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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM021328-11
Application #
3270400
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Project Start
1977-06-01
Project End
1986-05-31
Budget Start
1985-06-01
Budget End
1986-05-31
Support Year
11
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Utah
Department
Type
Schools of Arts and Sciences
DUNS #
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Choi, Seoung-Ryoung; Seo, Jin-Soo; Bohaty, Rochelle F H et al. (2014) Regio- and chemoselective immobilization of proteins on gold surfaces. Bioconjug Chem 25:269-75
Seo, Jin-soo; Poulter, C Dale (2014) Sandwich antibody arrays using recombinant antibody-binding protein L. Langmuir 30:6629-35
Choi, Seoung-ryoung; Breugst, Martin; Houk, Kendall N et al. (2014) ?-Deuterium isotope effects as probes for transition-state structures of isoprenoid substrates. J Org Chem 79:3572-80
Viswanathan, Rajesh; Labadie, Guillermo R; Poulter, C Dale (2013) Regioselective covalent immobilization of catalytically active glutathione S-transferase on glass slides. Bioconjug Chem 24:571-7
Rudolf, Jeffrey D; Wang, Hong; Poulter, C Dale (2013) Correction to multisite prenylation of 4-substituted tryptophans by dimethylallyltryptophan synthase. J Am Chem Soc 135:10879
Bellesia, Franco; Choi, Seoung-ryoung; Felluga, Fulvia et al. (2013) Novel route to chaetomellic acid A and analogues: serendipitous discovery of a more competent FTase inhibitor. Bioorg Med Chem 21:348-58
Rudolf, Jeffrey D; Poulter, C Dale (2013) Tyrosine O-prenyltransferase SirD catalyzes S-, C-, and N-prenylations on tyrosine and tryptophan derivatives. ACS Chem Biol 8:2707-14
Seo, Jin-soo; Lee, Sungwon; Poulter, C Dale (2013) Regioselective covalent immobilization of recombinant antibody-binding proteins A, G, and L for construction of antibody arrays. J Am Chem Soc 135:8973-80
Rudolf, Jeffrey D; Wang, Hong; Poulter, C Dale (2013) Multisite prenylation of 4-substituted tryptophans by dimethylallyltryptophan synthase. J Am Chem Soc 135:1895-902
Heaps, Nicole A; Poulter, C Dale (2011) Synthesis and evaluation of chlorinated substrate analogues for farnesyl diphosphate synthase. J Org Chem 76:1838-43

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