Enzyme on Ice: Kinetic and EPR Spectroscopic Characterization of the CoII-Substrate Radical Decay Reaction in Coenzyme B12-Dependent Ethanolamine Ammonia-Lyase Open Access
Zhu, Chen (2010)
Abstract
The transient decay reaction kinetics of 1,1,2,2-1H4- and 1,1,2,2-2H4-aminoethanol-generated CoII-substrate radical pair intermediate in ethanolamine ammonia-lyase (EAL) have been measured by using time-resolved, X-band continuous-wave electron paramagnetic resonance spectroscopy in frozen aqueous solution from 190 to 223 K. The decay is biexponential at temperature T<214 K (1H) or <210 K (2H), with fast and slow phase first-order rate constants kobs,f and kobs,s , respectively. The decay becomes monoexponential at temperature T≥214 K, with rate constant kobs,m . The kobs,f and kobs,m values adhere to the same linear relation on a lnk versus T -1 (Arrhenius) plot, and therefore represent the same mechanism, which is proposed to be the native forward reaction of the substrate radical through the radical rearrangement step. The 1H/2H isotope effect (IE) on kobs,f of 1.4±0.1 at 190≤T≤207 K is assigned to an alpha-secondary hydrogen kinetic IE on the rearrangement step. The kobs,s values obey a different Arrhenius relation, and display an inverse kinetic IE (0.8±0.1). The slow decay phase is proposed to be associated with the forward reaction, but with a different rate determining step. The 1H/2H IE on kobs,m increases continuously at T>210 K, to 2.1±0.1 at 223 K. A three-state (substrate radical, product radical, diamagnetic products), two-step [rearrangement, and subsequent hydrogen transfer, (HT)] model is used to generate a consistent fit to the temperature dependence of the k obs,f, k obs,m values and IEs at low temperature with k cat values and IEs at 277 K (IE=5.5) and 293 K (IE=7.8). The model shows that the four decade-old paradox of 1H/2H and 1H/3H IEs in EAL, and the temperature dependent IE, are caused by a significant negative activation entropy for the HT step, relative to rearrangement. The bifurcation of the decay kinetics at 207<T<214 K is addressed by measuring the detailed (1 K intervals) temperature dependence of samples with slow phase population. The steep lnk versus T -1 dependence is discontinuous with the fast and slow phase relations. The origin of the kinetic bifurcation is proposed to arise from a protein dynamical transition, which is coupled to the core adiabatic reaction in EAL.
Table of Contents
Table of Contents
Page Chapter I: Introduction 11.1 Coenzyme B12-dependent Enzymes 2
1.1.1 The Coenzyme B12-dependent Enzyme Superfamily ……………….. 2
1.1.2 Coenzyme B12 (Adenosylcobalamin) ……………………………….. 3
1.1.3 Survey of Coenzyme B12-dependent Enzyme, Ethanolamine Ammonia-lyase (EAL) ………………………………………………. 4
1.2 EPR Spectroscopic Studies of EAL 8
1.2.1 Continuous-wave (CW) EPR Spectroscopy ………………………… 9
1.2.2 Continuous-wave EPR Studies of EAL …………………………….. 11
1.2.3 Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy .. 12
1.2.4 ESEEM Studies of EAL ……………………………………............ 15
1.3 The "Glass Transition" in Protein Dynamics 17
1.4 Outline of Dissertation 20
Chapter II: Characterization of the CoII-[1H]-substrate Radical Pair Decay Kinetics in Frozen Aqueous Solution from 190 to 223 K 23
2.1 Background and Introduction 242.1.1 Survey of Synchronization of Biomolecular Reactions ……………. 24
2.1.2 Temperature Step Initiation of the Relaxation of Cryotrapped CoII-substrate Radical Pair ……………………………………………… 25
2.1.3 Sample Preparations, Instrument Setup and Data Analysis ……….. 25
2.2 Results 302.2.1 EPR Spectrum of the CoII-substrate Radical Pair ……...................... 30
2.2.2 Decay of the CoII-substrate Radical Pair ……................................... 31
2.2.3 Evaluation of Three-state, Two-step Model for the Substrate Radical Decay Reaction …………………………………………………….. 37
2.2.4 Evaluation of the Multi-population, Single-step Model …………… 43
2.2.5 Two-population, Single-step Model of Substrate Radical Decay ….. 44
2.3 Kinetic Transition of Substrate Radical Decay at T > 207 K 45
2.4 Proposed Origins on Kinetic Decay Phases 49 2.5 Conclusion 51Chapter III: Characterization of Substrate Hydrogen Isotope Effects on the CoII-substrate Radical Pair Decay Kinetics 54
3.1 Background and Introduction 553.2 Kinetic Characterization of Substrate Radical Decay 56
3.2.1 Kinetics of 1,1,2,2-2H4-aminoethanol-generated Substrate Radical Decay ………………………………………………………………. 56
3.2.2 Characterization of the Fast Phase of Decay of Substrate Radical … 59
3.2.3 Characterization of the Slow Phase of Decay of Substrate Radical .. 65
3.3 Conclusion 66
Chapter IV: Resolution of the Steady-state Hydrogen Isotope Effect Paradox in EAL 69
4.1 Background and Introduction 704.2 Resolution of 2H/3H Steady-state IE Paradox 71
4.2.1 Kinetics of 1H4- and 2H4-aminoethanol-generated Substrate Radical Decay from 190 to 223 K …………………………………………... 71
4.2.2 Temperature Dependence of Rate Limiting Step …………………... 73
4.2.3 Temperature and Isotope Dependent Three-state, Two-step Model .. 76
4.2.3.1 Derivation of Three-state, Two-step Model …………………... 76
4.2.3.2 Relative Activation Entropy Values of the HT2 and Reverse Radical Rearrangement Steps …………………………………. 83
4.3 Conclusion 87
Chapter V: Decay Kinetics of the CoII-substrate Radical Pair in the Transition Region 90
5.1 Background and Introduction 91
5.2 Two-temperature Annealing of the CoII-substrate Radical Pair 93
5.3 Three-temperature Annealing of the CoII-substrate Radical Pair 96
5.4 Conclusion 98
Chapter VI: Trapping of the CoII-product Radical Pair Intermediate and Kinetic Characterization of Recombination Reaction Steps 101
6.1 Background and Introduction 1026.2 Experimental Procedures 104
6.3 Results 106
6.4 Conclusion 109
Bibliography 112 Appendix: Manuals and Protocols 119A: Instruction for Oxford Cryostat System with Bruker E560 Console 120
B: Instruction for Pulse EPR Console 125
C: Protocol of Cell Growth 129
D: Protocol of EAL Isolation and Purification 135
E: List of Coded Programs 138
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