Flavoenzymes catalyze a wide variety of biochemical reactions and are commonly observed as electron transport proteins. The redox reactive portion of the enzymes is the isoalloxazine ring system of the flavin cofactor. It is known that the protein environment modulates the redox potential of the flavin, for example, "tuning" its redox potential to favor either a one-electron transfer (electron transfer proteins) or a two-electron transfer (oxidation reactions). This thesis presents an in depth structural study of the flavoenzyme, cholesterol oxidase (EC 1.1.3.6) from Streptomyces sp. SA-COO (SCOA) a multifunctional enzyme that oxidizes and isomerizes 3-beta-hydroxysteroids. This work was pursued in order to further our understanding of the mechanisms through which the protein interacts with the isoalloxazine system and modulates reactivity. Previous kinetic experiments have identified an active site asparagine (N485) and a histidine residue (H447) both of which are critical to the oxidative activity of the enzyme. On an atomic scale the role of the asparagine residue was unknown. Using mutagensis and crystallographic techniques we have characterized this novel N-H ··· pi protein-flavin interaction. SCOA crystals diffract to sub-atomic resolution providing us with a unique view of the protein bound isoalloxazine system. These atomic resolution maps have revealed unexpected structural features that were not previously apparent in the 1.5 A resolution of SCOA. For example, a second narrow pathway leading directly to the isoalloxazine system was discovered, which has provided a more complete mechanistic understanding of the reactions catalyzed by SCOA. Five atomic resolution structures of SCOA at varying pH values are reported. Differences among these structures provide insight into the affect of pH on protein structure and have revealed structural differences resulting from an inadvertent reduction of the cofactor. For example, these stru
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.82909 |
| Date | January 2002 |
| Creators | Lario, Paula I. |
| Contributors | Vrielink, Alice (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Biochemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001973820, proquestno: AAINQ88505, Theses scanned by UMI/ProQuest. |
Page generated in 0.0129 seconds