Hydrogen atom transfer reactions are fundamental to a large class of enzymes that catalyze a diverse array of important metabolic reactions in which carbon-based radicals are key intermediates. Radical abstraction of a non-acidic hydrogen atom from the substrate is the key activation step in a variety of unusual and chemically difficult transformations. In many enzymes 5'-deoxyadenosyl radical, generated from either adenosylcobalamin or reduction of S-adenosylmethionine, serves as the cofactor for hydrogen abstraction;in others a protein-based radical, often a cysteinyl radical, serves as the radical cofactor. Compared to their reactivity in free solution, free radicals generated in enzymes appear to be stabilized to a remarkable degree. The mechanisms by which enzymes generate and stabilize reactive free radicals remain poorly understood.
Our aim i s to investigate in detail how hydrogen atom transfer, the key step in substrate activation, is catalyzed with the goal of illuminating the general principles by which enzymes catalyze radical reactions. We will focus on understanding hydrogen atom transfer in two radical enzymes that serve as model systems. Glutamate mutase is an adenosylcobalamin-dependent enzyme in which hydrogen atom transfer serves to generate a substrate radical that then undergoes a carbon skeleton rearrangement. Benzylsuccinate synthase is a glycyl-radical enzyme in which hydrogen transfer from toluene to an active site cysteine is a key mechanistic feature. We will integrate experimental measurements on enzymes and non-enzymatic model reactions with computational studies of these reactions to provide a framework within which to understand the differences between enzyme-catalyzed hydrogen atom transfer reactions and non-enzymatic reactions. Among the experiments we will conduct are kinetic isotope effect measurements that aim to determine to what extent quantum tunneling of the migrating hydrogen atom is important in the reaction catalyzed by glutamate mutase. We will compare these results with those obtained for mutant enzymes and model, non- enzymatic B12 reactions. By modeling both enzymatic and non-enzymatic reactions using state-of-the-art QM/MM computational methods we will gain insights into catalysis by the enzyme, and, in particular, whether the enzyme actually increases the amount of quantum tunneling in a reaction to enhance catalysis. We will test whether enzyme-catalyzed hydrogen atom transfer reactions can rationalized by linear free energy relationships such as the extended Evans-Polanyi equation and Hammett analysis that successfully predict the rates of a large number of non-enzymatic hydrogen transfer reactions based on simple empirically determined parameters. We will measure the rates of hydrogen transfer in benzylsuccinate synthase and glutamate mutase when reacted with substrate analogs containing substituents capable of stabilizing the resultant substrate radicals by different amounts. These experiments should provide insights into the relative importance of enthalpic, steric and polar effects in enzyme-catalyzed hydrogen transfer reactions.

Public Health Relevance

Although reactive free radicals are generally perceived as harmful to living organisms, there are many biochemical reactions that involve free radicals that are essential for life. We are studying how the enzymes that use these free radicals control them and harness their reactivity for "good" purposes. A better understanding of these enzymes may help in the design of drugs to fight diseases such as cancer and arthritis, and in engineering micro-organisms to clean up toxic chemicals in the environment.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM093088-04
Application #
8423809
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Anderson, Vernon
Project Start
2010-04-01
Project End
2015-01-31
Budget Start
2013-02-01
Budget End
2015-01-31
Support Year
4
Fiscal Year
2013
Total Cost
$242,763
Indirect Cost
$60,316
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
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Waugh, Matthew W; Marsh, E Neil G (2014) Solvent isotope effects on alkane formation by cyanobacterial aldehyde deformylating oxygenase and their mechanistic implications. Biochemistry 53:5537-43
Das, Debasis; Ellington, Benjamin; Paul, Bishwajit et al. (2014) Mechanistic insights from reaction of *-oxiranyl-aldehydes with cyanobacterial aldehyde deformylating oxygenase. ACS Chem Biol 9:570-7
Roman-Melendez, Gabriel D; von Glehn, Patrick; Harvey, Jeremy N et al. (2014) Role of active site residues in promoting cobalt-carbon bond homolysis in adenosylcobalamin-dependent mutases revealed through experiment and computation. Biochemistry 53:169-77
Funk, Michael A; Judd, Evan T; Marsh, E Neil G et al. (2014) Structures of benzylsuccinate synthase elucidate roles of accessory subunits in glycyl radical enzyme activation and activity. Proc Natl Acad Sci U S A 111:10161-6
Paul, Bishwajit; Das, Debasis; Ellington, Benjamin et al. (2013) Probing the mechanism of cyanobacterial aldehyde decarbonylase using a cyclopropyl aldehyde. J Am Chem Soc 135:5234-7
Khara, Basile; Menon, Navya; Levy, Colin et al. (2013) Production of propane and other short-chain alkanes by structure-based engineering of ligand specificity in aldehyde-deformylating oxygenase. Chembiochem 14:1204-8
Marsh, E Neil G; Waugh, Matthew W (2013) Aldehyde Decarbonylases: Enigmatic Enzymes of Hydrocarbon Biosynthesis. ACS Catal 3:
van der Kamp, Marc W; Mulholland, Adrian J (2013) Combined quantum mechanics/molecular mechanics (QM/MM) methods in computational enzymology. Biochemistry 52:2708-28
Eser, Bekir E; Das, Debasis; Han, Jaehong et al. (2011) Oxygen-independent alkane formation by non-heme iron-dependent cyanobacterial aldehyde decarbonylase: investigation of kinetics and requirement for an external electron donor. Biochemistry 50:10743-50

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