Rl. Edelstein et Md. Distefano, PHOTOAFFINITY-LABELING OF YEAST FARNESYL-PROTEIN TRANSFERASE AND ENZYMATIC-SYNTHESIS OF A RAS PROTEIN INCORPORATING A PHOTOACTIVE ISOPRENOID, Biochemical and biophysical research communications, 235(2), 1997, pp. 377-382
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.