CHANGING THE REACTION SPECIFICITY OF A PYRIDOXAL-5'-PHOSPHATE-DEPENDENT ENZYME

The electron distribution in the coenzyme-substrate adduct of aspartat e aminotransferase was changed by replacing active-site Arg386 with al anine and introducing a new arginine residue nearby. [Y225R, R386A]Asp artate aminotransferase decarboxylates L-aspartate to L-alanine (k(cat ) = 0.04 s(-1)), while its transaminase activity towards dicarboxylic amino acids is decreased by three orders of magnitude (k(cat) = 0.19 s (-1)). Molecular-dynamics simulations based on the crystal structure o f the mutant enzyme suggest that a new hydrogen bond to the imine N at om of the pyridoxal-5'-phosphate-aspartate adduct and an altered elect rostatic potential around its beta-carboxylate group underlie the 6500 00-fold increase in the ratio of beta-decarboxylase/transaminase activ ity.