Students will have the option of choosing to work on a variety of different investigations of organic reaction mechanisms and enzyme catalysis. This work may involve limited chemical synthesis; kinetic analyses by UV, HPLC or 1H NMR; product analyses by HPLC, enzyme purification and the characterization of the mechanism of enzyme action.

A representative problem is the determination of the free energy reaction profile for transamination of glycine catalyzed by pyridoxal 5'-phosphate (PLP, Scheme). PLP is an essential cofactor for enzyme-catalyzed transamination of amino acids and a variety of other enzymatic reactions of amino acids.1 A recent paper noted that "There is no available experimental information on the energies of the carbanionic [quinonoid] intermediate for the uncatalyzed or PLP-catalyzed reactions (or their existence)".2 This is a significant gap in our understanding of the chemistry of this important cofactor; and, we have therefore initiated experiments to the determine values for the rate constants shown in Scheme 1 that are needed for the construction of a free-energy profile for transamination.

The experimental protocol is similar thematically to that used in an earlier determination of the free energy profile for aldose-ketose isomerization of glyceraldehydes.3 The project will require the determination of pseudo first-order rate constants for deprotonation of the aldimine and ketimine intermediates of transamination using conventional kinetic methods; the determination of so-called product rate constant ratios for partitioning the quinonoid intermediate generated by an independent pathway of PLP-catalyzed decarboxylation of a dicarboxylic amino acid; and, the development of a diffusion-controlled clock for protonation of the quinonoid.4 The undergraduate student will be given a clearly defined set of kinetic measurements and will work closely with a graduate student or postdoctoral fellow.

References

1. Sukhareva, B.S. (1986) Vitamin B6: Pyridoxal Phosphate, Chemical, Biochemical and Medical Aspects Part B. New York, John Wiley & Sons.
2. Spies, M.A., J.J. Woodward, M.R. Watnik and M.D. Toney (2004). "Alanine Racemase Free Energy Profile from Global Analysis of Progress Curves." J. Am. Chem. Soc. 126:
3. Richard, J.P. (1984). "Acid-Base Catalysis of the Elimination and Isomerization Reactions of Triose Phosphates." J. Am. Chem. Soc. 106:4926-4936.
4. Tapuhi, E. and W.P. Jencks (1982). "Base-catalyzed Halogenation of Acetone." J. Am. Chem. Soc. 104:5758-5765.