Entropic Origin for Catalysis of Cobalt-Carbon Bond Cleavage in Coenzyme B12 (Adenosylcobalamin) in Ethanolamine Ammonia-Lyase 公开

Wang, Miao (2009)

Permanent URL: https://etd.library.emory.edu/concern/etds/vm40xs27b?locale=zh
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Abstract

The formation of the CoII-substrate radical pair intermediate in coenzyme B12 (adenosylcobalamin)-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium has been studied in a 41% (v:v) DMSO/water cryosolvent system in the temperature range of 230-250 K by using X-band electron paramagnetic resonance (EPR) spectroscopy. For the first time, a stable (>4 hr at 230 K) ternary complex of enzyme, coenzyme and substrate is formed in a coenzyme B12-dependent enzyme, which allows temperature-step initiation of the reaction to form the CoII-substrate radical pair, and monitoring by time-resolved, full-spectrum EPR spectroscopy. A three-state (intact AdoCbl, substrate bound initial state; CoII-5'-deoxyadenosyl radical pair intermediate state; CoII-substrate radical pair state), two-step mechanism is used to treat the CoII-substrate radical pair formation reaction kinetics and equilibria with consideration of the intermediate radical pair state. By using this model, the absence of an EPR-detectable intermediate yields a limit of >3.3 kcal/mol for the free energy of the CoII-5'-deoxyadenosyl radical pair relative to the ternary complex. The free energy difference between the Coll-substrate radical pair state and the initial state is approximately 0 kcal/mol for 230-250 K, and has an extrapolated value of -2.6 kcal/mol at 298 K. The absence of a substrate hydrogen isotope effect on the rate of CoII-substrate radical pair formation indicates that the Co-C bond cleavage is rate determining at 230-250 K. This allows the first-time determination, by using Eyring analysis, of the Co-C bond cleavage activation enthalpy and entropy in a coenzyme B12-dependent enzyme. The 16 kcal/mol decrease in the activation free energy for Co-C bond cleavage in EAL relative to solution is contributed almost entirely by a large, positive activation entropy. The activation parameters offer a quantitative description for the coupling between the Co-C bond cleavage and hydrogen transfer steps in EAL. The UV-visible spectroscopy is used to obtain optical spectrum from the ternary complex. The result indicates that the ground state destabilization (enthalpic strain) is not a significant catalysis contribution, and supports the findings from the EPR studies. These results provide a new paradigm for catalysis of Co-C bond cleavage in coenzyme B12-dependent enzymes.

Table of Contents

Chapter One: Introduction............................................................1

