SITE SPECIFIC MUTAGENESIS OF ISOMERASES

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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.