D-Amino acid transaminase (EC 184.108.40.206), from Bacillus sp. YM-1, a thermostable enzyme with pyridoxal 5'-phosphate as coenzyme and a target for the design of novel antimicrobial agents, catalyzes the reversible transfer of an amino group between D-alanine and alpha-ketoglutarate to form pyruvate and D-glutamate, respectively. To explore the catalytic role of Lys-145, which binds the coenzyme, a site-specific mutant enzyme, K145Q (in which Lys-145 had been mutated to glutamine) constructed earlier (Futaki, S., Ueno, H., Martinez del Pozo, A., Pospischil, M. A., Manning, J. M., Ringe, D., Stoddard, B., Tanizawa, K., Yoshimura, T., and Soda, K. (1990) J. Biol. Chem. 265, 22306-22312) was compared to the wild-type enzyme for its kinetic parameters. Initial velocity studies and partial reaction isotope exchange experiments showed that the low activity of the mutant enzyme (about 1.5% the activity of the wild-type enzyme with saturating substrates) is an intrinsic property, confirming that contaminating enzymes do not account for the low activity of the K145Q mutant enzyme. The rates of the forward reaction for both wild-type and mutant enzymes were 30-40 times higher than the rates of the reverse reaction. KM values for the four substrates were 10 to 100 higher for the mutant compared to the wild-type enzyme. Whereas D-alanine is preferred over L-alanine by the wild-type enzyme (10(3) higher kcat/KM for D- over L-alanine), the K145Q enzyme does not efficiently discriminate between L- and D-alanine. Both wild-type and mutant enzymes also catalyze the slow racemization of L- and D-alanine. Proton NMR studies showed that wild-type enzyme catalyzed a time-dependent exchange of the C alpha proton of D-alanine with solvent D2O and a slow exchange of the alpha proton of L-alanine; the latter slow exchange rate is the same for the C alpha proton of both L- and D-alanine with the K145Q mutant enzyme. Thus, in addition to binding pyridoxal 5'-phosphate, the active-site Lys-145 of D-amino acid transaminase is involved in several other important functions, i.e. it optimizes catalytic efficiency and it maintains stereochemical fidelity. The steady-state kinetic results on the K145Q mutant enzyme together with the findings on the relative racemization rates and the NMR protein exchange data suggest that an alternate base catalyzes abstraction of the alpha proton of substrate in this mutant D-amino acid transaminase.