Contributions of Protein and Coupled Solvent Dynamics to Ethanolamine Ammonia-Lyase Enzyme Catalysis: An EPR Perspective Restricted; Files Only

Abstract

Adenosylcobalamin (B12)-dependent ethanolamine ammonia-lyase (EAL) is involved in intestinal microbiome homeostasis, and in disease conditions caused by pathogenic strains of Salmonella enterica and Escherichia coli. Multiple electron paramagnetic resonance (EPR) techniques are used to resolve the solvent-protein-function coupling for EAL from S. typhimurium in a tunable low-temperature(T) mesodomain system, above and below the temperature of the order-disorder transition (ODT) in the protein hydration layer (PAD), identified by the spin probe (TEMPOL) rotational dynamics. Time-resolved spin trap (DEPMPO) measurements detect the EAL local sidechain motions through a non-native hydroxyl radical (HO•) generation reaction. Time- resolved, T-initiated EPR reports on the native rate-limiting substrate radical rearrangement (RR) reaction in the EAL active site. The suppression of PAD characteristic nanosecond scale dynamics upon ODT, obstructs the native product-forming coordinate for protein configurational changes between microstates S1• and S2•, which leads to the two isolated, first-order decay reactions (kinetic bifurcation). The vectorial sequence of entropy-related atomic configurations and connecting fluctuations through the S1• microstate minima, revealed from heterogeneous sucrose-hydrate confinements, are proposed to be prerequisites for attaining the RR reaction-enabled state, S2•. The S2• microstate is enabled for the native RR reaction, pre-optimized for the subsequent hydrogen transfer step, and it is singular, relative to the distribution of configurations in S1•. In contrast to the kinetic bifurcation, the HO• generation rate is found to have the Arrhenius dependence through the same wide-T range, that includes the ODT. These results lead the proposal that the large-scale cooperative α relaxation (together with the Johari-Goldstein β relaxation) in the solvent actuates the configuration change coordinates for the S1•<->S2• interconversion, while the exploration inside the S1• microstate is driven by the intrinsic local incremental fluctuations of the hydrated EAL. Overall, the results show how adiabatic chemical-bond breaking/making events, that are critical for the enzyme catalytic cycle, utilize the full bandwidth of the hierarchy of global, solvent-coupled protein dynamics. 

Table of Contents

CHAPTER 1. GENERAL INTRODUCTION ...........................................................................................................1

1.1. PROTEIN MOTIONS AND SOLVENT DYNAMICS .....................................................................................................1

1.1.1. Energy Landscape of a Folded Protein.......................................................................................................1

1.1.2. Protein Dynamics........................................................................................................................................3

1.1.3. Enzyme Dynamics and Catalysis.................................................................................................................3

1.1.4. Solvent Contribution to Protein Dynamics .................................................................................................6

1.2. COENZYME B12-DEPENDENT ETHANOLAMINE AMMONIA-LYASE ........................................................................7

1.2.1. Structure of EAL..........................................................................................................................................8

1.2.2. Catalytic Reaction Cycle of EAL.................................................................................................................9

1.2.3. Two Dynamical Regimes of Substrate Radical Rearrangement Reaction in EAL....................................11

1.3 ELECTRON PARAMAGNETIC RESONANCE ...........................................................................................................12

1.3.1. Spin Probe TEMPOL ................................................................................................................................14

1.3.2. Spin Trap DEPMPO..................................................................................................................................16

1.3.3. Mesodomain and Protein-Associated Solvent Phases Surround EAL ......................................................17

1.4 OVERVIEW ..........................................................................................................................................................19

CHAPTER 2. CONFINEMENT DEPENDENCE OF PROTEIN-ASSOCIATED SOLVENT DYNAMICS AROUND DIFFERENT CLASSES OF PROTEINS, FROM THE EPR SPIN PROBE PERSPECTIVE.......21

2.1. INTRODUCTION ..................................................................................................................................................21

2.2. MATERIALS AND METHODS ................................................................................................................................23

2.2.1. Sample preparation ...................................................................................................................................23

2.2.2. Continuous-wave EPR spectroscopy.........................................................................................................24

2.2.3. TEMPOL spectrum simulations ................................................................................................................24

2.3. RESULTS ............................................................................................................................................................25

2.3.1. T dependence of the TEMPOL EPR line shapes in frozen aqueous solution under weak confinement....25

2.3.2. T dependence of the TEMPOL EPR line shapes in frozen aqueous solution under strong confinement..27

2.3.3. T dependence of the TEMPOL rotational correlation times and normalized component weights under weak and strong confinement conditions............................................................................................................28

2.4. DISCUSSION .......................................................................................................................................................31

2.4.1. Model for protein-PAD-mesophase systems: features from the weak confinement, aqueous cryosolvent mesodomain condition.........................................................................................................................................31

2.4.2. Model for protein-PAD-mesophase systems: model features from the strong confinement condition, in absence of cryosolvent........................................................................................................................................33

2.4.3. Soluble globular proteins display uniform dependence of PAD solvent dynamics on confinement.........35

2.4.4. Condensate-forming proteins display distinct TEMPOL detected solvent dynamics ...............................37

2.5. CONCLUSIONS ....................................................................................................................................................38

CHAPTER 3. PHYSICAL MECHANISMS OF SOLVENT-PROTEIN DYNAMICAL COUPLING IN ENZYME CATALYSIS REVEALED BY CONTROLLED CONFINEMENT..................................................40

