Yeast Protein Farnesyltransferase Research Articles

Regioselective Covalent Immobilization of Catalytically Active Glutathione S-Transferase on Glass Slides

The high selectivity of protein farnesyltransferase was used to regioselectively append farnesyl analogues bearing bioorthogonal alkyne and azide functional groups to recombinant Schistosoma japonicum glutathione S-transferase (GSTase) and the active modified protein was covalently attached to glass surfaces. The cysteine residue in a C-terminal CVIA sequence appended to N-terminally His(6)-tagged glutathione S-transferase (His(6)-GSTase-CVIA) was post-translationally modified by incubation of purified protein or cell-free homogenates from E. coli M15/pQE-His(6)-GSTase-CVIA with yeast protein farnesyltransferase (PFTase) and analogues of farnesyl diphosphate (FPP) containing ω-azide and alkyne moieties. The modified proteins were added to wells on silicone-matted glass slides whose surfaces were modified with PEG units containing complementary ω-alkyne and azide moieties and covalently attached to the surface by a Cu(I)-catalyzed Huisgen [3 + 2] cycloaddition. The wells were washed and assayed for GSTase activity by monitoring the increase in A(340) upon addition of 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GT). GSTase activity was substantially higher in the wells spotted with alkyne (His(6)-GSTase-CVIA-PE) or azide (His(6)-GSTase-CVIA-AZ) modified glutathione-S-transferase than in control wells spotted with farnesyl-modified enzyme (His(6)-GSTase-CVIA-F).

Farnesyl Diphosphate Analogues with ω-Bioorthogonal Azide and Alkyne Functional Groups for Protein Farnesyl Transferase-Catalyzed Ligation Reactions

Eleven farnesyl diphosphate analogues, which contained omega-azide or alkyne substituents suitable for bioorthogonal Staudinger and Huisgen [3 + 2] cycloaddition coupling reactions, were synthesized. The analogues were evaluated as substrates for the alkylation of peptide cosubstrates by yeast protein farnesyl transferase. Five of the diphosphates were good alternative substrates for farnesyl diphosphate (FPP). Steady-state kinetic constants were measured for the active compounds, and the products were characterized by HPLC and LC-MS. Two of the analogues gave steady-state kinetic parameters (kcat and Km) very similar to those of the natural substrate.

P44 Non small cell lung cancer xenografts as preclinical models for investigations with tyrosine kinase inhibitors

Didehydrofarnesyl diphosphate (ΔΔFPP), a fluorescent pentaene analogue of farnesyl diphosphate (FPP), was synthesized using stereoselective Wittig reactions. Although ΔΔFPP was not an alternative substrate for yeast protein farnesyltransferase (FTase), the fluorescent analogue was a potent competitive inhibitor with a Ki value of 8.8 μM (Km(FPP)=27 μM).

Didehydrofarnesyl diphosphate: an intrinsically fluorescent inhibitor of protein farnesyltransferase

Didehydrofarnesyl diphosphate (ΔΔFPP), a fluorescent pentaene analogue of farnesyl diphosphate (FPP), was synthesized using stereoselective Wittig reactions. Although ΔΔFPP was not an alternative substrate for yeast protein farnesyltransferase (FTase), the fluorescent analogue was a potent competitive inhibitor with a Ki value of 8.8μM (Km(FPP)=27μM).

Yeast protein farnesyltransferase. pKas of peptide substrates bound as zinc thiolates.

Protein farnesyltransferase (PFTase) is a zinc metalloenzyme that catalyzes the posttranslational alkylation of the cysteine in C-terminal -Ca(1)a(2)X sequences by a 15-carbon farnesyl residue, where C is cysteine, a(1) and a(2) are normally aliphatic amino acids, and X is an amino acid that specifies selectivity for the farnesyl moiety. Formation of a Zn(2+) thiolate in the PFTase. peptide complex was detected by the appearance of an absorbance at 236 nm (epsilon = 15 000 M(-1) cm(-1)), which was dependent on the concentration of peptide, in a UV difference spectrum in a solution of PFTase and the peptide substrate RTRCVIA. We developed a fluorescence anisotropy binding assay to measure the dissociation constants as a function of pH for peptide analogues by appending a 2',7'-difluorofluorescein to their N-terminus. The electron-withdrawing fluorine atoms allowed us to measure peptide binding down to pH 5.5 without having to correct for the changes in fluorescence intensity that accompany protonation of the fluorophore. Measurements of the pK(a)s for thiol groups in free and bound peptide indicate that peptide binding is accompanied by formation of a zinc thiolate and that binding to PFTase lowers the pK of the peptide thiol by 3 units. In similar studies with the betaY310F mutant, the pK(a) of the thiol moiety was lowered by 2 units upon binding, indicating that the hydroxyl group in the conserved tyrosine helps stabilize the bound thiolate.

