This proposal will determine the structure of conjugated ligands bound to enoyl-CoA hydratase and acyl-CoA dehydrogenase, two enzymes in the fatty acid n-oxidation pathway. Two questions will be addressed: (1) Can the molecular basis for the alterations in substrate electronic structure required for efficient catalysis be determined and (2) can the enzyme-substrate interactions responsible for preferential binding of one substrate conformer over another be identified? These questions stem from two proposals, namely: (1) that changes in the electronic structure of conjugated substrate/product analogs that occur upon binding to the enzymes reflect enzyme-substrate interactions in place to stabilize charge rearrangement as the reaction proceeds and (2) that the stereospecificity of the enzyme catalyzed reaction results from the preferential reaction of one bound conformer rather than the binding of a single substrate conformer to the enzyme. The experimental approaches involve modulating the size of the ligand or active site and modulating specific enzyme-ligand interactions using enzyme mutagenesis or ligand synthesis. The impact of these alterations on the distribution of bound ligand conformers, the stereochemistry of the reaction and changes in structure-reactivity will be assessed using vibrational and NMR spectroscopy in combination with enzyme kinetics. The identification and quantitation of enzyme-substrate interactions that correctly orient the substrate and stabilize charge redistribution will provide a basis for inhibitor design. Inhibitors of beta-oxidation enzymes have potential application as novel therapeutics for treating pathological conditions such as reperfusion-injury of the ischemic heart following a myocardial infarction and myocardial dysfunction in diabetes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063121-02
Application #
6520497
Study Section
Biochemistry Study Section (BIO)
Program Officer
Preusch, Peter C
Project Start
2001-04-01
Project End
2005-03-31
Budget Start
2002-04-01
Budget End
2003-03-31
Support Year
2
Fiscal Year
2002
Total Cost
$218,601
Indirect Cost
Name
State University New York Stony Brook
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Malo, Gabrielle D; Wang, Meitian; Wu, Di et al. (2008) Crystal structure and Raman studies of dsFP483, a cyan fluorescent protein from Discosoma striata. J Mol Biol 378:871-86
Bell, Alasdair F; Stratton, Christopher F; Zhang, Xujie et al. (2007) Evidence from Raman spectroscopy that InhA, the mycobacterial enoyl reductase, modulates the conformation of the NADH cofactor to promote catalysis. J Am Chem Soc 129:6425-31
Callahan, Brian P; Bell, Alasdair F; Tonge, Peter J et al. (2006) A Raman-active competitive inhibitor of OMP decarboxylase. Bioorg Chem 34:59-65
Doll, Charles; Bell, Alasdair F; Power, Nicholas et al. (2005) Procatalytic ligand strain. Ionization and perturbation of 8-nitroxanthine at the urate oxidase active site. Biochemistry 44:11440-6
Wu, Jiaquan; Bell, Alasdair F; Jaye, Andrew A et al. (2005) Ring current effects in the active site of medium-chain Acyl-CoA dehydrogenase revealed by NMR spectroscopy. J Am Chem Soc 127:8424-32
Bell, Alasdair F; Stoner-Ma, Deborah; Wachter, Rebekka M et al. (2003) Light-driven decarboxylation of wild-type green fluorescent protein. J Am Chem Soc 125:6919-26
Wu, Jiaquan; Bell, Alasdair F; Luo, Lian et al. (2003) Probing hydrogen-bonding interactions in the active site of medium-chain acyl-CoA dehydrogenase using Raman spectroscopy. Biochemistry 42:11846-56
He, Xiang; Bell, Alasdair F; Tonge, Peter J (2003) Ground state isomerization of a model green fluorescent protein chromophore. FEBS Lett 549:35-8
Feng, Yuguo; Hofstein, Hilary A; Zwahlen, Jacque et al. (2002) Effect of mutagenesis on the stereochemistry of enoyl-CoA hydratase. Biochemistry 41:12883-90
He, Xiang; Bell, Alasdair F; Tonge, Peter J (2002) Synthesis and spectroscopic studies of model red fluorescent protein chromophores. Org Lett 4:1523-6

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