9727613 Manning The enzyme, D-amino acid transaminase (DAT), has intrinsic interest for its enzymological and its bacteriological properties, which are related. This research will focus on its stereochemical relationship to an enzyme that acts only on L-amino acids. The investigators have both structures (which are remarkably similar overall) on their Silicon Graphics workstation and they have compiled tables of sequence homologies and differences, which will be used for site-directed mutagenesis studies in order to convert one enzyme specificity to the other. The bonding between the subunits will be studied by a new procedure using very high resolution gel filtration. Studies on enzyme-catalyzed side reactions, coenzyme binding, and the stabilization of a reaction intermediate by mutant enzymes will be continued. This is a project to study the stereochemistry and structure of D-amino acid transaminase (DAT), a vitamin B6 (pyridoxal phosphate or PLP) containing enzyme. DAT supplies the building blocks for biosynthesis of peptidoglycans, constituents of bacterial cell walls. It has unusual features, such as inactivation by substrate under certain conditions. Understanding how physiological compounds mediate substrate inactivation could be predictive of in vivo regulation of the enzyme. Using this enzyme to understand how to alter stereospecificity in enzymic reactions is a potential payoff. The mechanistic studies proposed should be generalizable to other enzymes in the transaminase family.
