Radical Rearrangement Catalysis in an Enzyme at 190-207 K: Mechanistic Features Revealed by Substrate $^{1}$H/$^{2}$H Isotope Effects

POSTER

Abstract

The decay kinetics of both the natural abundance and [1,1,2,2-$^{2}$H$_{4}$]-aminoethanol generated Co$^{II}$-substrate radical pair catalytic intermediate in ethanolamine ammonia-lyase (EAL) from \textit{Salmonella typhimrium} have been measured by using time-resolved, full-spectrum X-band continuous-wave electron paramagnetic resonance (EPR) spectroscopy in frozen aqueous solution from 190 to 207 K. The decay reaction proceeds through sequential radical covalent rearrangement and hydrogen atom transfer (HT) steps. In the temperature range from 190 to 207 K, the decay is biexponential, and the two phases correspond to distinct populations [1]. The $^{1}$H/$^{2}$H isotope effects (IE) on the fast phase and slow phase are 1.3 and 0.8, respectively. These IE are not caused by a primary kinetic IE. Therefore, HT is rapid, relative to rearrangement. We propose that the fast phase is rate-determined by the rearrangement step, and that the slow phase is rate-determined by a step after rearrangement that is associated with protein guidance of the reactions. The results reveal microscopic features of the core reaction chemistry and protein dynamics participation in the reaction, which are not accessible at ambient temperatures.\\[4pt] [1] Zhu, C., Warncke, K. \textit{Biophys. J. 95}, 5890 (2008). Supported by grant DK54514 from NIDDK/NIH.

Authors

  • Milind Purohit

    Univ of South Carolina, Benedict College, Sc 29204, Univ. South Carolina, Benedict College, Univ South Carolina, GA Tech, Central Microscopy Research Facility, University of Iowa, Iowa City, Iowa 52242, USA, Department of Chemistry, University of Sri Jayewardenepura, Nugegoda, Sri Lanka, Department of Physics and Astronomy, Georgia State University, Atlanta, Georgia 30303, USA, Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL 35294, Department of Physics, University of Alabama at Birmingham, Birmingham, AL 35294, College of William \& Mary, Harvard University, Benedict College, SC 29204, Massachusetts Institute of Technology, University of Applied Science, Bielefeld, Germany, Francis Marion University, Physics Dept., Emory University, Emory University, Formerly Emory University, currently UCLA, Physics Department, Georgia State University, Univ. of Georgia, Dept. of Chemistry, Universidad Nacional de Quilmes, Argentina, Pr, Dr, Derpartment of Physics, Florida A\&M University, Tallahassee, FL-32307, Department of Physics, Emory University, Georgia Institute of Technology, Naval Research Laboratory, University of Alabama, Tsinghua University, Lawrence Berkeley National Laboratory, Vanderbilt University, Jagellonian Univ., Univ. of Bonn, North Carolina A\&T State Univ., North Carolina Central Univ., Duke Univ. and TUNL, Georgia State University, Dept of Physics, Emory University, Cell Biology Department, Emory University, Physics Department, Emory University, University of South Carolina

  • Milind Purohit

    Univ of South Carolina, Benedict College, Sc 29204, Univ. South Carolina, Benedict College, Univ South Carolina, GA Tech, Central Microscopy Research Facility, University of Iowa, Iowa City, Iowa 52242, USA, Department of Chemistry, University of Sri Jayewardenepura, Nugegoda, Sri Lanka, Department of Physics and Astronomy, Georgia State University, Atlanta, Georgia 30303, USA, Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL 35294, Department of Physics, University of Alabama at Birmingham, Birmingham, AL 35294, College of William \& Mary, Harvard University, Benedict College, SC 29204, Massachusetts Institute of Technology, University of Applied Science, Bielefeld, Germany, Francis Marion University, Physics Dept., Emory University, Emory University, Formerly Emory University, currently UCLA, Physics Department, Georgia State University, Univ. of Georgia, Dept. of Chemistry, Universidad Nacional de Quilmes, Argentina, Pr, Dr, Derpartment of Physics, Florida A\&M University, Tallahassee, FL-32307, Department of Physics, Emory University, Georgia Institute of Technology, Naval Research Laboratory, University of Alabama, Tsinghua University, Lawrence Berkeley National Laboratory, Vanderbilt University, Jagellonian Univ., Univ. of Bonn, North Carolina A\&T State Univ., North Carolina Central Univ., Duke Univ. and TUNL, Georgia State University, Dept of Physics, Emory University, Cell Biology Department, Emory University, Physics Department, Emory University, University of South Carolina