There are six known branches of the isoprene biosynthetic pathway in man. Metabolites from two of the branches are directly related to two leading causes of death in the United States. High levels of serum cholesterol, the major metabolite of isoprene metabolism, is a major factor in coronary heart disease. Approximately 0.2% of the U.S. population suffers from familial hypercholesterolemia, a genetic predisposition to high blood cholesterol, which must be regulated by medication. These individuals have a 20-fold increase in risk for early heart attack. Several cancers, including 90% of pancreatic tumors, 50% of colorectal tumors, and 50% of lung adenocarcinomas, are linked to Ras oncoproteins. Normal Ras proteins are carefully regulated GTP-binding proteins in the signal transduction network for cell division. Certain oncogenic mutations shift the Ras proteins into a permanently activated state. The transforming activity of RAs oncoproteins requires their association with the inner surface of the plasma membrane. This association depends on posttranslational modification of a carboxy-terminal cysteine with an isoprenoid farnesyl residue. When farnesylation of human Ras oncoprotein is blocked, the protein no longer induces transformation. This proposal outlines a study of prenyltransferases, a family of enzymes responsible for the major building reactions in the trunk of the isoprene pathway and for all of its branch point reactions. Several of these enzymes are logical therapeutic targets for coronary heart disease and some forms of cancer. The enzymes listed in this proposal will be studied at the genetic, structural, and mechanistic levels. We plan to establish the chemistry of the prenyltransfer reaction for several known substrates, identify common structural motifs in prenyltransferases responsible for binding and catalysis, and develop potent inhibitors based on mechanistic and structural concepts. Seven enzymes will be studied - farnesyl diphosphate synthase, squalene synthase, hexaprenyl diphosphate synthase, dimethylallyladenosyl-tRNA synthase, protein prenyltransferase, dimethylallyltryptophan synthase, and geranylgeranylglyceryl phosphate synthase. Specific goals to be achieved include (i) identification and characterization of genes, (ii) development of systems for overproduction, (iii) determination of kinetic properties, (iv) definition of the chemistry of the reactions, (v) identification of important residues in the catalytic site, and (vi) synthesis and evaluation of new, selective inhibitors for prenyltransferases.
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