SITE-DIRECTED MUTAGENESIS OF THE AMINO-ACID-RESIDUES IN BETA-STRAND-III [VAL(30)-VAL(36)] OF D-AMINO-ACID AMINOTRANSFERASE OF BACILLUS SP YM-1

The beta-strand III formed by amino acid residues Val(30)-Val(36) is l ocated across the active site of the thermostable D-amino acid aminotr ansferase (D-AAT) from thermophilic Bacillus sp, YM-1, and the odd-num bered amino acids (Tyr(31), Val(33), Lys(35)) in the strand are reveal ed to be directed toward the active site, Interestingly, Glu(32) is al so directed toward the active site, We first investigated the involvem ent of these amino acid residues in catalysis by alanine scanning muta genesis. The Y31A and E32A mutant enzymes showed a marked decrease in k(cat) value, retaining less than 1% of the wild-type enzyme activity, The k(cat) values of V33A and K35A were changed slightly, but the K-m of K35A for alpha-ketoglutarate was increased to 35.6 mM, compared to the K-m value of 2.5 mM for the wild-type enzyme, These results sugge sted that the positive charge at Lys(35) interacted electrostatically with the negative charge at the side chain of cl-ketoglutarate, Site-d irected mutagenesis of the Glu(32) residue was conducted to demonstrat e the role of this residue in detail, From the kinetic and spectral ch aracteristics of the Glu(32)-substituted enzymes, the Glu(32) residue seemed to interact with the positive charge at the Schiff base formed between the aldehyde group of pyridoxal 5'-phosphate (PLP) and the eps ilon-amino group of the Lys(145) residue.