The major goal of this research program is to develop a fundamental understanding of the specificity, catalytic and control mechanisms, active site structures, and biological functions of key enzymes involved in terpene and sterol biosynthesis. Syntheses and applications of isotope-labelled substrates and intermediates, transition state-analog inhibitors, and related compounds are proposed to elucidate mechanistic aspects of the reactions, structures and conformations of intermediates, and 3-dimensional interactions in enzyme-substrate (or intermediate) analog complexes. Enzymatic experiments and X-ray diffraction analyses of the enzyme complexes will be carried out by collaborations with several biochemists and protein crystallographers. The stereochemistry and mechanistic characteristics of alkylations, cyclizations, and rearrangements catalyzed by squalene, taxadiene, abietadiene, epi-aristolochene, and vetispiradiene synthases as well as protein prenyl transferases will be elucidated to identify factors responsible for product specificity. Experiments with substrate analogs will probe active site tolerances, enable identification of enzyme-bound intermediates, and lead to novel isoprenoid products. Transition-state inhibitors as well as heavy-atom and other analogs of substrates and intermediates are proposed for use as active site and reaction pathway markers in X-ray crystallographic studies of isoprenoid synthases, or as photo-activatable agents for investigation of prenylated protein complexes. Fundamental knowledge concerning the substrate specificity, mechanisms, and catalytic functionality of enzymes such as epi-aristolochene, vetispiradiene, and taxadiene synthases may lead to more efficient production of the phytoalexins capsidiol and solavetivone for agricultural uses, or the chemotherapeutic agent taxol for cancer treatment, or lead to discovery of more active derivatives. High affinity inhibitors of squalene synthase have the potential to block cholesterol biosynthesis without deleterious side effects on the formation of other essential isoprenoid metabolites. Specific prenyl transferase inhibitors would have significant biochemical applications in the study of regulatory and signal-transducing proteins, as well as potential in cancer chemotherapy.
