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

Kohne, Meghan (Spring 2018)

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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

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