Faculty

Mechanisms of Flavin-Containing Enzymes. We study mechanisms of redox reactions occurring in the biologically critical pyrimidine interconversions catalyzed by flavin-dependent enzymes. By knowing the mechanisms in great detail, we will learn how enzymes accelerate reactions and should be able to design specific inhibitors that may be of therapeutic value. Our studies are guided by the philosophy that enzymes should be studied as reactants, at substrate-level concentrations, rather than as catalysts. Direct observation of events at the active site provides us with detailed chemical information.

Dihydroorotate Dehydrogenase (DHOD) - DHODs catalyze the oxidation of dihydroorotate (DHO) to orotate in the de novo biosynthesis of pyrimidines. They are targets, or are being considered as targets, in the treatment of a large number of diseases. We are currently studying DHODs from Homo sapiens, Escherichia coli, and two forms from Lactococcus lactis. We are investigating the mechanism of flavin reduction in these enzymes in order to determine the transition state structure. We are also studying the mechanisms of flavin oxidation by a variety of substrates and hope to learn the factors that cause a given enzyme to prefer one oxidizing substrate (e.g., fumarate) over another (e.g., ubiquinone).

Dihydrouridyl-tRNA Synthase (DUS) - After transcription, tRNA is modified extensively in a variety of ways. One of the most common is the reduction of specific uracil moieties to form dihydrouracil. These reactions are catalyzed by DUSs. We have shown that yeast DUS2 uses FMN, prefers NADPH over NADH, and transfers the proR-hydride of NADPH to the flavin. Surprisingly, we found that rapid reduction of RNA requires some other - as yet unidentified - modification, suggesting that the many tRNA maturation reactions are more ordered than commonly assumed.

Thymidylate Synthase (TS) - We are studying a newly discovered flavin-dependent form of TS. TSs methylate 2'-deoxyuridine monophosphate with the methylene group of methylenetetrahydrofolate. Classically, the methylene group is reduced to the methyl oxidation level by the folate. However, the "new" TS accomplishes this reduction by another mechanism, using an FAD prosthetic group and NAD(P)H as the source of reducing equivalents. Because the "new" thymidylate synthase occurs in a number of pathogens, it is an excellent drug target; because the "new" reaction involves a prosthetic group and an additional substrate, a novel mechanism reaction appears likely.

Representative Publications

1. Fagan, R.L., and Palfey, B.A., "Roles in Binding and Chemistry for Conserved Active Site Residues in the Class 2 Dihydroorotate Dehydrogenase from E. coli", Biochem, 2009, 48, 7169.

2. Koehn, E.M., Fleischmann, T., Conrad, J.A., Palfey, B.A., Lesley, S.A., Mathews, I.I., and Khoen, A., "An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene", Nature, 2009, 458, 919.

3. Wolfe, A.E., Thymark, M., Gattis, S.G., Fagan, R.L., Hu, Y.C., Johansson, E., Arent, S., Larsen, S. and Palfey, B.A., "The Interaction of Benzoate Pyrimidine Analogs with the Class 1A Dihydroorotate Dehydrogenase from Lactococcus lactis", Biochem., 2007, 46, 5741.

4. Fagan, R.L., Jensen, K.F., Björnberg, O. and Palfey, B.A., "Mechanism of Flavin Reduction in the Class 1A Dihydroorotate Dehydrogenase from Lactococcus lactis", Biochem., 2007, 46, 4028.

5. Palfey, B.A. and Fagan, R.L., "An Analysis of the Kinetic Isotope Effects on Initial Rates in Transient Kinetics", Biochem., 2006, 45, 13631.

6. Shi, J., Dertouzos, J., Gafni, A., Steel, D. and Palfey, B.A., "Single-Molecule Kinetics Reveals New Signatures of Half-Sites Reactivity in Dihydroorotate Dehydrogenase A in Catalysis", Proc. Nat. Acad. Sci., 2006, 103, 5775.

7. Frederick, K.K. and Palfey, B.A., "Kinetics of Proton-linked Flavin Conformational Changes in p-Hydroxybenzoate Hydroxylase", Biochemistry, 2005, 44, 13304.

8. Gattis, S.G. and Palfey, B.A., "Direct Observation of the Participation of Flavin in Product Formation by thyX-Encoded Thymidylate Synthase", J. Am. Chem. Soc., 2005.

Representative Book Chapters

1. Fagan, R.L. and Palfey, B.A., "Flavin-Dependent Enzymes", in Comprehensive Natural Products Chemistry II (Begley, T., ed.), 2008, Elsevier, Oxford, UK, in press.

2. Palfey, B.A., "Mechanisms", Ch. 2A in Enzymes and Their Inhibition: Drug Development (Smith, H.J. and Simons, C., eds.), 2005, CRC Press, 43-66.

3. Palfey, B.A., "Time Resolved Spectral Analysis", Ch. 9 in Kinetic Analysis of Macromolecules: A Practical Approach (Johnson, K.A., ed.), 2003, Oxford University Press, pp. 203-228.

4. Palfey, B.A. and Massey, V., "Flavin-Dependent Enzymes", Ch. 29 in Comprehensive Biological Catalysis, volume III / Radical Reactions and Oxidation/Reduction (Sinnott, M., ed.), 1998, Academic Press, pp. 83-154.

5. Palfey, B.A., Ballou, D.P. and Massey, V., "Oxygen Activation by Flavins and Pterins", Ch. 2 in Active Oxygen: Reactive Oxygen Species in Biochemistry (Valentine, J.S., Foote, C.S., Greenburg, A. and Leiberman, J.F., eds.), 1995, Chapman-Hall, pp. 37-83.