<p> Cytochrome P450s (P450s) are a superfamily of enzymes that catalyze oxidation of unactivated hydrocarbons. However, the means by which P450s control (1) regioselectivity of their activity and (2) specificity in their molecular recognition remain largely elusive. Toward investigation of the role of dynamics in the regioselectivity of the archetypal cytochrome P450cam (P450cam), two-dimensional infrared spectroscopy has been applied with heme-bound carbon monoxide (CO) as an infrared probe of the active site. The data support a model for P450cam regioselectivity in which binding of different substrates to P450cam variably stabilizes the active site into two distinct states, each associated with different dynamics linked to different levels of regioselectivity. To investigate the role of conformational heterogeneity in P450cam substrate specificity, infrared spectoscopy was combined with the site-specific incorporation of nitrile probes at distinct P450cam microenvironments. This approach enabled differentiation of changes experienced at each of those environments when <i> d</i>-camphor and/or CO binds to the active site. Finally, the impact of conformational heterogeneity on the affinity of substrate molecular recognition by wild-type and mutant P450cam was evaluated using both CO and nitrile probes. This study suggests that the nature of the conformations populated in the unbound states influences the affinity for different substrates. Collectively, these studies provide new insight into the roles of conformational heterogeneity and dynamics in P450cam activity. Furthermore, these studies help to lay the foundation for efforts toward understanding the roles of conformational heterogeneity and dynamics in the function of human P450s, for which unraveling the mechanisms involved in Phase I metabolism is a topic of great pharmacological concern. </p><p>
| Identifer | oai:union.ndltd.org:PROQUEST/oai:pqdtoai.proquest.com:10604874 |
| Date | 10 October 2017 |
| Creators | Basom, Edward J. |
| Publisher | Indiana University |
| Source Sets | ProQuest.com |
| Language | English |
| Detected Language | English |
| Type | thesis |
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