This application seeks to capitalize upon advances made in this project under the currently funded R29 award. The intention is to replace this award with an expanded research program funded by an R01 award. Free radicals are generally perceived as highly reactive species that are harmful to the cell. There is, however, a growing number of enzymes known that use carbon-based radicals to catalyze a variety of important metabolic reactions. Adenosylcobalamin (coenzyme B12) serves as a """"""""masked"""""""" form of free radical that is liberated by homolysis of the coenzyme cobalt-carbon bond. The radical is used to remove a hydrogen atom from the substrate, thereby activating the substrate towards reaction. We are studying the adenosylcobalamin-dependent isomerization of glutamate to 3-methylaspartate, catalyzed by glutamate mutase, as a model system to investigate several fundamental aspects of enzyme- mediated radical catalysis. a) How do enzymes generate radicals? b) How is the removal of hydrogen, the key step in substrate activation, catalyzed? c) How does the enzyme control the rearrangement of reactive substrate-radical intermediates? When bound by the enzyme, a histidine residue coordinates cobalt trans- axially to the cobalt-carbon bond; the histidine, in turn, participates in a hydrogen bond with an aspartate residue. To probe the role of these residues in catalysis, we will examine the ability of imidazole and other exogenous ligands to rescue activity in mutants in which the histidine and aspartate have been deleted. We will determine whether changes the pKa of the ligand correlate with the ability to rescue enzyme activity. We will complete our analysis of the free energy profile of the glutamate mutase reaction. Stopped flow spectroscopy, rapid quenched flow techniques, and tritium partioning experiments will be used to measure the rates of hydrogen transfer between substrate, coenzyme and product, the rate of product formation on the enzyme, and the rates of substrate-radical rearrangement. These measurements will provide a more detailed description of a radical reaction than has been possible previously. To test mechanistic hypotheses concerning the rearrangement of the substrate-radical we will examine the ability of substrate analogs to function as alternative substrates and/or mechanism-based inhibitors of glutamate mutase. Thermodynamic aspects of the interactions of the protein with coenzyme, substrates and reaction intermediates will be studied by isothermal titration microcalorimetry. These studies aim to provide insight into how binding energy may contribute to activate the coenzyme towards homolysis. Finally, we will continue x-ray crystallography and protein NMR studies to elucidate the three- dimensional structure of the enzyme.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry Study Section (BIO)
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Jones, Warren
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University of Michigan Ann Arbor
Schools of Arts and Sciences
Ann Arbor
United States
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Yoon, Miri; Song, Hangtian; HÃ¥kansson, Kristina et al. (2010) Hydrogen tunneling in adenosylcobalamin-dependent glutamate mutase: evidence from intrinsic kinetic isotope effects measured by intramolecular competition. Biochemistry 49:3168-73
Marsh, E Neil G (2009) Insights into the mechanisms of adenosylcobalamin (coenzyme B12)-dependent enzymes from rapid chemical quench experiments. Biochem Soc Trans 37:336-42
Li, Lei; Patterson, Dustin P; Fox, Christel C et al. (2009) Subunit structure of benzylsuccinate synthase. Biochemistry 48:1284-92
Lee, Hyang-Yeol; Yoon, Miri; Marsh, E Neil G (2007) Synthesis of Mono- and Di-Deuterated (2S, 3S)-3-Methylaspartic Acids to Facilitate Measurement of Intrinsic Kinetic Isotope Effects in Enzymes. Tetrahedron 63:4663-4668
Yoon, Miri; Kalli, Anastasia; Lee, Hyang-Yeol et al. (2007) Intrinsic deuterium kinetic isotope effects in glutamate mutase measured by an intramolecular competition experiment. Angew Chem Int Ed Engl 46:8455-9
Patwardhan, Anjali; Marsh, E Neil G (2007) Changes in the free energy profile of glutamate mutase imparted by the mutation of an active site arginine residue to lysine. Arch Biochem Biophys 461:194-9
Cheng, Mou-Chi; Marsh, E Neil G (2007) Evidence for coupled motion and hydrogen tunneling of the reaction catalyzed by glutamate mutase. Biochemistry 46:883-9
Li, Lei; Marsh, E Neil G (2006) Deuterium isotope effects in the unusual addition of toluene to fumarate catalyzed by benzylsuccinate synthase. Biochemistry 45:13932-8
Yoon, Miri; Patwardhan, Anjali; Qiao, Chunhua et al. (2006) Reaction of adenosylcobalamin-dependent glutamate mutase with 2-thiolglutarate. Biochemistry 45:11650-7
Li, Lei; Marsh, E Neil G (2006) Mechanism of benzylsuccinate synthase probed by substrate and isotope exchange. J Am Chem Soc 128:16056-7

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