1.1 Problem Statement....................................................................2 1.2 Literature Review .....................................................................6 1.2.1 Cofactor B12 Dependent Enzymes………………………………....................6 1.2.2 Electron Paramagnetic Resonance………………………………...................13 1.2.3 Photochemistry of coenzyme B12 ………………………………...................22 1.3 Outline of Dissertation ..............................................................25 Chapter Two: Development of Low Temperature Cryosolvent System 28 2.1 Survey of Cryosolvents 29 2.1.1 Cryoenzymology at Subzero Temperatures ………...........................29 2.1.2 VS41A and VS41A-G …………………………………………...........................32 2.1.3 41% DMSO/water ………………………………………………...........................34 2.1.4 Concluding Remarks ………….......................................................36 2.2 Kinetic Arrest of Ternary Complex at 230 K 37 2.2.1 Physical Property of 41% DMSO/water ………………………...................38 2.2.2 pH-balancing at Subzero Temperature ………………………….................41 2.2.3 Procedure to Introduce Substrate at 230 K ……………………...............43 2.2.4 Spectroscopic Evidence for Ternary Complex …………………….............45 2.2.5 Concluding Remarks ……………………………………………..........................46 2.3 Substrate Radical Pair Formation at Subzero Temperatures 46 2.3.1 EPR Study for the Free Radical Formed at 242K .……………................47 2.3.2 Characterization of the Substrate Radical Reaction to Form the Product Radical ………………………………………………………….......................50 2.3.3 Viscosity Dependence on EAL's Kinetics ……………………....................52 2.3.4 Concluding Remarks ……………………………………………..........................54 2.4 Instrument Preparation 54 2.4.1 Capillary Tube Packaging ………………………………………........................55 2.4.2 Instrument Setup ………………………………………………...........................56 2.4.3 Concluding Remarks ………………………………………….............................56 Chapter Three: Kinetic and Thermodynamic Studies of CoII-substrate Radical Pair in Cryosolvent System ...............................................57 3.1 Temperature Dependence of Substrate Radical Pair Formation ...........59 3.1.1 Time-resolved, Full Spectrum Continuous Wave Electron Paramagnetic Resonance ……………………………………………………....................59 3.1.2 Time-dependence of CoII-substrate radical pair formation …….........60 3.1.3 Temperature-dependence of CoII-substrate Radical Pair Formation between 234 - 248 K ………………………………………………………….....................62 3.1.4 Attempted Detection of Paramagnetic Intermediate States …….......65 3.1.5 Concluding Remarks ……………………………………………..........................66 3.2 Kinetic Evidence for the Formation of Ternary Complex ....................67 3.2.1 Substrate Concentration Variation Experiment ………………….............68 3.2.2 Concluding Remarks ……………………………………………..........................71 3.3 Three-state, Two-step Reaction Model ........................................71 3.3.1 Kinetic Model Setup ……………………………………………..........................71 3.3.2 Temperature-dependence of the first-order rate and equilibrium constants ……………………………………………………………………...........................75 3.4 Retrieving Thermodynamic Parameters for CoII-substrate Radical Pair Formation 76 3.4.1 Equilibrium Perturbation Experiments ………………………….....................76 3.4.2 Relations among Experimental Observables and Microscopic Rate Constants ……………………………………………………………………............................80 3.4.3 Thermodynamics of CoII-substrate Radical Pair Formation ….............82 3.4.4 Concluding Remarks …………………………………………..............................89 Chapter Four: Activation Parameters of Cobalt-Carbon Bond Cleavage 90 4.1 Synthesis of Deuterated Substrate 92 4.1.1 Synthesis Protocol …………………………………………...............................93 4.1.2 Product Analysis ………………………………………………..............................93 4.2 Substrate Radical Formation with 2H2-Substrate ..............................98 4.2.1 EPR lineshape of CoII-substrate Radical Pair in Aqueous and Cryosolvent Systems with 2H2-Substrate ……………………………………..............98 4.2.2 Time-dependence of CoII-substrate radical Pair Formation with 2H2-Substrate ……………………………………………………………………......................100 4.2.3 Temperature-dependence of kobs with 2H-Substrate……………............101 4.2.4 Temperature-dependence of v with 2H-Substrate ………………............102 4.3 Revisiting the Three-step, Two-state Reaction Model .......................107 4.4 Retrieving Co-C Bond Cleavage Activation Parameters ......................110 4.5 Other Possible Reaction Scheme ...................................................114 4.6 Solvent Effect Study ..................................................................121 4.7 Concluding Remarks ....................................................................122 Chapter Five: Optical Absorption Studies of the Ternary Complex .....127 5.1 Instrument Design and Setup .......................................................128 5.2 Temperature-dependence of Cofactor B12 UV-visible Spectrum ..........130 5.3 Ternary Complex Preparation Protocol ............................................133 5.4 UV-visible spectrum of the Ternary Complex and Holoenzyme .............134 5.5 Concluding Remarks ....................................................................140 Chapter Six: Investigation of Photo-induced CoII-substrate Radical Pair Formation .....................................................................................141 6.1 Instrument Setup .......................................................................144 6.2 Photolysis of Cofactor B12 ............................................................146 6.3 Photolysis of the Holoenzyme and Ternary Complex ...........................147 6.4 Application of Spin Trap in Photolysis .............................................150 6.5 Concluding Remarks ....................................................................151 Appendix: Manuals and Protocols ...................................................152 A: Instruction for Oxford Cryostat System with Bruker E560 Console .........153 B: Ternary Complex Preparation Procedure for EPR Studies ......................158 C: Synthesis of [1,1-2H2]-aminopropanol .............................................161 D: Ternary Complex Sample Preparation for UV-Visible Abs ......................163 E: Cary 100 Basic Operation Manual ....................................................165 F: List of Coded Programs .................................................................167 Bibliography .................................................................................168

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