Bacterial resistance to beta-lactam antibiotics continues to become more prevalent and more clinically important. A large part of the resistance can be understood and investigated experimentally in terms of the chemistry of the interactions of beta-lactam antibiotics with the active sites of two groups of bacterial enzymes, the beta-lactamases on one hand, which catalyze the hydrolysis of the antibiotics, and the D-alanyl-D-alanine transpeptidase/carboxypeptidases on the other, which catalyze the synthesis and maintenance of the peptide cross-links of bacterial cell walls, and which are inhibited by beta-lactam antibiotics. There is now good reason to believe that all of these beta-lactam binding sites have much in common. An understanding of the structure and function of these sites and of the relationship between them is fundamental to future antibiotic design --both beta-lactam and otherwise. The object of the proposed research is to explore further the chemical functionality and the substrate binding properties of a series of these active sites, using a number of modified substrates, novel inhibitors, and potential effecters. In particular, a novel series of acyclic substrates and derived inhibitors will be employed. A mechanistic study of these sites, designed to determine the role of the functional groups present and the relationship between the mechanism of action of these enzymes and that of other more closely studied enzymes, e.g. serine proteinases, will be performed. Structure-based methods will be employed in the design of new transition-state analog inhibitors. In order to understand the structural and mechanistic basis of bacterial beta- lactam-resistance through mutation of transpeptidases, a study of one such beta-lactam-resistant enzyme, penicillin binding protein 2a of methicillin- resistant Staphylococcus aureus (MRSA), will be commenced. These studies will lead to a clearer view of the chemistry of beta-lactamase and transpeptidase active sites, and thus to new directions in antibiotic design.