dc.creator | Sohl, Christal Dyane | |
dc.date.accessioned | 2020-08-22T17:06:37Z | |
dc.date.available | 2010-06-23 | |
dc.date.issued | 2010-06-23 | |
dc.identifier.uri | https://etd.library.vanderbilt.edu/etd-06152010-090554 | |
dc.identifier.uri | http://hdl.handle.net/1803/12580 | |
dc.description.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. | |
dc.format.mimetype | application/pdf | |
dc.subject | global fitting | |
dc.subject | cooperativity | |
dc.subject | aromatase | |
dc.subject | enzymes | |
dc.subject | pre-steady-state kinetics | |
dc.subject | kinetics | |
dc.subject | P450s | |
dc.title | Kinetic Analysis of the Multi-Step Cytochrome P450 1A2 and 19A1 Enzymes | |
dc.type | dissertation | |
dc.contributor.committeeMember | David Hachey | |
dc.contributor.committeeMember | Michael Waterman | |
dc.contributor.committeeMember | Claus Schneider | |
dc.contributor.committeeMember | Richard Armstrong | |
dc.type.material | text | |
thesis.degree.name | PHD | |
thesis.degree.level | dissertation | |
thesis.degree.discipline | Biochemistry | |
thesis.degree.grantor | Vanderbilt University | |
local.embargo.terms | 2010-06-23 | |
local.embargo.lift | 2010-06-23 | |
dc.contributor.committeeChair | F. Peter Guengerich | |