Please use this identifier to cite or link to this item: http://hdl.handle.net/10125/100889

Mechanistic studies of biotin synthase using isotopically-labeled dethiobiotin

File Description Size Format  
Fugate Corey r.pdf Version for non-UH users. Copying/Printing is not permitted 22.9 MB Adobe PDF View/Open
Fugate Corey uh.pdf Version for UH users 22.89 MB Adobe PDF View/Open

Item Summary

dc.contributor.author Fugate, Corey
dc.date.accessioned 2016-02-19T22:09:30Z
dc.date.available 2016-02-19T22:09:30Z
dc.date.issued 2012-12
dc.identifier.uri http://hdl.handle.net/10125/100889
dc.description Ph.D. University of Hawaii at Manoa 2012.
dc.description Includes bibliographical references.
dc.description.abstract Biotin synthase is a S-adenosylmethionine radical enzyme that catalyzes the conversion of dethiobiotin to biotin by replacing two hydrogen atoms in dethiobiotin with a sulfur atom to form the thiophane ring of biotin. Key questions remain unanswered in the mechanism of conversion including the role of the [2Fe-2S] coordinating cysteine residues, the structure of the intermediate state, and the rate-determining step of the reaction. The [2Fe-2S] coordinating cysteine residues have been found to be essential for conversion of DTB to biotin. Mutation of any of the coordinating cysteine residues to aspartate abolished activity. Mutation of the coordinating cysteine residues to aspartate causes a significant shift in the redox potential of the [2Fe-2S] cluster. This shift in redox potential is likely the cause of inactivity as the [2Fe-2S] cluster is likely destroyed once reduced by an external electron donor. It is necessary to produce isotopically labeled DTB in order to probe various aspects of the mechanism of biotin synthase. The biotin biosynthetic pathway has successfully been co-opted to generate various isotopically labeled compounds. d3-9-dethiobiotin was used to determine if hydrogen atom abstraction from the C9 position of DTB is rate limiting. A KIE of 7.95 was measured that confirms that hydrogen atom abstraction is in fact rate limiting. The intermediate state of catalysis is proposed to exist as DTB covalently bound to an intact reduced [2Fe-2S] cluster. HYSCORE spectroscopy was used to examine the intermediate state generated using 13C-(9-methyl)-dethiobiotin and provided the first direct evidence for the existence of the proposed intermediate. In summation, this body of would has provided many insights into the mechanism of sulfur insertion catalyzed by biotin synthase.
dc.language.iso eng
dc.publisher [Honolulu] : [University of Hawaii at Manoa], [December 2012]
dc.relation Theses for the degree of Doctor of Philosophy (University of Hawaii at Manoa). Chemistry.
dc.subject biotin synthase
dc.subject dethiobiotin
dc.title Mechanistic studies of biotin synthase using isotopically-labeled dethiobiotin
dc.type Thesis
dc.type.dcmi Text
Appears in Collections: Ph.D. - Chemistry


Please email libraryada-l@lists.hawaii.edu if you need this content in ADA-compliant format.

Items in ScholarSpace are protected by copyright, with all rights reserved, unless otherwise indicated.