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

Wang, Miao (2009)
Dissertation (193 pages)
Committee Chair / Thesis Adviser: Warncke, Kurt
Committee Members: Huynh, B H Vincent ; Finzi, Laura ; Rasnik, Ivan ; Heaven, Michael
Research Fields: Biophysics, General
Keywords: EPR; Cryoenzymology; B12; coenzyme B12-dependent enzyme; Photolysis; Enzymology
Program: Laney Graduate School, Physics
Permanent url: http://pid.emory.edu/ark:/25593/195px

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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