The proposed research involves studies on b lactamase catalysis and inhibition, using a combination of site directed mutagenesis and structural probes, especially attenuated total reflectance (ATR) FTIR. b Lactamases are the major source of resistance to penicillin antibiotic therapy, an increasingly common health problem, and are thus potential targets for the design of new antibiotics. In spite of considerable study the mechanism of action of the b lactamases remains somewhat of an enigma. Although the active site residues have been identified through structural, chemical and mutagenesis studies, and several possible mechanisms have been proposed, there is no consensus on the catalytic mechanism, and many outstanding fundamental questions remain. Through detailed kinetic and structural characterization of selected active site mutants we hope to answer a number of specific questions regarding the b lactamase catalytic mechanism, including which ionizing groups are responsible for the bell shaped pH rate profiles; the role of the conserved Lys73; why some substrates with wild type enzy me, and some mutants with all substrates, show a branched path mechanism (reflected in substrate induced inactivation or reversible suicide inhibition), and the nature of the resulting inactive acyl enzyme species; and the reason why the deacylation rate is so much faster in the Class A B lactamases compared to the penicillin target enzymes (transpept-idases and D Ala,D Ala carboxypeptidases). We anticipate that the inform-ation learned in this investigation will be very valuable in understanding how b lactamase catalysis works and in the design of new inhibitors which would be potential antibiotics. %%% Lactamases are the major source of resistance to penicillin antibiotic therapy, an increa ingly common health problem, and are thus potential targets for the design of new antibiotics. In spite of considerable study, the mechanism of action of the b lactamases remains somewhat of an enigma. Through detailed kinetic and strutural characterization of selected active site mutants we hope to answer a number of specific questions regarding the B lactamase catalytic mechanism. The mutants will be characterized using both kinetic and stnuctural probes. We propose a novel approach to obtaining information about the structure of enzyme-substrate complexes (and enzyme-inhibitor complexes), namely the use of attenuated total reflectance (ATR) FTIR (Fourier Transform Infrared spectroscopy),in conjunction with immobilized enzyme in a flow cell. Normally transient intermediates will be stabilized either with low temperatures, or by using relatively inactive mutants. The information obtained in the proposed studies should facilitate the development of improved b lactam antibiotics.
