Wei Li

Graduation Semester and Year

2014

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry

Department

Chemistry and Biochemistry

First Advisor

Brad Pierce

Abstract

Cysteine dioxygenase (CDO) is an non-heme mononuclear iron enzymes that catalyzes the O2-dependent oxidation of L-cysteine (Cys) to produce cysteine sulfinic acid (CSA). CDO controls cysteine levels in cells and is a potential drug target for some diseases such as Parkinson's and Alzhermer's. Several crystal structures of CDO have been determined and they reveal a ferrous iron active site coordinated by three histidine residues. This feature is divergent from the monoanionic 2-histidine-1-carboxylate coordination typically observed within the non-heme mononuclear iron super family of oxidase/oxygenase enzymes. Furthermore, within 3.3 Å of the CDO active site iron is an unusual covalently crosslinked cysteine-tyrosine pair (C93-Y157). To date, only 3 other enzymes have been identified with a similar Cys-Tyr post-transitional modification and the role of this modification in CDO is still unknown. Due to the lack of structural evidence of oxygen-bound intermediates, the mechanism of CDO remains unclear. In this work, a transient intermediate FeIII-superoxo was discovered by chemical rescue reaction and characterized using UV-vis, EPR and resonance Mossbauer. To probe the influence of second-sphere enzyme-substrate interaction, the steady-state kinetics and O2/CSA coupling were measured for wild-type CDO and selected active site variants (Y157F, C93A, H155A). In additional, using CN- as a probe, the influence of the C93-T157 pair to the active site is investigated on EPR. Key substrate-enzyme interaction was also investigated by substrate specificity of CDO. Selected thiol-containing compounds were incubated with CDO for steady-state kinetic analysis using NMR. LC-MS confirmed the presence of products and dioxygenase activity.

Disciplines

Chemistry | Physical Sciences and Mathematics

Comments

Degree granted by The University of Texas at Arlington

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