Influence of Protein and Solvent Dynamics on Reactions in Ethanolamine Ammonia-Lyase addressed by using Time-Resolved Electron Paramagnetic Resonance and UV-visible Spectroscopy Open Access

Ucuncuoglu, Neslihan (2016)

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The radical-rearrangement step in the reaction of the (S)-2-aminopropanol-generated Co(II)-substrate radical pair in coenzyme B12 (adenosylcobalamin)-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium has been studied by using time-resolved, full-spectrum, continuous-wave electron paramagnetic resonance (EPR) spectroscopy over the temperature range of 210 - 250 K. This approach addresses the kinetics of the radical rearrangement reaction step in EAL with a single step resolution. We identified that the substrate assumes two sequential inter-converting configurations, one of which is proposed to be reconfigured by the protein to form the native Co(II)-substrate radical pair, and the other, relatively high entropy/enthalpy state, which reacts by a non-native pathway to form an magnetically-uncoupled Co(II) and radical species. The emergence of the unprecedented, non-native pathway of deleterious radical reaction enabled us to assess whether the protein dynamic effect is generic, or specific to the native reaction. The free energy curve profiled a different T-dependency for the native and non-native reaction pathways at T > 240 K which could indicate the presence of protein configurational fluctuations that guide the native reaction pathway. In the second part of the dissertation, we presented the temperature dependence of the spin probe (TEMPOL) mobility in aqueous solution of EAL and (S)-2-aminopropanol to investigate the solvent-dynamical contributions to the enzyme kinetics. In the final chapter of the this study, a new reaction step; the conversion of adenosylcob(III)alamin to cob(II)alamin through the Co-C bond homolysis is investigated by using optical absorption spectroscopy, using ethylene glycol as the substrate. The EAL single step kinetics under these conditions were unraveling at temperature and solvent conditions that are close to the physiological conditions. The decay of the adenosylcob(III)alamin absorption signal is used as a measure to quantify the conversion, and it is proposed that the cob(II)alamin formation reaction is at least partially rate-determined by the hydrogen transfer-1 step. Overall, the results provide insight into the mechanisms and protein and solvent dynamical contributions to the substrate radical formation and rearrangement reactions in EAL.

Table of Contents

1 Introduction 1

1.1 Literature Review 2

1.1.1 Vitamin B12: Structure and derivatives 2

1.1.2 Ethanolamine Ammonia-Lyase: an AdoCbl-dependent enzyme 4

1.2 EPR Theory 10

1.2.1 Continuous wave (CW) EPR spectroscopy 11

1.2.2 Nitroxide Spin Labels 25

1.3 The EPR spectrum of Co(II)-substrate radical pair 27

The scope of the project 29

2 Chapter 2 32

2.1 Background 33

2.2 Materials and Methods 35

2.2.1 Enzyme Preparation 35

2.2.2 Sample Preparation 35

2.2.3 Continuous-wave EPR spectroscopy 36

2.2.4 Time-resolved EPR Spectroscopy 36

2.2.5 Kinetic Analysis 37

2.2.6 Optical Measurements 38

2.3 Results 39

2.3.1 Time-dependence of the Co(II)-substrate radical pair EPR spectrum following temperature-step reaction initiation 39

2.3.2 Deconvolution of the time-dependent Co(II)-radical EPR spectra 42

2.3.3 Time and Temperature dependence of the Co(II)-substrate radical pair decay 44

2.3.4 Time and temperature dependence of the uncoupled signal formation 53

2.4 Discussion 60

2.4.1 Observed kinetics of the Co(II)-substrate radical decay 60

2.4.2 Introduction of the Kinetic Model 64

2.5 Simulation of the kinetic model and data-fitting 66

2.6 Conclusions 76

3 Chapter 3 79

3.1 Introduction 80

3.2 Materials and Methods 82

3.3 Results 83

3.3.1 EPR Spectroscopy of TEMPOL 83

3.3.2 The EasySpin Simulations of the EPR spectra of TEMPOL 86

3.4 Discussion 91

3.5 Conclusions 95

4 Chapter 4 98

4.1 Introduction 99

4.1.1 Literature Review 99

4.1.2 Basics of the UV-Vis Absorption Spectroscopy 101

4.1.3 Optical Features of AdoCbl 102

4.1.4 Project Overview 104

4.2 Materials and Methods 105

4.2.1 Sample Preparation 105

4.2.2 UV-visible Spectroscopy 106

4.3 Results 107

4.3.1 Preliminary spectral measurement 107

4.3.2 Treatment of the time-coursed kinetics data of single-wavelength absorption 110

4.3.3 Dependence of the rate on the substrate concentration 111

4.3.4 Time-Course Kinetics Results 112

4.4 Discussion 114

4.4.1 Comparison of the results with the previous studies 115

4.4.2 The Co-C bond cleavage kinetics using the deuterated substrate 118

4.4.3 Arrhenius relations of the EG and EG-d4 119

4.5 Conclusions 123

Chapter 5 125

Conclusions 125

References 131

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