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Author Notes:

Kurt Warncke, Department of Physics, N201 Mathematics and Science Center, 400 Dowman Drive, Emory University, Atlanta, GA 30322-2430, Phone: 404-727-2974, Fax: 404-727-0873, kwarncke@physics.emory.edu.

We thank Dr. Li Sun, Dr. Adonis M. Bovell, and Professor Vincent Huynh for helpful discussions.

Subjects:

Research Funding:

Research reported in this publication was supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under Award Number R01 DK054514.

The purchase of the Bruker E500 EPR spectrometer was funded by the National Center for Research Resources of the National Institutes of Health under Award Number RR17767, and by Emory University.

Keywords:

  • Science & Technology
  • Physical Sciences
  • Chemistry, Multidisciplinary
  • Chemistry
  • CO-C BOND
  • RIBONUCLEOSIDE TRIPHOSPHATE REDUCTASE
  • HYDROGEN-ATOM ABSTRACTION
  • COENZYME B-12
  • CONFORMATIONAL ENTROPY
  • DISSOCIATION ENERGY
  • HOMOLYSIS
  • SUBSTRATE
  • ACTIVATION
  • PROTEINS

Entropic Origin of Cobalt-Carbon Bond Cleavage Catalysis in Adenosylcobalamin-Dependent Ethanolamine Ammonia-Lyase

Tools:

Journal Title:

Journal of the American Chemical Society

Volume:

Volume 135, Number 40

Publisher:

, Pages 15077-15084

Type of Work:

Article | Post-print: After Peer Review

Abstract:

Adenosylcobalamin-dependent enzymes accelerate the cleavage of the cobalt-carbon (Co-C) bond of the bound coenzyme by >1010-fold. The cleavage-generated 5′-deoxyadenosyl radical initiates the catalytic cycle by abstracting a hydrogen atom from substrate. Kinetic coupling of the Co-C bond cleavage and hydrogen-atom-transfer steps at ambient temperatures has interfered with past experimental attempts to directly address the factors that govern Co-C bond cleavage catalysis. Here, we use time-resolved, full-spectrum electron paramagnetic resonance spectroscopy, with temperature-step reaction initiation, starting from the enzyme-coenzyme-substrate ternary complex and 2H-labeled substrate, to study radical pair generation in ethanolamine ammonia-lyase from Salmonella typhimurium at 234-248 K in a dimethylsulfoxide/water cryosolvent system. The monoexponential kinetics of formation of the 2H- and 1H-substituted substrate radicals are the same, indicating that Co-C bond cleavage rate-limits radical pair formation. Analysis of the kinetics by using a linear, three-state model allows extraction of the microscopic rate constant for Co-C bond cleavage. Eyring analysis reveals that the activation enthalpy for Co-C bond cleavage is 32 ± 1 kcal/mol, which is the same as for the cleavage reaction in solution. The origin of Co-C bond cleavage catalysis in the enzyme is, therefore, the large, favorable activation entropy of 61 ± 6 cal/(mol·K) (relative to 7 ± 1 cal/(mol·K) in solution). This represents a paradigm shift from traditional, enthalpy-based mechanisms that have been proposed for Co-C bond-breaking in B12 enzymes. The catalysis is proposed to arise from an increase in protein configurational entropy along the reaction coordinate.

Copyright information:

© 2013 American Chemical Society.

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