Yeast protein farnesyltransferase. Binding of S-alkyl peptides and related analogues.

[reaction: see text] Protein farnesyltransferase (PFTase) catalyzes alkylation of cysteine residues by farnesyl diphosphate (FPP). The dissociation constants for the PFTase-peptide analogue complexes for the series of analogues fl-RTRC(X)VIA (X = H, methyl, dodecyl, farnesyl) were measured by fluorescence anisotropy. The results indicate that an ionizable sulfhydryl moiety is important for substrate binding and the farnesyl group in the product facilitates binding.

ChemInform Abstract: Stereochemical Analysis of the Reaction Catalyzed by Yeast Protein Farnesyltransferase.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Stereochemical Analysis of the Reaction Catalyzed by Yeast Protein Farnesyltransferase

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTStereochemical Analysis of the Reaction Catalyzed by Yeast Protein FarnesyltransferaseRebecca L. Edelstein, Valerie A. Weller, Mark D. Distefano, and Jay S. TungView Author Information Department of Chemistry, University of Minnesota, Minnepolis, Minnesota 55455 Athena Neurosciences, Inc., South San Francisco, California 94080 Cite this: J. Org. Chem. 1998, 63, 16, 5298–5299Publication Date (Web):July 23, 1998Publication History Received23 February 1998Published online23 July 1998Published inissue 1 August 1998https://doi.org/10.1021/jo980304sCopyright © 1998 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views216Altmetric-Citations23LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (35 KB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information SUBJECTS:Chemical synthesis,Molecular structure,Nuclear magnetic resonance spectroscopy,Peptides and proteins,Stereochemistry Get e-Alerts

Amino Acid Residues That Define Both the Isoprenoid and CAAX Preferences of the Saccharomyces cerevisiae Protein Farnesyltransferase: CREATING THE PERFECT FARNESYLTRANSFERASE

Studies of the yeast protein farnesyltransferase (FTase) have shown that the enzyme preferentially farnesylates proteins ending in CAAX (C = cysteine, A = aliphatic residue, X = cysteine, serine, methionine, alanine) and to a lesser degree CAAL. Furthermore, like the type I protein geranylgeranyltransferase (GGTase-I), FTase can also geranylgeranylate methionine- and leucine-ending substrates both in vitro and in vivo. Substrate overlap of FTase and GGTase I has not been determined to be biologically significant. In this study, specific residues that influence the substrate preferences of FTase have been identified using site-directed mutagenesis. Three of the mutations altered the substrate preferences of the wild type enzyme significantly. The ram1p-74D FTase farnesylated only Ras-CIIS and not Ras-CII(M,L), and it geranylgeranylated all three substrates as well or better than wild type. The ram1p-206DDLF FTase farnesylated Ras-CII(S,M,L) at wild type levels but could no longer geranylgeranylate the Ras-CII(M,L) substrates. The ram1p-351FSKN FTase farnesylated Ras-CIIS and Ras-CIIM but not Ras-CIIL. The ram1p-351FSKN FTase was not capable of geranylgeranylating the Ras-CII(M,L) substrates, giving this mutant the attributes of the dogmatic FTase that only farnesylates non-leucine-ending CAAX substrates and does not geranylgeranylate any substrate. These results suggest that the isoprenoid and protein substrate specificities of FTase are interrelated. The availability of a mutant FTase that lacked substrate overlap with the protein GGTase-I made possible an analysis of the role of substrate overlap in normal cellular processes of yeast, such as mating and growth at elevated temperatures. Our findings suggest that neither farnesylation of leucine-ending CAAX substrates nor geranylgeranylation by the FTase is necessary for these cellular processes.

Yeast protein farnesyltransferase. Site-directed mutagenesis of conserved residues in the beta-subunit.

Protein prenyltransferases catalyze the posttranslational modification of cysteines by isoprenoid hydrocarbon chains. A protein farnesyltransferase (PFTase) and a protein geranylgeranyltransferase (PGGTase-I) alkylate cysteines in a CaaX C-terminal tetrapeptide sequence, where a is usually an aliphatic amino acid and X is an amino acid that specifies whether a C15 farnesyl or C20 geranylgeranyl moiety is added. A third enzyme, PGGTase-II, adds geranylgeranyl groups to both cysteines at the C-terminus of Rab proteins. All three enzymes are Zn2+ metalloproteins and also require Mg2+ for activity. The protein prenyltransferases are heterodimers. PFTase and PGGTase I contain identical alpha-subunits and distinctive beta-subunits, which are responsible for the differences in substrate selectivity seen for the two enzymes. The subunits in PGGTase-II are similar, but not identical, to their counterparts in the other two enzymes. An alignment of amino acid sequences for the beta-subunits of all three enzymes shows five regions of high similarity. Thirteen of the conserved polar and charged residues in yeast PFTase were selected for substitution by site-directed mutagenesis. Kinetic studies revealed a subset of five enzymes, R211Q, D307A, C309A, Y310F, and H363A, with substantially reduced catalytic constants (kcat). Metal analyses of wild-type enzyme and the five least reactive mutants showed that the substitutions had compromised Zn2+ binding in the D307A, C309A, and H363A enzymes.

