title: Photoaffinity labeling of yeast farnesyl protein transferase and enzymatic synthesis of a Ras protein incorporating a photoactive isoprenoid.
abstract: Farnesyl protein transferase (FPTase) catalyzes the covalent attachment of a farnesyl (C15) group from farnesyl pyrophosphate (FPP) to a specific cysteine residue of Ras and several other proteins. In this report, photoactive farnesyl and geranylgeranyl pyrophosphate analogs 2-diazo-3,3,3-trifluoropropionyloxy-geranyl pyrophosphate (DATFP-GPP) and 2-diazo-3,3,3-trifluoropropionyloxy-farnesyl pyrophosphate (DATFP-FPP) were used to study the active site of Saccharomyces cerevisiae FPTase. Both analogs are substrates for the enzyme, and upon irradiation, DATFP-GPP inhibits FPTase activity in a time-dependent manner. Photoinactivation by DATFP-GPP is prevented by the presence of the natural substrate FPP. Photolysis of radiolabeled DATFP-GPP results in preferential labeling of the beta subunit of FPTase, suggesting that this subunit is involved in recognition of FPP. Of particular importance, DATFP-GPP and DATFP-FPP were used to enzymatically transfer the photoactive isoprenoid moieties to peptides and to Ras; such molecules should be useful for identifying cellular components which specifically recognize farnesylated Ras and other prenylated proteins.
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis 55455, USA.
authors.forename: R L
authors.initials: RL
authors.lastname: Edelstein
authors.forename: M D
authors.initials: MD
authors.lastname: Distefano
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis / Q000378:metabolism*; D019883:Alkyl and Aryl Transferases; D001665:Binding Sites; D003619:Dansyl Compounds / Q000138:chemical synthesis / Q000378:metabolism; D003979:Diazonium Compounds / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D008027:Light; D015394:Molecular Structure; D010782:Photolysis; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D017368:Protein Prenylation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012717:Sesquiterpenes; D014166:Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*
chemical_list: C095155:2-diazo-3,3,3-trifluoropropionyloxy-farnesyl diphosphate; D000345:Affinity Labels; D003619:Dansyl Compounds; D003979:Diazonium Compounds; D004791:Enzyme Inhibitors; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; C046509:2-diazo-3-trifluoropropionyloxy geranyl pyrophosphate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; D018631:ras Proteins; C002963:geranylgeranyl pyrophosphate