Prenyltransferases catalyze key condensation reactions in the terpene pathway during biosynthesis of sterols (including cholesterol), ubiquinones, and dolichols. We are studying the mechanisms of three prenyl transfer enzymes - farnesyl pyrophosphate synthetase(sterol, ubiquinone, and dolichol pathways), squalene synthetase (sterol pathway), dimethylallyl pyrophosphate:tryptophan synthetase (ergot alkaloid pathway) - to determine the chemistry of the carbon-carbon bond forming steps and how the enzymes catalyze their respective reactions. A fourth enzyme, isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase is being studied because the mechanism of the isomerization may be similar to prenyl transfer. Substrate analogues containing fluorine and sulfur are being synthesized as alternate substrates and inhibitors for initial velocity measurements. The binding mechanisms and individual kinetic constants will be evaluated by isotope partitioning, rapid quench, and isotope scrambling experiments. Linear free energy correlations will be used to determine the chemistry of the condensation step. The function of metal ion in prenyl transfer reactions will be studied using CD spectroscopy to monitor conformational changes in the protein and 31P NMR spectroscopy to monitor changes in the pyrophosphate moieties of the substrates. Analogues of the reactive carbocation intermediates proposed during rearrangement of presqualene pyrophosphate to squalene will be synthesized. It is anticipated that these compounds will be potent, selective inhibitors of squalene synthetase, the first pathway specific enzyme in cholesterol biosynthesis. Affinity columns for enzymes in the terpene pathway will be prepared with a pyrophosphonate moiety attached to the support. We anticipate this group will approximate the favorable binding characteristics of pyrophosphate but will be stable toward ubiquitous phosphatases. These columns will be used for purification of enzymes and separation of terpene and phosphatase activities during work with inhomogeneous, cell free systems. An assay will be developed for differentiating among the sterol, ubiquinone, and dolichol pathways based on chiral 2-fluoroisopentenyl pyrophosphate.
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