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.
<p> 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.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-06152010-090554 |
| Date | 23 June 2010 |
| Creators | Sohl, Christal Dyane |
| Contributors | David Hachey, Michael Waterman, Claus Schneider, F. Peter Guengerich, Richard Armstrong |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-06152010-090554/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
Page generated in 0.0184 seconds