Prenyltransferase catalyze key bond-forming reactions during the biosynthesis of hormones that stimulate cell division, sterols, ubiquinones, and dolichols. We are studying the prenyltransfer enzymes farnesyl pyrophosphate synthetase (sterol, ubiquinone, and dolichol pathways), squalene synthetase (branch point for sterol pathway) dimethylallyltryptophan synthetase (ergot alkaloid metabolism), 3-octaprenyl-4-hydroxy benzoate synthetase (ubiquinone metabolism), and i6A-tRNA synthetase (prenylation of tRNAs). A sixth enzyme under investigation, isopentenyl pyrophosphate:dimethylallyl-pyrophosphate isomerase, catalyzes the step in isoprene metabolism which chemically activates the biogenetic isoprene unit. The goals of this project are to secure reliable sources of enzymes for mechanistic and structural studies, to synthesize substrate analogs and inhibitors, and to characterize the chemistry of the reactions catalyzed by the six enzymes. Recombinant DNA techniques will be used to construct plasmids containing the genes for 3-octaprenyl-4-hydroxybenzoate synthetase, i6A-tRNA synthetase, and isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase for subsequent characterization of the genes and overproduction of the enzyme. Affinity columns will be constructed for purification of polyprenyl pyrophosphates. Substrate analogs containing fluorine, sulfur, and nitrogen (as an ammonium salt) are being synthesized to use as reversible inhibitors in linear free energy studies, as reactive intermediate analogs, as ion-pair traps for enzyme-bound carbocations, and as active-site directed irreversible inhibitors. Farnesyl pyrophosphate synthetase will be purified by affinity chromatography in sufficient quantities for characterization by X-ray, as will any of the cloned enzymes that can be crystallized. The orientation of substrates and metal ions in the catalytic site and conformational changes in the protein, which occurs during substrate binding, will be studied by 1H-13C 2D-NMR and 31P-NMR. Active site residues in isomerase have been covalently tagged with fluorinated irreversible inhibitors. The mechanism for the covalent attachment, and the amino acid(s) attached to the irreversible inhibitor will be determined.