The research program proposed here is designed to develop new low-temperature methods for the study of the reactions of enzymes in general, eventually using structural biophysical techniques such as nuclear magnetic resonance (NMR) spectroscopy. The ultimate goal is to develop a generally applicable method for the structural elucidation of enzyme-substrate and intermediate complexes. The reactions catalyzed by enzymes are usually extremely fast, and traditionally techniques for the characterization of enzyme-substrate and intermediate complexes have involved stopped- or quenched-flow often coupled with relatively low-resolution spectroscopic methods such as UV or CD spectroscopy. In this proposal, the idea is to mix rapidly enzyme and substrate solutions, and spray the mixture into a very cold liquid, such as liquid isopentane kept at liquid nitrogen temperatures. A commercially available device will be used to mix and freeze solutions into a very fine """"""""snow"""""""" in ca. 3 ms - 10 s, in steps of 3 ms. Although this snow will be examined eventually by solid-state NMR spectroscopy, allowing the structures of the intermediates on the reaction pathway to be essentially """"""""mapped"""""""" out in time, the present proposal will concentrate on characterizing the optimum conditions for the generation of enzyme frozen in an ice matrix which have uniform size and structure. Initial studies will focus on small molecules (e.g. penicillin G) examined by solid-state NMR spectroscopy under a wide range of conditions. Then the enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, which catalyses the condensation of shikimate-3-phosphate and phosphoenolpyruvate, will be used as a test case for the development of this novel technique of cryoenzymological solid-state NMR spectroscopy. Recent solid-state NMR methods for the measurement of internuclear distances will be used in conjunction with the rapid freezing technique in order to delineate the structure of the enzyme-intermediate complex of EPSP synthase bound to the enzyme active site.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM043215-01A2
Application #
3302204
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1991-08-01
Project End
1995-07-31
Budget Start
1991-08-01
Budget End
1992-07-31
Support Year
1
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Washington State University
Department
Type
Schools of Arts and Sciences
DUNS #
041485301
City
Pullman
State
WA
Country
United States
Zip Code
99164
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