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    Kinetic Analysis of the Multi-Step Cytochrome P450 1A2 and 19A1 Enzymes

    Sohl, Christal Dyane
    : https://etd.library.vanderbilt.edu/etd-06152010-090554
    http://hdl.handle.net/1803/12580
    : 2010-06-23

    Abstract

    The kinetic characterization of cytochrome P450s that catalyze multi-step, sequential reactions is the focus of this work. Two novel substrates were identified for P450 1A2, one of which showed a high degree of homotropic positive cooperativity. Structural modeling was used to explain why cooperativity was substrate dependent. Pre-steady-state kinetics were used to characterize substrate binding, and fitting of these and the sigmoidal rate vs. substrate concentration plots yielded a kinetic model for the cooperative, sequential reaction. A robust heterologous expression and purification strategy for P450 19A1 was developed. Steady-state and pre-steady-state kinetic parameters were measured for the substrate, intermediates, and product. Unlike many other P450s that catalyze multi-step reactions, P450 19A1 was shown to be a distributive enzyme in that the intermediates freely dissociated during the course of the reaction. Global fitting of kinetic experiments resulted in a kinetic model of the three-step reaction catalyzed by P450 19A1.
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