title: A versatile photoactivatable probe designed to label the diphosphate binding site of farnesyl diphosphate utilizing enzymes.
abstract: Farnesyl diphosphate (FPP) is a substrate for a diverse number of enzymes found in nature. Photoactive analogues of isoprenoid diphosphates containing either benzophenone, diazotrifluoropropionate or azide groups have been useful for studying both the enzymes that synthesize FPP as well as those that employ FPP as a substrate. Here we describe the synthesis and properties of a new class of FPP analogues that links an unmodified farnesyl group to a diphosphate mimic containing a photoactive benzophenone moiety; thus, importantly, these compounds are photoactive FPP analogues that contain no modifications of the isoprenoid portion of the molecule that may interfere with substrate binding in the active site of an FPP utilizing enzyme. Two isomeric compounds containing meta- and para-substituted benzophenones were prepared. These two analogues inhibit Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) with IC(50) values of 5.8 (meta isomer) and 3.0 microM (para isomer); the more potent analogue, the para isomer, was shown to be a competitive inhibitor of ScPFTase with respect to FPP with a K(I) of 0.46 microM. Radiolabeled forms of both analogues selectively labeled the beta-subunit of ScPFTase. The para isomer was also shown to label Escherichia coli farnesyl diphosphate synthase and Drosophila melanogaster farnesyl diphosphate synthase. Finally, the para isomer was shown to be an alternative substrate for a sesquiterpene synthase from Nostoc sp. strain PCC7120, a cyanobacterial source; the compound also labeled the purified enzyme upon photolysis. Taken together, these results using a number of enzymes demonstrate that this new class of probes should be useful for a plethora of studies of FPP-utilizing enzymes.
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, United States.
authors.forename: Olivier
authors.initials: O
authors.lastname: Henry
authors.forename: Fernando
authors.initials: F
authors.lastname: Lopez-Gallego
authors.forename: Sean A
authors.initials: SA
authors.lastname: Agger
authors.forename: Claudia
authors.initials: C
authors.lastname: Schmidt-Dannert
authors.forename: Stephanie
authors.initials: S
authors.lastname: Sen
authors.forename: David
authors.initials: D
authors.lastname: Shintani
authors.forename: Katrina
authors.initials: K
authors.lastname: Cornish
authors.forename: Mark D
authors.initials: MD
authors.lastname: Distefano
mesh_terms: D000818:Animals; D001665:Binding Sites; D019755:Carbon-Carbon Lyases / Q000737:chemistry / Q000378:metabolism; D004331:Drosophila melanogaster / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D051231:Farnesyltranstransferase / Q000737:chemistry* / Q000378:metabolism; D051228:Geranyltranstransferase / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D046937:Nostoc / Q000201:enzymology; D019920:Photoaffinity Labels / Q000032:analysis / Q000737:chemistry*; D011106:Polyisoprenyl Phosphates / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012717:Sesquiterpenes / Q000737:chemistry* / Q000378:metabolism
chemical_list: D019920:Photoaffinity Labels; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D051228:Geranyltranstransferase; D051231:Farnesyltranstransferase; D019755:Carbon-Carbon Lyases; C051130:trichodiene synthetase