The objective of this proposal is to use the technique of site-directed mutagenesis, combined with X-rays crystallography, to learn how enzymes are able to catalyze isomerization reactions efficiently. Isomerizations are the simplest metabolic reactions, and enzymes that catalyze them are important causative agents of a number of inherited metabolic diseases. Three isomerases have been chosen as systems to study in this proposal: the glycolytic enzyme triosephosphate isomerase, the glycolytic enzyme phosphoglucose isomerase (which is identical to the lymphokine neuroleukin), and the important food-processing enzyme xylose isomerase (also known as glucose isomerase). All of these enzymes catalyze a single substrate/single product equilibration, which allows direct crystallographic observation of the enzyme-substrate complex for any mutant. For triosephosphate isomerase, the specific aims of the proposal are to understand the roles of conserved amino acids in and away from the active site, and to dissect the mechanism of the substrate-induced conformational changes that causes a flexible loop to shield the active site from bulk solvent. Knowledge of the mechanism of action of triosephosphate isomerase is expected to add to understanding of the severe disease that results when mutations lead to a suboptimal amount of this enzyme. For xylose isomerase, the specific aims are to learn how the enzyme catalyzes sugar ring opening, to understand how the two metal ions in the active site cooperate in the hydride transfer mechanism, and to reengineer the enzyme to use the triosephosphate isomerase proton transfer mechanism. Such an altered xylose isomerase would be of great value for biotechnology. For phosphoglucose isomerase, the specific aims are to express the pig muscle enzyme in E. coli, determine its crystal structure with and without substrate bound, and identify the resides important for its mechanism, which involves ring opening followed by base-catalyzed proton transfer. Since phosphoglucose isomerase is identical to the potent lymphokine neuroleukin, knowledge of its structure and mechanism of action will have a significant impact on understanding of neurological disorders such as AIDS-related dementia, in which this enzyme has been shown to be involved.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032415-17
Application #
2734479
Study Section
Biochemistry Study Section (BIO)
Project Start
1990-01-16
Project End
1999-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
17
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Brandeis University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
616845814
City
Waltham
State
MA
Country
United States
Zip Code
02454
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

Showing the most recent 10 out of 24 publications