Enzymes that harness the extreme reactivity of electron-deficient free radical species carry out some of the most difficult chemical reactions in biology. The regio- and stereo-selectivity achieved by these enzymes defies long-held ideas that radical reactions are non-specific. This class includes the following: ribonucleotide reductases, which catalyze the first unique step in DNA biosynthesis, prostaglandin H-synthase, the target of aspirin and other non- steroidal anti-inflammatory drugs, the S-adenosylmethionine-dependent enzymes, with over six-hundred members that catalyze a wide range of radical- mediated redox reactions, and the coenzyme B12 (adenosylcobalamin)-dependent enzyme superfamily, whose members catalyze metabolite covalent bond rearrangements. The common primary step in these chemically-disparate catalyses is metal-assisted generation of an electron-deficient organic radical. This initiator radical, either by itself or through secondary radical species, promotes hydrogen atom abstraction from the substrate to form a substrate- based radical, opening a new reaction channel that facilitates transformation to the product. An outstanding issue is how the radical pair is stabilized against rapid recombination to achieve productive reaction in high yield. Elucidating the basic principles of how protein and cofactor guide radical generation, stabilization, intra-protein radical migration, and radical rearrangement will be sustained focuses of the proposed studies. The coenzyme B12-dependent enzymes, and ethanolamine ammonia-lyase specifically, have been selected for scrutiny. The contributions of molecular structure and dynamics to enzyme function will be studied by using techniques of pulsed-electron paramagnetic resonance and visible/near-infrared absorption spectroscopy, in cryotrapped and time-resolved systems on time scales ranging from picoseconds to hours. The fundamental insights and novel methods developed will promote identification and characterization of radical intermediates in other biological reactions, inform the design of programmed radical reactivity, and assist molecular-therapeutic efforts to combat pernicious free radical processes.

Public Health Relevance

Enzymes that harness the extreme reactivity of electron-deficient free radicals perform some of the most difficult reactions in biology. We seek to understand the contributions of molecular structure and dynamics to this productive radical reactivity in coenzyme B12-dependent enzymes by using spectroscopic techniques. The fundamental insights and novel approaches developed will promote characterization of radical intermediates in other biological systems, inform the design of programmed radical reactivity, and assist molecular- therapeutic efforts to combat pernicious free radical processes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK054514-14
Application #
8266392
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Sechi, Salvatore
Project Start
1998-03-15
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2014-04-30
Support Year
14
Fiscal Year
2012
Total Cost
$264,332
Indirect Cost
$93,795
Name
Emory University
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Wang, Miao; Zhu, Chen; Kohne, Meghan et al. (2015) 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. Methods Enzymol 563:59-94
Robertson, Wesley D; Bovell, Adonis M; Warncke, Kurt (2013) Cobinamide production of hydrogen in a homogeneous aqueous photochemical system, and assembly and photoreduction in a (βα)8 protein. J Biol Inorg Chem 18:701-13
Wang, Miao; Warncke, Kurt (2013) Entropic origin of cobalt-carbon bond cleavage catalysis in adenosylcobalamin-dependent ethanolamine ammonia-lyase. J Am Chem Soc 135:15077-84
Chen, Hanlin; Sun, Li; Warncke, Kurt (2013) Heterogeneous ordered-disordered structure of the mesodomain in frozen sucrose-water solutions revealed by multiple electron paramagnetic resonance spectroscopies. Langmuir 29:4357-65
Hernandez-Guzman, Jessica; Sun, Li; Mehta, Anil K et al. (2013) Copper(II)-bis-histidine coordination structure in a fibrillar amyloid *-peptide fragment and model complexes revealed by electron spin echo envelope modulation spectroscopy. Chembiochem 14:1762-71
Sun, Li; Savory, Joshua J; Warncke, Kurt (2013) Design and implementation of an FPGA-based timing pulse programmer for pulsed-electron paramagnetic resonance applications. Concepts Magn Reson Part B Magn Reson Eng 43:100-109
Bovell, Adonis Miguel; Warncke, Kurt (2013) The structural model of Salmonella typhimurium ethanolamine ammonia-lyase directs a rational approach to the assembly of the functional [(EutB-EutC)ýýý]ýýý oligomer from isolated subunits. Biochemistry 52:1419-28
Gunderson, William A; Hernández-Guzmán, Jessica; Karr, Jesse W et al. (2012) Local structure and global patterning of Cu2+ binding in fibrillar amyloid-β [Aβ(1-40)] protein. J Am Chem Soc 134:18330-7
Robertson, Wesley D; Wang, Miao; Warncke, Kurt (2011) Characterization of protein contributions to cobalt-carbon bond cleavage catalysis in adenosylcobalamin-dependent ethanolamine ammonia-lyase by using photolysis in the ternary complex. J Am Chem Soc 133:6968-77
Zhu, Chen; Warncke, Kurt (2010) Kinetic isolation and characterization of the radical rearrangement step in coenzyme B12-dependent ethanolamine ammonia-lyase. J Am Chem Soc 132:9610-5

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