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 an 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 o the structure and function of these sites and of the relationship between them is fundamental to future antibiotic design--both beta-lactam and otherwise. Th 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. A mechanistic study of these sites, designed to determine the role of the functional groups present and the relationship between the proteinases, will be performed. Computational methods will be employed in order to interpre the results in terms of available crystal structures of these enzymes and to thus establish new guidelines to inhibitor design. In order to understand the structural and mechanistic basis of bacterial beta-lactam-resistance through mutation of transpeptidases, one important example of such beta-lactam-resistant enzymes, penicillin binding protein 2a of the methicillin-resistant Staphylococcus aureus (MRSA), will be studied in detail. 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.
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