9730035 Fink The proposed research involves two related areas: (1) the use of Fourier Transform Infrared (FTIR) spectroscopy, in the attenuated total reflectance (ATR) mode, to study enzyme-substrate intermediates and enzyme-inhibitor complexes, and (2) studies on beta-lactamase catalysis and inhibition, especially using a combination of site-directed mutagenesis and structural probes, including ATR FTIR. Beta-lactamases are the major source of resistance to penicillin antibiotic therapy. In spite of considerable study there is no consensus on their catalytic mechanism. A detailed kinetic and structural characterization of selected active-site mutants is planned to answer a number of specific questions regarding the beta-lactamase catalytic mechanism. Infrared spectroscopy can reveal critical information about catalytic mechanisms, as well as the conformation and interactions of bound inhibitors and drugs. By immobilizing the enzyme to the internal reflectance element of an ATR flow cell it is possible to acquire the IR spectra of stabilized enzyme-substrate or enzyme-inhibitor complexes. The ATR FTIR studies will initially be applied to complexes of beta-lactamase, and subsequently extended to other enzyme systems. Beta-lactamases are enzymes made by many bacteria and are the major source of resistance to penicillin. The planned research is aimed at understanding how these enzymes work, and how they may be inhibited. A detailed kinetic and structural characterization of selected active-site mutants will be undertaken to answer a number of specific questions regarding the catalytic mechanism. The information learned will contribute to understanding how beta-lactamases work and to the design of potential inhibitors. Infrared spectroscopy can reveal critical information about bond changes during catalysis. A new procedure will be developed to study enzyme-substrate intermediates and enzyme-inhibitor complexes, especially those of beta-lactamase.
