Robert W. ShawAssociate Professor Ph.D., The Pennsylvania State University, 1976 Postdoctoral Study, Institute for Enzyme Research, University of Wisconsin, Madison
Phone: (806) 742-3055 Fax: (806) 742-1289 |
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Robert W. ShawAssociate Professor Ph.D., The Pennsylvania State University, 1976 Postdoctoral Study, Institute for Enzyme Research, University of Wisconsin, Madison
Phone: (806) 742-3055 Fax: (806) 742-1289 |
|
Professor Shaw's research program involves the study of the structure and function of physiologically important metalloenzymes. Structures of the metal binding sites of these biological molecules are under investigation largely through the use of optical and electron paramagnetic resonance (EPR) spectroscopy. Mechanistic details of the functions of metal ions in these proteins are being studied through the use of spectroscopy coupled to rapid kinetic techniques in order to detect short-lived species such as reaction intermediates and enzyme complexes with substrates or inhibitors. Enzyme structure-function relationships are analyzed by site-directed mutagenesis combined with presteady-state spectroscopy and steady-state enzyme kinetic techniques.
Current interest is focused on the metallo-ß-lactamases purified from the pathogenic organism Bacillus cereus. ß-Lactamases catalyze the hydrolytic inactivation of ß-lactam antibiotics such as penicillins and cephalosporins and thereby constitute the major mechanism by which pathogenic bacteria become resistant to such antibiotics. Clearly, understanding the reaction mechanisms of ß-lactamases is important to the pharmaceutical industry. Continuous-flow/freeze-quench techniques are being used to generate time-resolved EPR spectra on the millisecond time scale of the Co(II)-reconstituted ß-lactamase during its reaction with ß-lactam antibiotics. Such studies yield information concerning the number and chemical identity of reaction intermediates and hence are very useful in exploring enzymatic reaction mechanisms.
The structural gene for the B. cereus 5/B/6 ß-lactamase has been cloned into an Escherichia coli expression vector and techniques have been devised to purify the hyperexpressed B. cereus enzyme (both wild type and mutant forms) to homogeneity in very high (~90%) yield. Using site-directed mutagenesis techniques and this recombinant construct, a study of the relationship of protein structure to enzymatic function of this enzyme has begun. So far, 44 mutant forms of the enzyme have been expressed and 12 of these purified and characterized. These experiments are designed to identify those amino acid residues in the protein which are important in substrate specificity and catalysis.
Inhibition of metallo-ß-lactamases has proven to be problematic. In a collaborative project with Professor John Buynak's group at Southern Methodist University we have used a national drug design approach to test compounds as putative irreversible or reversible inhibitors of the B. cereus 5/B/6 ß-lactamase. In separate studies, we are using combinatorial chemistry methods to discover a new class of ß-lactamase inhibitors. The results of these experiments may yield further insight into the mechanism of the enzymatic reaction and be of importance to the pharmaceutical industry.
Selected Publications
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