This proposal outlines a research effort to delineate the roles of active site amino acids in human carbonic anhydrase II (CA II) in catalysis and drug (sulfonamide) binding. Sulfonamide inhibition of this enzyme has been used to treat glaucoma, epilepsy and ulcers. The effect of single-site mutations (prepared by oligonucleotide-directed mutagenesis) on CA II function, stability and structure will be measured. The goal of the proposed studies is to further our understanding of the importance of protein/substrate and protein/protein interactions for binding and catalysis. Experiments are designed to: 1) delineate the pathway and mechanism of CA II-catalyzed inter- and intramolecular proton transfer reactions; 2) measure the importance of general base catalysis or a proton shuttle mechanism in catalysis by CA II; 3) determine the effect of active site amino acids on the pKa of the enzyme-bound zinc-water ligand; 4) quantitatively evaluate the importance of hydrophobic interactions for binding and catalysis; 5) determine the effects of single amino acid changes on transition state structure; 6) quantitate the importance of isozymic active site variations; and 7) measure the effect of single site mutations on the structure of CA II using one- and two- dimensional NMR techniques. Information gained will impact on our understanding of the mechanism of carbonic anhydrases and other metalloenzymes, as well as the mechanism of proton transfers in all proteins. Quantitative dissection of the contribution of various residues to binding and catalysis for carbonic anhydrase hopefully can be generalized to other enzymes and will play a significant role in the design of active site inhibitors and new protein catalysts, as well as enlarge our understanding of enzymic catalysts.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
1R29GM040602-01
Application #
3467222
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1988-07-01
Project End
1993-06-30
Budget Start
1988-07-01
Budget End
1989-06-30
Support Year
1
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Duke University
Department
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
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