PHOTOAFFINITY-LABELING OF YEAST FARNESYL-PROTEIN TRANSFERASE AND ENZYMATIC-SYNTHESIS OF A RAS PROTEIN INCORPORATING A PHOTOACTIVE ISOPRENOID

Farnesyl protein transferase (FPTase) catalyzes the covalent attachmen t of a farnesyl (C-15) group from farnesyl pyrophosphate (FPP) to a sp ecific cysteine residue of Ras and several other proteins. In this rep ort, photoactive farnesyl and geranylgeranyl pyrophosphate analogs 2-d iazo-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 FPTas e. Both analogs are substrates for the enzyme, and upon irradiation, D ATFP-GPP inhibits FPTase activity in a time-dependent manner. Photoina ctivation by DATFP-GPP is prevented by the presence of the natural sub strate FPP. Photolysis of radiolabeled DATFP-GPP results in preferenti al labeling of the beta subunit of FPTase, suggesting that this subuni t is involved in recognition of FPP. Of particular importance, DATFP-G PP and DATFP-FPP were used to enzymatically transfer the photoactive i soprenoid moieties to peptides and to Ras; such molecules should be us eful for identifying cellular components which specifically recognize farnesylated Ras and other prenylated proteins. (C) 1997 Academic Pres s.