Characterizing the Configurational Fluctuations that Contribute to Enzyme Catalysis for Ethanolamine Ammonia Lyase Öffentlichkeit

Abstract

Ethanolamine Ammonia Lyase (EAL) is a coenzyme-B12 dependent enzyme found in Salmolella typhimurium that converts the substrate ethanolamine into two products: acetaldehyde and ammonia. This process is utilized by the bacterium to produce adenosine triphosphate (ATP) and is the sole source of carbon and nitrogen. The minimalistic mechanism for EAL catalysis consists of six steps. First-order rate constants of the rate limiting chemical reaction step (radical rearrangement) are determined by using temperature-step-triggered decay of the cryotrapped substrate radical intermediate, and time-resolved, full-spectrum electron paramagnetic resonance (EPR) spectroscopy over a wide temperature range. The piecewise-linear Arrhenius dependence shows a kinetic bifurcation (220 K) and kinks (217 K) in the decay components, which arise from the effective quenching of collective configurational fluctuations that are coupled to the radical rearrangement reaction. Below the temperature transition, the reaction persists by local protein fluctuations. The origins of the dynamical transitions are described by using a temperature-dependent free energy landscape (FEL) model. Experiments using deuterated ethanolamine and utilizing these same methods account for the kinetic isotope effects (KIE), determine the origin of the microscopic states from the FEL model, and reveal a distinct set of specific collective-atom protein motions that contribute to an additional step in the EAL catalytic cycle. These dynamical temperature transitions are shifted to higher temperatures through the introduction of varying sucrose concentrations, which quantify the influence of the solvent on the dynamics of the protein and protein function. The results and analysis established here reveal key insights in enzymology, specifically the role of configurational protein fluctuations in catalysis.

Table of Contents

1.1       Enzyme Catalysis and Dynamics                                                                               

1.1.1    Timescale of Enzyme Fluctuations                                                                           

1.1.2    Dielectric Relaxation Studies of Protein Fluctuations                                              

1.1.3    Categories of Protein Fluctuations                                                                            

1.1.4    Temperature Dependence on Protein Fluctuations                                                    

1.1.5    Solvent Contributions to Protein Dynamics                                                              

1.2       Ethanolamine Ammonia-Lyase                                                                                 

1.2.1    B12-Dependent Superfamily                                                                                      

1.2.2    Ethanolamine Catabolism Pathway                                                                            

1.2.3    Structure of EAL                                                                                                        

1.2.4    Minimal Catalytic Mechanism for EAL                                                                 

1.2.5    EAL Active Site                                                                                                      

1.2.6    Paramagnetic Species in the EAL Catalytic Cycle                                                  

1.3       Electron Paramagnetic Resonance                                                                          

1.3.1    Paramagnetic States of EAL                                                                                   

1.3.2    CW-EPR Spectral of the Substrate Radical State                                                   

1.4       Outline of Dissertation

About this Dissertation

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Physics
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biophysics, General
Stichwort	enzyme
Committee Chair / Thesis Advisor	Warncke, Kurt, Emory University
Committee Members	Roth, Connie, Emory University Boettcher, Stefan, Emory University Finzi, Laura, Emory University Weinert, Emily, Emory University

Zuletzt geändert

Primary PDF

Thumbnail	Title	Date Uploaded	Actions
	Characterizing the Configurational Fluctuations that Contribute to Enzyme Catalysis for Ethanolamine Ammonia Lyase ()	2018-04-05 21:12:06 -0400	Press to Select an action Download

Supplemental Files

Thumbnail	Title	Date Uploaded	Actions