We intend to covalently modify a-chymotrypsin with various aryl azides and diazocompounds. Crystals of the pure modified proteins will be photolyzed at -196 degrees. We will use ESR spectroscopy to directly detect the resultant enzyme bound nitrenes and carbenes. We will use ESR to measure the kinetics of reaction of the nitrenes and carbenes with the enzyme. We expect the reaction will involve hydrogen atom transfer by quantum mechanical tunneling to give a radical-pair. The kinetic barrier will be fit to an Eckart barrier to obtain the barrier width-the distance travelled by the tunneling hydrogen from an amino acid residue to the carbene or nitrene. We will detect the product radical pair by ESR. The spectrum of the radical pair will give a second measure of the separation of the pair. The nuclear hyperfine interaction present in the pair will allow us to identify the amino acid residue which is the hydrogen donor. We will study the products of the low temperature photochemistry. It is proposed that cryogenic conditions will improve the labelling efficiency. If successful this project will produce an important new method in photoaffinity labelling for identifying constituents of the active sites of biomolecues.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034823-02
Application #
3286476
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1986-07-01
Project End
1989-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
2
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Ohio State University
Department
Type
Schools of Arts and Sciences
DUNS #
098987217
City
Columbus
State
OH
Country
United States
Zip Code
43210