Reaction of Ethylene Glycol with Adenosylcobalamin-Dependent Ethanolamine Ammonia-Lyase 公开
Guidry, Bryce (Spring 2022)
Published
Abstract
Adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia lyase (EAL) is associated with gut microbe homeostasis and disease conditions in the bacteria, Escherichia coli and Salmonella typhimurium. The enzyme operates by homolytically cleaving the cobalt-carbon (Co-C) bond in AdoCbl to generate a short-lived radical state that is then used to trigger hydrogen atom transfers and radical rearrangement. When the Co-C bond is cleaved, the cobalt in cobalamin changes formal redox state from Co(III) to Co(II). Since the corresponding cobalamin states, Cbl(III) and Cbl(II), absorb different wavelengths of light, this project observed the progress of the AdoCbl-dependent EAL reaction by using optical spectroscopy. The long-term goal of this project is to characterize the reaction of the pseudo-substrate, ethylene glycol (EG), with the AdoCbl-EAL holoenzyme in the Salmonella system, toward understanding mechanisms for stabilizing and channeling highly-reactive radical species, and to develop a time-resolved reaction-measurement system in solution at room reaction, for kinetic studies of mechanism. The reaction of the AdoCbl-bound EAL holoenzyme with EG at different concentrations is studied in buffered aqueous solution at room temperature (295 K). Ultraviolet (UV)/visible absorbance spectroscopy was used to characterize the AdoCbl-dependent EAL reaction at room temperature. EG leads to the production of a distinct, non-native cobalt species in place of the cob(II)alamin produced by the native substrate. This unknown cobalt species has a distinct absorption feature in the range 476 nm to 525 nm and an absorption peak at 529 nm. Reaction in the presence of dithiothreitol showed that dioxygen is not involved in the reaction. The EG concentration dependence of the reaction was characterized, and a kinetic model proposed, that accounts for the first-order, exponential kinetics and concentration-dependence. Overall, visible absorption spectroscopy and time-resolution of the EG-EAL reaction in the S. typhimurium enzyme in solution at room temperature are developed, and the kinetic model is a foundation for future studies of the role of dynamics in EAL catalysis.
Table of Contents
I. Introduction 1.
II. Experimental Methods 8.
- Ultraviolet-Visible Absorption Spectroscopy 8.
- Preparation of Stock Solutions and Initial Sample Conditions 9.
- Protocol for Spectroscopy Experiments 10.
- Baseline Correction Analysis 12.
III. Results 13.
- Absorption Spectra of AdoCbl Samples Before and After Reaction 13.
- Absorption Spectra of AdoCbl Reaction Using DTT 14.
- Dependence of the EAL-Bound Cobalamin Absorption Spectra on EG Concentration 16.
IV. Discussion 21.
- Determination of Sonication as a Means of Reducing Sample Absorbance 21.
- Analysis of the Cobalamin Spectrum Following EG Addition to the EAL-AdoCbl Complex 21.
- Influence of DTT on the Cobalamin Spectrum Following EG Addition to the EAL-AdoCbl Complex 22.
- Concentration-Dependence of the Kinetics of the EG-Generated Cobalamin Species in EAL 23.
- Analysis of the Kinetics of Reaction Induced by EG in the EAL-AdoCbl Complex 24.
- Structure of the Modified AdoCbl, XCbl Product of EG Reaction in EAL 29.
- Summary and Conclusions 30.
V. References 32.
About this Honors Thesis
School | |
---|---|
Department | |
Degree | |
Submission | |
Language |
|
Research Field | |
关键词 | |
Committee Chair / Thesis Advisor | |
Committee Members |
Primary PDF
Thumbnail | Title | Date Uploaded | Actions |
---|---|---|---|
|
Reaction of Ethylene Glycol with Adenosylcobalamin-Dependent Ethanolamine Ammonia-Lyase () | 2022-04-11 21:43:41 -0400 |
|
Supplemental Files
Thumbnail | Title | Date Uploaded | Actions |
---|