Faculty

Bruce Palfey

Associate Professor of Biological Chemistry
Assistant Research Scientist, Biophysics Research Division
Associate Program Director

Ph.D., University of Michigan
Postdoctoral Fellow, University of Michigan

Research Focus: Enzyme Reaction Mechanisms in Pyrimidine Metabolism

Phone: 734.615.2452
E-mail: brupalf@umich.edu
Fax: 734.763.4581

Mechanisms of Flavin-Containing Enzymes. Flavin-dependent enzymes catalyze a large number of biologically important redox reactions. They are usually made of protein and a tightly-bound flavin prosthetic group (FAD or FMN); together these components catalyze the conversion of substrates to products with concomitant cycling of the flavin through oxidized and reduced states. In small-molecule model reactions, several very different reaction mechanisms can lead to reduced flavins, and even more can lead to flavin reoxidation. However, when flavins are bound to a particular protein, most reaction mechanisms are thwarted, while one or two are vastly accelerated. The overarching theme of our work is to learn how proteins control the chemistry available to flavins. How do they turn-off one mechanism while turbo-charging another? We approach this by studying reaction mechanisms of example flavoenzymes in detail using whatever means are available, especially transient kinetics, and interpreting the results in terms of three-dimensional structures.

Some enzymes currently being studied are flavin-dependent thymidylate synthase, dihydrouridine synthase, MICAL, and dihydroorotate dehydrogenases. Each plays an important role in biology, suggesting that uncovering their mechanisms will enable the design of specific inhibitors that may be of therapeutic value. More importantly, we will learn how enzymes accelerate reactions and control the reactivity of the flavin prosthetic group. 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 mind-boggling detailed chemical information.

Thymidylate Synthase (TS) - The TS described in your biochemistry textbook does not use a flavin prosthetic group. However, a new type of TS, discovered about a decade ago, requires an FAD prosthetic group in order to methylate 2'-deoxyuridine monophosphate with the methylene group of methylenetetrahydrofolate. Genomic analysis shows that the flavin-dependent TS is actually widespread in archea and bacteria, including some nasty pathogens. How does the flavin participate in this carbon-transfer? A novel mechanism is emerging for activating the deoxynucleotide and transferring the carbon. We are attacking the problem with a battery of methods, including transient kinetics, NMR spectroscopy, chemical synthesis, and site-directed mutagenesis. Because the flavin-dependent TS occurs in a number of pathogens, it is an excellent drug target; because the "new" reaction involves a prosthetic group, exciting enzymology is guaranteed.
 
Dihydrouridyl-tRNA Synthase (DUS) - After transcription, tRNA is modified extensively in a variety of ways. One of the most common modifications is the reduction of specific uracil moieties to form dihydrouracil, catalyzed by flavin-dependent enzymes, DUSs. The biological role of dihydrouridine in tRNA is not known, but it occurs in almost all organisms, indicating an important selective benefit. We are defining the substrate specificities of yeast and human DUSs. Some tRNA substrates react terribly slowly unless they have first been modified in other ways, while different tRNAs react relatively rapidly even without being modified after transcription. Kinetics, spectroscopy, and hydrodynamic methods are being applied to learn more about what controls the reactivity of this system.

Molecule Interacting with CasL (MICAL) - MICALs are large, multi-domain flavoenzymes involved in signal-transduction pathways. Their biological roles are still being defined. They appear to cause actin to depolymerize and are, therefore, intimately involved in many of the processes that control cell morphology, such as axon growth in neurons. The sequence and structure of the N-terminal domain of MICAL are very similar to those of classic flavin-dependent aromatic hydroxylases. We are studying the reactivity of the flavin of MICAL in order to see how similar the enzyme is to other flavin-dependent hydroxylases. Does MICAL make a C4a-flavin hydroperoxide intermediate? What substrates can it hydroxylate? Does its reaction with NADPH control the catalytic cycle? How are each of these reactions affected by the other domains? Answering these questions will help us understand how well-known flavin-chemistry has been adapted to a unique biological purpose.

Dihydroorotate Dehydrogenase (DHOD) - DHODs catalyze the oxidation of dihydroorotate (DHO) to orotate in the de novo biosynthesis of pyrimidines. They are 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).

 

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.), 2010, Elsevier, Oxford, UK, 37-144.

  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.

 

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