Free radicals are generally perceived as highly reactive species that are harmful to the cell. There are, however, a growing number of enzymes known that use carbon-based radicals to catalyze a variety of important metabolic reactions. We are studying two enzymes that use free radicals in quite different ways as model systems to investigate several fundamental aspects of enzyme-mediated radical catalysis, a) How do enzymes generate radicals? b) How is the removal of hydrogen, key step in substrate activation, catalyzed? c) How do enzymes control the rearrangement of reactive substrate-radical intermediates towards productive catalysis rather than harmful side reactions? One important class of radical enzymes uses adenosylcobalamin (coenzyme B12)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 paradigm for this class of enzymes.
We aim to use mutagenesis to investigate how the protein catalyzes homolysis of the coenzyme and controls radical species at the active site. The kinetic properties of various active site mutants will be examined in detail using a combination of rapid-reaction techniques and the crystal structures of selected mutants will be determined so that changes in structure can be correlated with changes in catalysis. We will also start to investigate the mechanism of the newly discovered glycyl radical enzyme, benzylsuccinate synthase, which is involved in the anaerobic degradation of toluene by various bacteria. The enzyme catalyzes a remarkable reaction - addition of toluene to the double bond of fumarate to form (R)-benzylsuccinate. It is believed to share structural and mechanistic similarities with ribonucleotide reductase and pyruvate formate-lyase, however the chemistry catalyzed is very different. We will examine the kinetic properties of the enzyme and attempt to identify and characterize various radical intermediates involved in the reaction. We will explore the substrate specificity of the enzyme to try and identify mechanism-based inhibitors of the protein and to evaluate the potential of the enzyme to detoxify various aromatic compounds.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059227-06
Application #
6838802
Study Section
Biochemistry Study Section (BIO)
Program Officer
Jones, Warren
Project Start
1999-04-01
Project End
2007-12-31
Budget Start
2005-01-01
Budget End
2005-12-31
Support Year
6
Fiscal Year
2005
Total Cost
$278,148
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
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) Mechanism of benzylsuccinate synthase probed by substrate and isotope exchange. J Am Chem Soc 128:16056-7
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

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