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Title page for ETD etd-06152010-090554


Type of Document Dissertation
Author Sohl, Christal Dyane
URN etd-06152010-090554
Title Kinetic Analysis of the Multi-Step Cytochrome P450 1A2 and 19A1 Enzymes
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
F. Peter Guengerich Committee Chair
Claus Schneider Committee Member
David Hachey Committee Member
Michael Waterman Committee Member
Richard Armstrong Committee Member
Keywords
  • global fitting
  • cooperativity
  • aromatase
  • enzymes
  • pre-steady-state kinetics
  • kinetics
  • P450s
Date of Defense 2010-06-08
Availability unrestricted
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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