Studies on bacteria D-amino acid transaminase will focus on the functional groups at its active site, its stereochemical mechanism, and its role in bacteria as a target for enzyme activated inhibitors. The gene that encodes this enzyme allows several hundred milligrams of pure enzyme to be prepared in one week. Several site-directed mutagenesis studies have been carried out; the most interesting is the change of the active site Lys-145, which binds pyridoxal-5'- phosphate, to Gin. This mutant turns over very slowly so that the kinetics of formation of several intermediated can be easily measured. Thus, the formation of the Schiff base with the substrate, D-alanine, takes place over a period of minutes at 27o. Exogenous (added) amines have a significant effect on the rate of Schiff base (aldimine formation). The rate of formation of the E-PMP and/or ketimine form takes place over a period of hours and the exogenous amines have no effect on this step with this mutant enzyme. The effect of a large number of amines with the K145Q mutant enzyme and the K145N mutant will be ascertained in the present proposal. The K145Q mutant enzyme is under less stringent stereochemical control than is the wild-type enzyme (see enclosed galley proofs of manuscript in press). This result will be extended with isotope studies to determine which proton of the C-4 of PMP is retained or released in the wild- type enzyme as well as in the K145Q and K145N mutant enzymes. L-Ala and L-Glu can undergo one turnover with D-amino acid transaminase in the half-reaction to yield the corresponding keto aid and the pyridoxamine-5'-phosphate form of the enzyme. However, in the second half of the reaction, i.e., transfer o the amino group to another keto acid only D-amino acids are formed. Therefore, stereochemical fidelity is maintained. These experiments will also be performed with the K145Q and K145N mutant enzyme to determine whether there is less stereochemical constraint as measured by this technique also. D-serine, a relatively slow substrate, generates a quinonoid- like intermediate band at 493 nm. The half-life of species is of the order of hours and its disappearance is correlated with inactivation of the enzyme. Preliminary studies by stopped-flow methods indicate that another quinonoid with a rapid turnover is formed. These studies will be continued. The mode of inactivation by D-serine by use of 14C-labeled compound and peptide mapping will be investigated. D-Amino acid transaminase inactivated by chloro-D alanine, cycloserine, or gabaculine will be investigated by difference maps once the structure of the wild-type enzyme is established. The inactivation of D-amino acid transaminase by various suicide substrates and the accumulation of intermediates during this process will be studied.
