This Application describes a program of research designed to provide a critical evaluation of the use of solvent isotope effects in H2O-D2O mixtures, the """"""""proton-inventory"""""""" method, in studies of enzyme-catalyzed reactions. Such an assessment is necessary because serious questions have been raised about the ability of the proton-inventory method to enumerate catalytically active hydrogens in enzymic reactions, a purpose for which it has been used extensively and through which much of what is believed to be known about acid-base catalysis in enzymic systems is based. The proposed research consists of three parts: (1) examination of cyclodextrin-based models, (2) accurate determination of isotopic fractionation factors, and (3) studies with enzymes modified by site-directed mutagenesis. The results obtained would provide fundamental understanding of solvent isotope effects in H2O-D2O mixtures, and that would furnish practical guidelines for the correct application of the proton-inventory method to enzymic systems, and also supply criteria by which to judge the validity of conclusions reached through previous applications of the method.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM047539-03
Application #
2185016
Study Section
Biochemistry Study Section (BIO)
Project Start
1992-05-01
Project End
1995-04-30
Budget Start
1994-05-01
Budget End
1995-04-30
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Toronto
Department
Type
DUNS #
259999779
City
Toronto
State
ON
Country
Canada
Zip Code
M5 1-S8