The reactions catalyzed by farnesyl pyrophosphate synthetase and squalene synthetase are the fundemental building steps in the cholesterol biosynthetic pathway. This proposal addresses mechanistic and topological questions for substrates and reactive intermediates in these two transformations. In both instances, molecules will be synthesized which covalently link partners present during catalysis but which are not normally covalently linked. A novel class of bisubstrate analogs with join homoallylic and allylic substrates for farnesyl pyrophosphate synthetase is being synthesized. These molecules are designed so the range of topologies which can bind and react is restricted. A study of the kinetic properties of the analogs and elucidation of products and stereochemistries should permit us to deduce permissible topologies for binding and provide information concerning the location of active-site functionality in the enzyme. The conversion of presqualene pyrophosphate to squalene is thought to be directed by the pyrophosphate anion during rearrangement of the presqualene cyclopropylcarbinyl cation. The substrate, presqualene pyrophosphate, is bound and the components of the ion pair are generated during catalysis. Kinetic studies show that squalene synthetase is inhibited when inorganic pyrophosphate and ammonium analogs of the carbocation are present in the buffer. It should be possible to eliminate entropic considerations in the binding of the two species by covalently linking the components of the ammonium-pyrophosphate ion pair. Ion pair analogs should be potent inhibitors of squalene synthetase. Squalene synthetase is an attractive target for inhibition of cholesterol biosynthesis because the enzyme lies at the branchpoint to sterols and regulation at that point permits expression of the other branches of the isoprene pathway. The research described in the proposal combines the traditional disciplines of enzymology (purification, kinetic methods) and organic chemistry (synthesis, isolation-identification) to study farnesyl pyrophosphate synthetase and squalene synthetase.
Showing the most recent 10 out of 72 publications
