physiological substrate, aspartate. A comparison of these two structures indicates that the poorer catalytic efficiency of R292D, when presented with arginine, is not due to a gross conformational difference, but is rather a consequence of both small side chain and main chain reorientations and the pre–existing active site polar environment, which greatly favors the wild type ion pair interaction.