Mechanistic Analysis of the Aspartate Aminotransferase Active Site Mutants—Y70F, K258A, and R292D

Summary

Three mutant enzymes produced by site-directed mutagenesis have been analyzed in some detail. Y70F retains substantial enzymatic activity, proving that Y70 is not an essential amino acid for catalysis. Rather, this residue does provide an important functional role in substantially reducing the rate constant for cofactor dissociation from the enzyme. The PMP form of K258A reacts with α-ketoglutarate to give the ketimine as a stable final product. The corresponding reaction with oxalacetate leads ultimately to the decarboxy lat ed, transaminated aldimine of alanine as final product. Earlier crystallographic analysis showed that the dicarboxylic amino acid substrate specificity is dictated by ion pairing with R292. The mutation R292D converts the title enzyme to a cationic amino acid transaminase, which preferentially catalyzes the transamination of Arg or Lys over Asp or Glu.