This proposal will support genetic, structural, and mechanistic studies of isopentenyl diphosphate isomerase and squalene synthase, two key enzymes in the cholesterol biosynthetic pathway. High serum cholesterol is a major factor in the progression of coronary heart disease, the leading cause of death in the United States. Although diet has a significant influence on serum cholesterol levels, approximately 0.2% of the worldwide population suffers from hypercholesterolemia, the most common of all simply inherited genetic disorders. Hypercholesterolemia heterozygotes account for 5% of heart attack victims under the age of 60. Homozygotes have extremely high levels of serum cholesterol and typically die of heart disease before the age of 15. Major therapeutic breakthroughs were recently achieved with inhibitors of hydroxymethylglutaryl reductase. However, the mode of action of reductase inhibitors is not from blocking biosynthesis of cholesterol but rather from stimulating clearance of serum cholesterol by up- regulation of the LDL receptor population. This mode of action is ineffective for treatment of homozygotes, and other therapeutic targets with a different mechanism for cholesterol lowering are being sought. This project seeks to establish the mechanism of the chemical transformations catalyzed by isopentenyl diphosphate synthase and squalene synthase, to determine the 3-dimensional structures of the enzymes, and to discover how they catalyze their respective reactions. These studies will facilitate the rational design of inhibitors. Recombinant forms of the enzymes will be produced in multimilligram quantities for mechanistic and structural studies. The mechanisms of the chemical transformations and the factors that control regio- and stereoselectivity will be studied, using substrate analogs and mutant enzymes. Specific goals for the coming period include i) active collaborations to obtain X-ray structures for isopentenyl diphosphate isomerase and squalene synthase; ii) measurements of individual rate constants for binding, catalysis, and dissociation by pre-steady state techniques; iii) location of catalytic residues by covalent modification and site-directed mutagenesis of highly conserved amino acids; (iv) establishment of the chemical mechanisms of the reactions and relating the conversion of farnesyl diphosphate to squalene to the larger question of biosynthesis of non-head-to-tail isoprenoids; and v) completion of a topological study of the active site of farnesyl diphosphate synthase using bisubstrate analogs.
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