The goal of this project is to use a combination of site-directed mutagenesis and X-ray crystallography to understand the structural basis of the catalytic power of yeast triosephosphate isomerase (TIM) and to unravel the mechanism of action of xylose isomerase (also called glucose isomerase and abbreviated XyI). Triosephosphate isomerase is the central enzyme in the glycolytic pathway and is extremely efficient: it operates at the diffusion-controlled limit. Xylose isomerase is the most widely-used industrial enzyme, is of paramount importance to the food industry, and is very inefficient. It operates over 100,000 times slower than TIM. Yet the two enzymes catalyse the same chemical transformation, the interconversion of an aldehyde and a ketone. Understanding the catalytic efficiency of TIM is important because there is a terrible human TIM deficiency disease. It is an inborn error of metabolism, inherited in an automosomal recessive fashion, and it leads to acute hemolytic anemia, severe neurological disfunction, increased susceptibility to infection, and propensity for sudden cardiac death. The lesion in several patients appears to be the mutation of a single amino acid, Glu 104, to an aspartic acid. One of the specific aims of this proposal is to duplicate this human TIM mutant in yeast TIM, characterize the kinetics and stability of the altered protein, and determine its three-dimensional structure. Comparison of this structure with that of the wild-type enzyme may lead to an under- standing of how this substitution leads to a deadly inherited metabolic disease. Other specific goals focus on understanding the roles of specific amino acids in the catalytic activity of both enzymes. The residues in question will be altered by sitedirected mutagenesis and the properties of the mutant proteins will be determined. One advantage of the TIM system is that the complete free-energy profile can be determined for the reaction catalysed by any interesting mutant, so the exact microsteps affected by the mutation can be discovered. Crystal structures will be obtained for every mutant in complex with a tight-binding inhibitor that is an analog of the intermediate in the reaction. For XyI, every mutant will be characterized in terms of its effect on the two separate stages of the reaction: catalytic opening of the sugar ring and isomerization. Every mutant will also be examined crystallographically, in complex with the actual substrate glucose.
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| 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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