Manning 9404332 Studies on the mechanism and stereochemistry of an enzyme that makes D-glutamate in bacteria will be continued. This enzyme, D-amino acid transaminase (DAT), is a target for thedesign of novel antibacterial agents since it is present in bacterial cells but not in the mammalian cells. Site-directed mutagenesis studies at the active site Lys that binds cofactor PLP suggest the presence of an alternate base for catalysis since these mutant (attenuated) enzymes retain intrinsic catalytic activity. The proposed studies will build on these findings. If the optimal base in the wild-type enzyme is replaced, then the results indicate that the alternate catalytic base can assume some of its function but with a reduced degree of efficiency. This alternate base has been identified as Lys-267 and the recent solution of the structure of the enzyme shows that it is indeed adjacent to the active site.The recent elucidation of the structure of this enzyme by our collaborator, Dagmar Ringe, has suggested many other candidate sites for site-directed mutagenesis. These include side chains that bind substrates, cofactor PLP, subunit contacts, or in catalysis. The mutant enzymes will be evaluated by multiple criteria in order to establish the role of the replaced sites. Studies on the identity of the natural endogenous amine that is released from the active site by substrates will be continued and any role in catalysis will be explored. Mechanistic studies on the slow inactivation of the enzyme by normal substrates will focus on the structure of the inactive enzyme with a fragment of substrate attached. The alternate catalytic base, Lys-267, which is operative in the active site mutant enzymes, has been shown to be required for the slow inactivation by normal substrates. On the other hand, when the active site Lys that binds PLP in the wild-type enzyme is replaced by Gln or Asn, these mutant enzymes are still inactivated by normal substrates. Hence, the active-site Ly s is not required for this inactivation. These studies will be expanded. The process of re-activation at mildly acidic pH will be studied. The structure of pseudo-substrates with the enzyme will also be evaluated by x-ray diffraction techniques. A general assay for D-amino acids employing DAT is being developed. The procedure can quantitate all of the D-amino acids except D-proline. The pool of D-amino acids needed for bacterial peptidoglycan synthesis will be determined after exposure of bacteria to suicide substrates of DAT. Studies on the anti-bacterial effects of ~-keto acids in gram-positive and gram-negative strains will be continued. %%% The enzyme D-amino acid transaminase is being studied with a dual objective. First, its mechanism will be elucidated in order to understand how it stereochemically prefers the D-isomers of amino acids rather than the L-isomers, which occur in humans. This goal is being addressed using the methods of recombinant DNA technology. Recent studies in our laboratory have provide evidence for a second catalytic base at the entrance of the active site of the enzyme. These studies will be continued in order to show how this enzyme works in the bacterial cell by providing the cells with the essential D-amino acids. The second objective is that with this information we hope to synthesize inhibitors for this enzyme that could be useful antibiotics, especially towards penicillin-resistant microorganisms. Since the enzyme occurs in bacteria but not in humans, this inherent selectivity could be advantageous. ***