Yeast protein farnesyltransferase: a pre-steady-state kinetic analysis.

Protein farnesyltransferase catalyzes alkylation of the cysteine in a carboxy-terminal CaaX motif where a is typically an aliphatic amino acid and X is alanine, methionine, serine, glutamine, or cysteine by a farnesyl residue. The modification enhances the lipophilicity of farnesylated proteins and promotes their association with membranes as part of their normal cellular function. Among the proteins modified by farnesyl residues is Ras, an important component in the signal transduction network for cell division that has been implicated in several forms of human cancer. In this paper, we describe isotope trapping, rapid quench, and single turnover experiments with the yeast enzyme using farnesyl diphosphate and the short peptide RTRCVIA as substrates. The kinetic constants for substrate binding, chemistry, and product release were determined from a fit of the differential equations describing the minimal catalytic mechanism to the kinetic data by numerical integration. Rate constants for chemistry and product release were 10.5 and 3.5 s(-1), respectively. The dissociation rate constant (33 s(-1)) for release of peptide from the ternary enzyme-substrate complex was three times larger than the rate constant for chemistry. The enthalpy of reaction, delta Hrxn = -17 +/- 1 kcal/mol for farnesylation of cysteine, was measured by microcalorimetry. Isotope trapping experiments revealed that the enzyme-farnesyl diphosphate complex was efficiently trapped by peptide but that the enzyme-peptide complex was not trapped by farnesyl diphosphate. These results are consistant with an ordered mechanism for formation of a catalytically competent ternary enzyme-farnesyl diphosphate-peptide complex.

ChemInform Abstract: Farnesyl and Geranylgeranyl Pyrophosphate Analogues Incorporating Benzoylbenzyl Ethers: Synthesis and Inhibition of Yeast Protein Farnesyltransferase.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Farnesyl and geranylgeranyl pyrophosphate analogs incorporating benzoylbenzyl ethers: Synthesis and inhibition of yeast protein farnesyltransferase

The syntheses of two photoactive analogs ( 1a and 1b) of farnesyl pyrophosphate that incorporate stable ether-linked benzophenones are described. Compounds 1a and 1b were prepared from geraniol in 17% overall yield. Both 1a and 1b are competitive inhibitors of yeast protein famesyltransferase with respect to farnesyl pyrophosphate and have K 1 values of 45 nM and 49 nM. Upon photolysis for twelve hours, 1a and 1b both inactivate the enzyme by 40%

Analogs of farnesyl pyrophosphate incorporating internal benzoylbenzoate esters: Synthesis, inhibition kinetics and photoinactivation of yeast protein farnesyltransferase

The syntheses of two analogs ( 1a and 1b) of farnesyl pyrophosphate incorporating photoactive benzoylbenzoate esters are described. Both 1a and 1b are competitive inhibitors of yeast protein farnesyltransferase with respect to farnesyl pyrophosphate and have Ki values of 3300 nM and 880 nM, respectively. Upon photolysis for two hours, 1a and 1b inactivate the enzyme by 46% and 11%, respectively. These compounds should be useful for a variety of studies of protein prenyltransferases.

Photoactive Analogs of Farnesyl Pyrophosphate Containing Benzoylbenzoate Esters: Synthesis and Application to Photoaffinity Labeling of Yeast Protein Farnesyltransferase.

Farnesyl pyrophosphate (FPP) is involved in a large number of cellular processes including the prenylation of transforming mutants of Ras proteins implicated in cancer. Photoactive analogs could provide useful information about enzyme active sites that bind farnesyl pyrophosphate; however, the availability of such compounds is extremely limited. Molecules that incorporate benzophenone moieties are attractive photoaffinity labeling reagents because of their useful photochemical properties. Here, the syntheses of two compounds, 3a and 3b, containing para- and meta-substituted benzoylbenzoates are described. Compounds 3a and 3b are competitive inhibitors (with respect to FPP) of yeast protein farnesyltransferase (PFTase) with K(i) values of 910 and 380 nM, respectively. Both compounds inactivate PFTase upon photolysis, resulting in as much as 44% inactivation of enzyme activity. Photolysis of PFTase in the presence of [(32)P]3a or of [(32)P]3b results in preferential labeling of the beta subunit, suggesting that this subunit is involved in prenyl group recognition. These compounds should be valuable tools for studying enzymes that utilize FPP as a substrate.

