Protein farnesyltransferase isoprenoid substrate discrimination is dependent on isoprene double bonds and branched methyl groups

Farnesylation is a posttranslational lipid modification in which a 15-carbo n farnesyl isoprenoid is linked via a thioether bond to specific cysteine r esidues of proteins in a reaction catalyzed by protein farnesyltransferase (FTase). We synthesized the benzyloxyisoprenyl pyrophosphate (BnPP) series of transferable farnesyl pyrophosphate (FPP) analogues (la-e) to test the l ength dependence of the isoprenoid substrate on the FTase-catalyzed transfe r of lipid to protein substrate. Kinetic analyses show that pyrophosphates la-e and geranyl pyrophosphate (GPP) transfer with a lower efficiency than FPP whereas geranylgeranyl pyrophosphate (GGPP) does not transfer at all. W hile a correlation was found between K-m and analogue hydrophobicity and le ngth, there was no correlation between k(cat) and these properties. Potenti al binding geometries of FPP, GPP, GGPP, and analogues la-e were examined b y modeling the molecules into the active site of the FTase crystal structur e. We found that analogue ld displaces approximately the same volume of the active site as does FPP, whereas GPP and analogues la-c occupy lesser volu mes and le occupies a slightly larger volume. Modeling also indicated that GGPP adopts a different conformation than the farnesyl chain of FPP, partia lly occluding, the space occupied by the Ca(1)a(2)X peptide in the ternary X-ray crystal structure. Within the confines of the FTase pocket, the doubl e bonds and branched methyl groups of the geranylgeranyl chain significantl y restrict the number of possible conformations relative to the more flexib le lipid chain of analogues la-e. The modeling results also provide a molec ular explanation for the observation that an aromatic ring is a good isoste re for the terminal isoprene of FPP.