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.
