Protein Farnesyltransferase Isoprenoid Substrate Discrimination Is Dependent on Isoprene Double Bonds and Branched Methyl Groups

Abstract

Farnesylation is a posttranslational lipid modification in which a 15-carbon farnesyl isoprenoid is linked via a thioether bond to specific cysteine residues of proteins in a reaction catalyzed by protein farnesyltransferase (FTase). We synthesized the benzyloxyisoprenyl pyrophosphate (BnPP) series of transferable farnesyl pyrophosphate (FPP) analogues (1a-e) to test the length dependence of the isoprenoid substrate on the FTase-catalyzed transfer of lipid to protein substrate. Kinetic analyses show that pyrophosphates 1a-e and geranyl pyrophosphate (GPP) transfer with a lower efficiency than FPP whereas geranylgeranyl pyrophosphate (GGPP) does not transfer at all. While a correlation was found between K(m) and analogue hydrophobicity and length, there was no correlation between k(cat) and these properties. Potential binding geometries of FPP, GPP, GGPP, and analogues 1a-e were examined by modeling the molecules into the active site of the FTase crystal structure. We found that analogue 1d displaces approximately the same volume of the active site as does FPP, whereas GPP and analogues 1a-c occupy lesser volumes and 1e occupies a slightly larger volume. Modeling also indicated that GGPP adopts a different conformation than the farnesyl chain of FPP, partially 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 double bonds and branched methyl groups of the geranylgeranyl chain significantly restrict the number of possible conformations relative to the more flexible lipid chain of analogues 1a-e. The modeling results also provide a molecular explanation for the observation that an aromatic ring is a good isostere for the terminal isoprene of FPP.

A tagging-via-substrate technology for detection and proteomics of farnesylated proteins

“…Previous studies have indicated that minor modification of FPP has little effect on protein farnesylation (11,12). Therefore, we anticipated that addition of azide would not affect the enzymatic farnesylation reaction.…”

Farnesyl Diphosphate Analogues with Aryl Moieties Are Efficient Alternate Substrates for Protein Farnesyltransferase

“…K M isoprenoid was measured by varying the FPP or analogue concentration in the presence of saturating peptide, and k cat was measured by varying the isoprenoid concentration in the presence of saturating peptide, or by varying the peptide concentration in the presence of saturating analogue. (43) The dns-GCVLS peptide corresponds to the well-characterized Ca 1 a 2 X sequence of H-Ras. (21, 39, 44)…”

Organophosphorus Compounds: Intervention in Mechanisms of Signal Transduction Relevant to Proliferative, Immunological and Circulatory Disorders

“…p-Biphenyl structure 220, representing an , -bisisoprene-surrogate analogue of FPP proved an effective substrate for mammalian FTase [266], while aniline derivative 221 was as efficient a substrate for transfer to ras as 211 itself [267]. While the monoterpenes limonene 222 and perillyl alcohol 223 are anticancer agents capable of inhibiting mammalian FTase, derivatisation to the -hydroxyphosphonates failed to generate inhibitory structures [263].…”