One of the holy grails in contemporary enzymology is to identify and characterize enzyme motions at the femtosecond time scale and their relationship to the reorganization and distance sampling motions that determine the rate of the chemical step. The objective of this application is to characterize the enzyme active site dynamics at the femtosecond to picosecond time scale (using 2D IR vibrational spectroscopy) and relate them to the catalyzed H-transfer reaction (using temperature dependence of the intrinsic kinetic isotope effects - KIEs). The central hypothesis is that the spectroscopically measured enzyme dynamic motions and the temperature dependence of KIEs can be correlated within the framework of the Marcus-like models, yielding a unified model that relates the enzyme's dynamics and functionality. We plan to test our central hypothesis and accomplish the objective of this application using the enzyme formate dehydrogenase (FDH) as a model system by pursuing the following three specific aims: 1) Establish the dynamic signatures of an optimized tunneling-ready configuration. The working hypothesis for this aim is that our recent discoveries that the active- site dynamics of FDH in a transition-state-analog complex are unusually rigid and its intrinsic KIEs are temperature independent reflect the formation of a well organized, tunneling-ready configuration. We will test this hypothesis by measuring the temperature dependence of the intrinsic KIEs and the frequency- frequency time correlation function (FFCF) for the antisymmetric stretch of the azide anion in transition state analog complexes of site-specific mutants of FDH. 2) Characterize the time scales for active-site motions that reflect donor-acceptor distance sampling. The working hypothesis is that the promoting vibrations that have been invoked in connection with temperature dependent KIEs occur on the time scale of hundreds of femtoseconds. We will test this hypothesis by measuring the temperature dependence of the enzyme dynamics using 2D IR spectroscopy and correlating that temperature dependence with that of the intrinsic KIEs. 3) Determine whether the active site dynamics of FDH are localized or collective. Our working hypothesis is that the dynamic motions of the enzyme that contribute to donor acceptor distance sampling are collective motions of the active site. We will test this hypothesis by measuring the dynamics of the active site using a second vibrational chromophore, azo-NAD+, in the ternary complex of FDH with azide to compare the dynamics measured at this second location with those for the azide. The proposed research will identify the relationships between the various components of the active site dynamics at the femtosecond to picosecond time scale and the intrinsic KIEs measured with the azo-NAD+. These outcomes are expected to have significant overall impact because identifying the relationship between active-site dynamics and the kinetic properties of the catalyzed reaction will allow us to exploit this relationship to address the controversy surrounding the role of such dynamics in enzyme catalyzed H-transfer reactions.
There is the promise that the insights gained from this research will clarify the influence of enzyme motions on the chemical step contributing to a comprehensive theory of enzyme-catalyzed reactions. The outcomes of this research will enable efforts to incorporate an understanding of the role of enzyme motions in structure-based rational drug design efforts improving the potential for success in developing new pharmaceuticals to treat an array of diseases.
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