title: Resolution and Characterization of Chemical Steps in Enzyme Catalytic Sequences by Using Low-Temperature and Time-Resolved, Full-Spectrum EPR Spectroscopy in Fluid Cryosolvent and Frozen Solution Systems.
abstract: Approaches to the resolution and characterization of individual chemical steps in enzyme catalytic sequences, by using temperatures in the cryogenic range of 190-250 K, and kinetics measured by time-resolved, full-spectrum electron paramagnetic resonance spectroscopy in fluid cryosolvent and frozen solution systems, are described. The preparation and performance of the adenosylcobalamin-dependent ethanolamine ammonia-lyase enzyme from Salmonella typhimurium in the two systems exemplifies the biochemical and spectroscopic methods. General advantages of low-temperature studies are (1) slowing of reaction steps, so that measurements can be made by using straightforward T-step kinetic methods and commercial instrumentation, (2) resolution of individual reaction steps, so that first-order kinetic analysis can be applied, and (3) accumulation of intermediates that are not detectable at room temperatures. The broad temperature range from room temperature to 190 K encompasses three regimes: (1) temperature-independent mean free energy surface (corresponding to native behavior); (2) the narrow temperature region of a glass-like transition in the protein, over which the free energy surface changes, revealing dependence of the native reaction on collective protein/solvent motions; and (3) the temperature range below the glass transition region, for which persistent reaction corresponds to nonnative, alternative reaction pathways, in the vicinity of the native configurational envelope. Representative outcomes of low-temperature kinetics studies are portrayed on Eyring and free energy surface (landscape) plots, and guidelines for interpretations are presented.
authors.affiliation: Department of Physics, Emory University, N201 Mathematics and Science Center, Atlanta, Georgia, USA.
authors.forename: Miao
authors.initials: M
authors.lastname: Wang
authors.affiliation: Department of Physics, Emory University, N201 Mathematics and Science Center, Atlanta, Georgia, USA.
authors.forename: Chen
authors.initials: C
authors.lastname: Zhu
authors.affiliation: Department of Physics, Emory University, N201 Mathematics and Science Center, Atlanta, Georgia, USA.
authors.forename: Meghan
authors.initials: M
authors.lastname: Kohne
authors.affiliation: Department of Physics, Emory University, N201 Mathematics and Science Center, Atlanta, Georgia, USA. Electronic address: kwarncke@physics.emory.edu.
authors.forename: Kurt
authors.initials: K
authors.lastname: Warncke
mesh_terms: D002384:Catalysis; D003038:Cobamides / Q000378:metabolism; D003080:Cold Temperature; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D004981:Ethanolamine Ammonia-Lyase / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000737:chemistry / Q000201:enzymology*; D012997:Solvents / Q000737:chemistry; D014805:Vitamin B 12 / Q000378:metabolism
chemical_list: D003038:Cobamides; D012997:Solvents; D004981:Ethanolamine Ammonia-Lyase; C000913:cobamamide; D014805:Vitamin B 12
keywords: Adenosylcobalamin; Cryosolvent; EPR; Enzyme; Free energy surface; Glass transition; Protein dynamics; Reaction kinetics; Time-resolved; Vitamin B(12)