The objective of this project is to use X-ray crystallography, computer simulations, site-directed mutagenesis and kinetic measurements to determine what structural features in an enzyme active site promote efficient catalysis of hydrogen ion transfer. Transfer of a hydrogen, usually in the form of either a proton or a hydride ion is the most common chemical reaction in biology, and is also perhaps the simplest, yet it is not always an easy reaction to carry out rapidly. When the donor and acceptor atoms are unactivated carbons or oxygens, the uncatalyzed reactions are very slow, because the -C-H and -O-H groups are not very acidic (both typically have pKa values near 20). Yet enzymes are able to accelerate the transfer of H+ and H- from -C-H and -O-H by more than 15 orders of magnitude in some cases, achieving turnover numbers exceeding 1000 per second. Establishing how enzymes activate substrates, cofactors and their own catalytic groups to effect such catalysis is the goal of this project. We have selected several model systems for study. For proton transfer catalysis: mutarotase (GalM), which catalyses both ring opening and proton transfer; and ketosteroid isomerase (KSI), which utilizes a prototypical acid/base mechanism. For hydride transfer: xylose isomerase (XyI), which catalyzes sugar ring opening followed by metal-mediated 1,2-hydride transfer and dihydrofolate reductase (DHFR), where the hydride donor is NAD. For mutarotase and KSI our aim is to understand how the enzymes increase the basicity of the catalytic base and lower the pKa of the carbon acid and how the transition states are stabilized. For XyI, we aim to learn how the two metal ions in the active site cooperate to promote hydride transfer and how the ring-opening reaction is catalyzed. For DHFR we are studying the orientation and distance requirements for hydride donor and substrate positioning, and the possible role of coenzyme and substrate strain. The protic environment in the active sites and the role of protein dynamics in catalysis are also being probed for all these enzymes computationally and by ultra-high resolution (beyond approximately IA resolution) X-ray diffraction.
| Naffin-Olivos, Jacqueline L; Daab, Andrew; White, Andre et al. (2017) Structure Determination of Mycobacterium tuberculosis Serine Protease Hip1 (Rv2224c). Biochemistry 56:2304-2314 |
| Huang, Yu-Hwa; Zhu, Chen; Kondo, Yasuyuki et al. (2015) CEACAM1 regulates TIM-3-mediated tolerance and exhaustion. Nature 517:386-90 |
| Keedy, Daniel A; van den Bedem, Henry; Sivak, David A et al. (2014) Crystal cryocooling distorts conformational heterogeneity in a model Michaelis complex of DHFR. Structure 22:899-910 |
| Auclair, Jared R; Somasundaran, Mohan; Green, Karin M et al. (2012) Mass spectrometry tools for analysis of intermolecular interactions. Methods Mol Biol 896:387-98 |
| Brodkin, Heather R; Novak, Walter R P; Milne, Amy C et al. (2011) Evidence of the participation of remote residues in the catalytic activity of Co-type nitrile hydratase from Pseudomonas putida. Biochemistry 50:4923-35 |
| Somarowthu, Srinivas; Brodkin, Heather R; D'Aquino, J Alejandro et al. (2011) A tale of two isomerases: compact versus extended active sites in ketosteroid isomerase and phosphoglucose isomerase. Biochemistry 50:9283-95 |
| Lazar, Louis M; Fisher, S Zoe; Moulin, Aaron G et al. (2011) Time-of-flight neutron diffraction study of bovine ?-chymotrypsin at the Protein Crystallography Station. Acta Crystallogr Sect F Struct Biol Cryst Commun 67:587-90 |
| Sigala, Paul A; Caaveiro, Jose M M; Ringe, Dagmar et al. (2009) Hydrogen bond coupling in the ketosteroid isomerase active site. Biochemistry 48:6932-9 |
| Novak, Walter R P; Moulin, Aaron G; Blakeley, Matthew P et al. (2009) A preliminary neutron diffraction study of gamma-chymotrypsin. Acta Crystallogr Sect F Struct Biol Cryst Commun 65:317-20 |
| Sigala, Paul A; Kraut, Daniel A; Caaveiro, Jose M M et al. (2008) Testing geometrical discrimination within an enzyme active site: constrained hydrogen bonding in the ketosteroid isomerase oxyanion hole. J Am Chem Soc 130:13696-708 |
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