Low Temperature Trapping of Intermediates in Enzyme- Catalyzed Reactions

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This research involves a combination of method development for the acquisition of structural information concerning enzyme- substrate or enzyme-inhibitor complexes, and application to the study of slow-binding transition-state analog inhibitors, early events in protease catalysis and intermediates in glycosidase catalysis. The focus of the investigation is on the application of attenuated total reflectance (ATR) Fourier transfer infrared spectroscopy to study enzyme-substrate intermediates, stabilized at low temperatures, and enzyme- inhibitor complexes, and the use of immobilized enzymes and stopped-flow kinetics in cryoenzymology studies. FT-IR has the potential to reveal not only new bonds formed in enzyme-ligand complexes, but also bond deformations and polarization. The importance of enzymes in numerous areas of science, medicine and industry needs no elaboration. Recent developments in molecular biology and biotechnology have led to a greatly increased interest in all aspects of enzymology. A major problem in determining the details of an enzyme. Catalytic mechanism is the short lifetime of enzyme - substrate complexes and intermediates, making it particularly difficult to obtain structural information. The lifetimes of normal transient species can be significantly increased at sintably low temperatures. The development of some methods which should facilitate the acquisition of structural data are underway here.