Studies will be conducted to determine the mechanism of action of chalcone isomerase and the factors which are responsible for catalysis. The isomerization of chalcones to flavanones is an intramolecular reaction and a member of the class of biochemical reactions involving addition to a double bond conjugated to a carbonyl group. The proposal that the enzyme utilizes covalent catalysis will be tested using a suicide substrate designed to trap the putative covalent enzyme intermediate. The metal content of the enzyme will be determined using atomic absorption analysis to evaluate the possibility of electrophilic catalysis. The hypothesis that an active site histidine is essential for catalysis will be tested by investigating the diethylpyrocarbonate modification of the enzyme. Affinity labels will be designed to identify a putative enzymic base. The equilibrium constant for enzyme bound chalcone/flavanone species will be determined using C13 NMR spectroscopy. The nature of the rate-limiting step will be studied using deuterium isotopes. Isotope exchange studies will be performed to determine if the enzyme catalyzes proton exchange from C-3 of the flavanone product. The stereochemistry at C-2 and C-3 of the flavanone product will be determined and its implications evaluated. The hypothesis that the enzyme utilizes a """"""""one base-mechanism"""""""" will be evaluated using isotope transfer and NMR saturation transfer. Chalcones have a number of biological activities. They have an anti-inflammatory activity and are useful in the treatment of allergies and gastrointestinal ulcers. As a result of our studies, it is inevitable that we will learn more about the chemistry and reactivity of this class of compounds.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034832-03
Application #
3286508
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1985-09-05
Project End
1988-09-18
Budget Start
1987-09-01
Budget End
1988-09-18
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794