Publications

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

  Micali E., Chehade K.A., Isaacs R.J., Andres D.A., Spielmann H.P.

  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) serie ... >> More

  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. << Less

  Biochemistry 40:12254-12265(2001) [PubMed] [EuropePMC]

• Protein farnesyltransferase: kinetics of farnesyl pyrophosphate binding and product release.

  Furfine E.S., Leban J.J., Landavazo A., Moomaw J.F., Casey P.J.

  Protein farnesyltransferase (FTase) catalyzes the prenylation of Ras and several other key proteins involved in cell regulation. The mechanism of the FTase reaction was elucidated by pre-steady-state and steady-state kinetic analysis. FTase catalyzed the farnesylation of biotinylated peptide subst ... >> More

  Protein farnesyltransferase (FTase) catalyzes the prenylation of Ras and several other key proteins involved in cell regulation. The mechanism of the FTase reaction was elucidated by pre-steady-state and steady-state kinetic analysis. FTase catalyzed the farnesylation of biotinylated peptide substrate (BiopepSH) by farnesyl pyrophosphate (FPP) to an S-farnesylated peptide (BiopepS-C15). The steady-state kinetic mechanism was ordered. FTase bound FPP in a two-step process with an effective dissociation rate constant of 0.013 s-1 and an overall Kd of 2.8 nM. BiopepSH reacted with FTase.FPP irreversibly, with a second-order rate constant of 2.2 x 10(5) M-1 s-1, to form FTase.BiopepS-C15. Because most of the FPP in FTase.FPP was trapped as FTase.BiopepS-C15 at high concentrations of BiopepSH, FPP dissociated slowly from the ternary complex relative to catalysis, so that the commitment to catalysis was high. The maximal rate constant for formation of FTase.BiopepS-C15 (enzyme-bound product) is much larger than kcat (0.06 s-1), indicating that product release is the rate-determining step in the reaction mechanism. << Less

  Biochemistry 34:6857-6862(1995) [PubMed] [EuropePMC]

• Protein prenyltransferases.

  Casey P.J., Seabra M.C.

  J Biol Chem 271:5289-5292(1996) [PubMed] [EuropePMC]

• Cocrystal structure of protein farnesyltransferase complexed with a farnesyl diphosphate substrate.

  Long S.B., Casey P.J., Beese L.S.

  Protein farnesyltransferase (FTase) catalyzes the transfer of the hydrophobic farnesyl group from farnesyl diphosphate (FPP) to cellular proteins such as Ras at a cysteine residue near their carboxy-terminus. This process is necessary for the subcellular localization of these proteins to the plasm ... >> More

  Protein farnesyltransferase (FTase) catalyzes the transfer of the hydrophobic farnesyl group from farnesyl diphosphate (FPP) to cellular proteins such as Ras at a cysteine residue near their carboxy-terminus. This process is necessary for the subcellular localization of these proteins to the plasma membrane and is required for the transforming activity of oncogenic variants of Ras, making FTase a prime target for anticancer therapeutics. The high-resolution crystal structure of rat FTase was recently determined, and we present here the X-ray crystal structure of the first complex of FTase with a FPP substrate bound at the active site. The isoprenoid moiety of FPP binds in an extended conformation in a hydrophobic cavity of the beta subunit of the FTase enzyme, and the diphosphate moiety binds to a positively charged cleft at the top of this cavity near the subunit interface. The observed location of the FPP molecule is consistent with mutagenesis data. This binary complex of FTase with FPP leads us to suggest a "molecular ruler" hypothesis for isoprenoid substrate specificity, where the depth of the hydrophobic binding cavity acts as a ruler discriminating between isoprenoids of differing lengths. Although other length isoprenoids may bind in the cavity, only the 15-carbon farnesyl moiety binds with its C1 atom in register with a catalytic zinc ion as required for efficient transfer to the Ras substrate. << Less

  Biochemistry 37:9612-9618(1998) [PubMed] [EuropePMC]

• Reaction path of protein farnesyltransferase at atomic resolution.

  Long S.B., Casey P.J., Beese L.S.

  Protein farnesyltransferase (FTase) catalyses the attachment of a farnesyl lipid group to numerous essential signal transduction proteins, including members of the Ras superfamily. The farnesylation of Ras oncoproteins, which are associated with 30% of human cancers, is essential for their transfo ... >> More

  Protein farnesyltransferase (FTase) catalyses the attachment of a farnesyl lipid group to numerous essential signal transduction proteins, including members of the Ras superfamily. The farnesylation of Ras oncoproteins, which are associated with 30% of human cancers, is essential for their transforming activity. FTase inhibitors are currently in clinical trials for the treatment of cancer. Here we present a complete series of structures representing the major steps along the reaction coordinate of this enzyme. From these observations can be deduced the determinants of substrate specificity and an unusual mechanism in which product release requires binding of substrate, analogous to classically processive enzymes. A structural model for the transition state consistent with previous mechanistic studies was also constructed. The processive nature of the reaction suggests the structural basis for the successive addition of two prenyl groups to Rab proteins by the homologous enzyme geranylgeranyltransferase type-II. Finally, known FTase inhibitors seem to differ in their mechanism of inhibiting the enzyme. << Less

  Nature 419:645-650(2002) [PubMed] [EuropePMC]