3.1. INTRODUCTION ..................................................................................................................................................40

3.2. MATERIALS AND METHODS ...............................................................................................................................42

3.2.1. Enzyme and sample preparation. ..............................................................................................................42

3.2.2. Time-Resolved EPR Measurement and Analysis ......................................................................................42

3.2.3. TEMPOL spin probe measurements and simulation.................................................................................42

3.2.4. EAL-Bounded Co(II)alamin Amplitude Measurements and Analysis.......................................................43

3.3. RESULTS ............................................................................................................................................................46

3.3.1. T-dependence of spin probe rotational mobility in the solvent domains around EAL in frozen solution.46

3.3.2. T-dependence of relative cavity quality factor q(ε") from protein-bound Co(II)alamin ..........................48

3.3.3. T-dependence of the substrate radical decay reaction in different aqueous cryosolvent systems............49

3.4. DISCUSSION .......................................................................................................................................................51

3.4.1. The PAD solvent order-disorder transition, kinetic bifurcation, and substrate radical reactions are coupled................................................................................................................................................................51

3.4.2. Congruence of the shifted Arrhenius relations indicates universal solvent-protein-reaction coupling. ..51

3.4.3. The protein configurational transition and reactions display preferential coordinates...........................53

3.5. CONCLUSIONS ....................................................................................................................................................54

CHAPTER 4. REACTIVITY TRACKING OF AN ENZYME REACTION PROGRESS COORDINATE THROUGH CORE CHEMICAL STEPS................................................................................................................57

4.1. INTRODUCTION ..................................................................................................................................................57

4.2. MATERIALS AND METHODS ...............................................................................................................................60

4.2.1. Enzyme and EPR sample preparation.......................................................................................................60

4.2.2 Time-resolved EPR measurement ..............................................................................................................60

4.2.3. Fitting of substrate radical decay kinetics ................................................................................................60

4.3. RESULTS ............................................................................................................................................................62

4.3.1. Effect of sucrose on substrate radical decay kinetics ...............................................................................62

4.3.2. Sucrose does not perturb EAL active site structure ..................................................................................64

4.3.3 Sucrose dependence of 1H/2H isotope effects on substrate radical decay kinetics ....................................65

4.4. DISCUSSION .......................................................................................................................................................66

4.4.1 Sucrose selectively perturbs the decay kinetics of S1• relative to S2•..........................................................66

4.4.2. Sucrose effect on S1• microstate: Solvent perspective ...............................................................................66

4.4.3. Sucrose effect on S1• microstate: Protein perspective...............................................................................68

4.4.4. Selective effect of sucrose on internal reaction steps: RR and HT2 .........................................................69

4.5. CONCLUSIONS ....................................................................................................................................................72

CHAPTER 5. HYDROGEN PEROXIDE PROJECT.............................................................................................74

5.1. INTRODUCTION ..................................................................................................................................................74

5.2. MATERIALS AND METHODS ...............................................................................................................................76

5.2.1 Materials ....................................................................................................................................................76

5.2.2. Continuous-wave EPR spectroscopy.........................................................................................................76

5.2.3. Simulation for EPR spectrum of spin probe TEMPOL .............................................................................77

5.2.4. CD Measurement.......................................................................................................................................77

5.2.5. Initial Rate of DEPMPO Spin Adduct Formation.....................................................................................77

5.2.6. Arrhenius Relation ....................................................................................................................................79

5.3. RESULTS ............................................................................................................................................................80

5.3.1. T-Dependence of the TEMPOL Rotation Correlation Time and Normalized Weights in Frozen Aqueous Solution, in the presence of EAL and added 2% H2O2.......................................................................................80

5.3.2. Time-Resolved, Full-Spectrum EPR Measurements of DEPMPO-HO• Formation .................................81

5.3.3. H2O2 Concentration-Dependence of ν0 for HO• Formation.....................................................................83

5.3.4. Trapping HO• Radical in Frozen Aqueous Solution of BSA or LYZ, and 2% H2O2 ................................83

5.4. DISCUSSION .......................................................................................................................................................85

5.4.1. H2O2 Concentrates in between Protein Surface and Ice Boundary ..........................................................85

5.4.2. H2O2 Permeates the Protein Hydration Layer ..........................................................................................85

5.4.3. Microscopic Kinetic Mechanism for HO• Generation..............................................................................88

5.4.4. Physical interpretation of the T-dependence of ν0 for SHP reaction in protein and solution ..................91

5.5. CONCLUSIONS ....................................................................................................................................................92

CHAPTER 6. SUMMARY.........................................................................................................................................94

APPENDIX S1.............................................................................................................................................................96

APPENDIX S2.............................................................................................................................................................97

APPENDIX S3...........................................................................................................................................................113

APPENDIX S4...........................................................................................................................................................129

APPENDIX S5...........................................................................................................................................................141 REFERENCES..........................................................................................................................................................156 

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School	Laney Graduate School
Department	Physics
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Physics, Molecular Chemistry, Physical Biophysics, Biomechanics
Keyword	Solvent Dynamics Electron Paramagnetic Resonance Protein Dynamics Enzyme Catalysis Ethanolamine Ammonia-Lyase
Committee Chair / Thesis Advisor	Kurt Warncke, Emory University
Committee Members	Laura Finzi, Emory University Katherine Davis, Emory University Justin Burton, Emory University Connie Roth, Emory University

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