title: Protein farnesyltransferase: kinetics of farnesyl pyrophosphate binding and product release.
abstract: 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.
authors.affiliation: Division of Experimental Therapy, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709, USA.
authors.forename: E S
authors.initials: ES
authors.lastname: Furfine
authors.forename: J J
authors.initials: JJ
authors.lastname: Leban
authors.forename: A
authors.initials: A
authors.lastname: Landavazo
authors.forename: J F
authors.initials: JF
authors.lastname: Moomaw
authors.forename: P J
authors.initials: PJ
authors.lastname: Casey
mesh_terms: D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001665:Binding Sites; D001710:Biotin; D002384:Catalysis; D003545:Cysteine / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D017368:Protein Prenylation; D012717:Sesquiterpenes; D013050:Spectrometry, Fluorescence; D014166:Transferases / Q000378:metabolism*
chemical_list: D010455:Peptides; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D001710:Biotin; C004808:farnesyl pyrophosphate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; D003545:Cysteine