title: Modulation of the zinc(II) center in protein farnesyltransferase by mutagenesis of the zinc(II) ligands.
abstract: Protein farnesyltransferase (PFTase) is a zinc-containing metalloenzyme that catalyzes the alkylation of cysteine (C) in protein substrates containing a C-terminal "CaaX" motif by farnesyl diphosphate (FPP). In yeast PFTase Zn(II) is coordinated to D307, C309, and H363 in the beta-subunit. The inner coordination sphere of the metal also contains a water molecule to give a net charge of 0 for the tetracoordinate Zn(II) center. When the protein substrate binds, the water molecule is replaced by the thiol of the cysteine residue, and the thiol is deprotonated to generate a Zn(II)-stabilized thiolate in the PFTase.FPP.protein ternary complex for the ensuing prenyl transfer reaction. An expression system was constructed for yeast PFTase containing a His(6) tag at the C-terminus of the beta-subunit to facilitate purification of the wild-type enzyme and site-directed mutants. The amino acids that coordinate Zn(II) were substituted to give a series of mutant PFTases with net charges of +1, 0, -1, and -2 at the Zn(II) center of the ternary enzyme.substrate complexes. Wild-type PFTase and the site-directed mutants were purified as alpha,beta-heterodimers, and each was found to contain an equivalent of Zn(II). All of the mutants were less reactive than wt PFTase (net charge of -1), with the greatest losses of activity seen for the mutants with net charges of 0 and +1. Equilibrium binding experiments with dGCVIA peptide and an unreactive analogue of FPP, (E,E)-2-[2-oxo-2-[[(3,7,11-trimethyl-2,6,10-dodecatrienyl)oxy]amino]ethyl]phosphonate (FNP), established that all of the mutants bound an equivalent of the peptide substrate. Like wt PFTase, the pH dependence of K(D) for the mutants did not change significantly between pH 5 and pH 9, indicating that pK(A)s for the thiol moiety in the (mutant PFTase).FNP.peptide complexes were <5. dGSVIA and dG(beta-NH2-Ala)VIA, where the sulfhydryl moiety was replaced by hydroxyl and amino groups, respectively, were not substrates. These experiments suggest a direct relationship between the net charge of the Zn(II) center in PFTase and the reactivity of the peptide thiolate that is alkylated by FPP.
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Harris
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Derdowski
authors.forename: C Dale
authors.initials: CD
authors.lastname: Poulter
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D005454:Fluorescence Polarization; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009584:Nitrogen / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids / Q000138:chemical synthesis / Q000235:genetics; D017368:Protein Prenylation / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D015032:Zinc / Q000032:analysis / Q000737:chemistry*
chemical_list: D008024:Ligands; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; D015032:Zinc; D009584:Nitrogen; D010100:Oxygen