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-labeled substrates and intermediates, transition state-analog inhibitors, and modified substrates are proposed to elucidate mechanistic aspects of the reactions, structures and conformations of intermediates, enzyme tolerance to structural modifications, 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 through collaborations with several biochemists and protein crystallographers. ? ? The stereochemistry and mechanistic characteristics of alkylations, cyclizations, and rearrangements catalyzed by taxadiene, abietadiene, epi-aristolochene, delta-cadinene and other terpene synthases as well as protein prenylation pathway enzymes will be elucidated to identify factors responsible for product specificity. Experiments with substrate analogs will probe active site tolerances, will enable identification of enzyme-bound intermediates, and will lead to novel isoprenoid products. Transition-state inhibitors and other substrate/intermediate analogs will be used as crystallization aids and as active site and reaction pathway markers in X-ray analyses of these biologically important proteins. ? ? Fundamental knowledge concerning the specificity, mechanisms, and catalytic functionality of enzymes such as epi-aristolochene, delta-cadinene, and taxadiene synthases may lead to more efficient production of the phytoalexin capsidiol, the antifertility agent gossypol, or the chemotherapeutic agent taxol, or lead to discovery of new derivatives of these natural products. The understanding gained about pyrophosphate binding and carbocation-pyrophosphate ion pairs will aid the design of new pharmaceutical agents having the potential to block cholesterol biosynthesis without side effects on other essential isoprenoid metabolites. Specific inhibitors of prenyl protein proteases have significant biochemical applications in the study of regulatory and signaltransducing proteins, as well as potential in cancer chemotherapy. A new synthesis of deoxyxylulose pathway intermediates and inhibitors will afford access to useful mechanistic tools and compounds having significant potential for treatment of malaria and other parasitic diseases. ? ?