Yeast protein farnesyltransferase: steady-state kinetic studies of substrate binding.

Protein farnesyltransferase (PFTase) catalyzes the alkylation of cysteine in C-terminal CaaX sequences of a variety of proteins, including Ras, nuclear lamins, large G-proteins, and phosphodiesterases, by farnesyl diphosphate (FPP). These modifications enhance the ability of the proteins to associate with membranes and are essential for their respective functions. The binding mechanism for yeast PFTase was deduced from a combination of steady-state kinetic and equilibrium studies. Rates for prenylation were measured by a continuous assay based on an enhancement in the fluorescence of the dansyl moiety in pentapeptide dansyl-GCVIA upon farnesylation by FPP. Unreactive substrate analogs for FPP and dansyl-GCVIA gave steady-state inhibition patterns for the dead-end inhibitors typical of an ordered sequential mechanism in which FPP adds to the enzyme before the peptide. The kinetic analysis was complicated by substrate inhibition for dansyl-GCVIA. The substrate inhibition was reversed at high concentrations of FPP, indicating that formation of the nonproductive enzyme--peptide complex is competitive with respect to FPP. Progress curves were fitted to an integrated form of the rate expression to determine the catalytic constant, kcat = 4.5 +/- 1.9 s-1, and the Michaelis constant for dansyl-GCVIA, KMD = 0.9 +/- 0.1 microM. The dissociation constant for FPP, KD = 75 +/- 15 nM, was measured using a membrane retention assay.

Yeast Protein Geranylgeranyltransferase Type-I: Overproduction, Purification, and Characterization

Protein geranylgeranyltransferase type-I (PGGTase-I) catalyzes alkylation of the cysteine residue in proteins containing a consensus C-terminal CaaX sequence ending in leucine by the C 20 hydrocarbon moiety in geranylgeranyl diphosphate (GGPP). The Saccharomyces cerevisiae genes encoding the α ( RAM2) and β ( CDC43) subunits of PGGTase-I were translationally coupled by overlapping the RAM2- CDC43 stop-start codons and by locating a ribosome-binding site near the 3′ end of RAM2. Recombinant PGGTase-I was overproduced in Escherichia coli to give ∼8% of total cellular protein and purified 12-fold to >95% homogeneity in two steps by ion-exchange and immunoaffinity chromatography. The purified heterodimer contained α- and β-subunits with molecular masses of 34 and 42 kDa, respectively. A continuous fluorescence assay was developed to measure PGGTase-I activity. The recombinant enzyme showed maximal activity at pH 7.5 and required both Mg 2+ and Zn 2+. Michaelis constants for GGPP (1.0 μM) and dansyl-Gly-Cys-Ile-Ile-Leu (2.4 μM) were similar to those reported for yeast protein farnesyltransferase (PFTase) with farnesyl diphosphate and dansyl-Gly-Cys-Val-Ile-Ala; V max = 0.20 μmol min −1 mg −1 for recombinant yeast PGGTase-I was similar to that reported for yeast PFTase.

A mechanism for posttranslational modifications of proteins by yeast protein farnesyltransferase.

Protein farnesyltransferase catalyzes the alkylation of cysteine in C-terminal CaaX sequences of a variety of proteins, including Ras, nuclear lamins, large G proteins, and phosphodiesterases, by farnesyl diphosphate (FPP). These modifications enhance the ability of the proteins to associate with membranes and are essential for their respective functions. The enzyme-catalyzed reaction was studied by using a series of substrate analogs for FPP to distinguish between electrophilic and nucleophilic mechanisms for prenyl transfer. FPP analogs containing hydrogen, fluoromethyl, and trifluoromethyl substituents in place of the methyl at carbon 3 were evaluated as alternative substrates for alkylation of the sulfhydryl moiety in the peptide dansyl-GCVIA. The analogs were alternative substrates for the prenylation reaction and were competitive inhibitors against FPP. A comparison of kcat for FPP and the analogs with ksolv, the rate constants for solvolysis of related p-methoxybenzenesulfonate derivatives, indicated that protein prenylation occurred by an electrophilic mechanism.

The inhibition of protein prenyltransferases by oxygenated metabolites of limonene and perillyl alcohol.

The monoterpenes limonene and perillyl alcohol are effective therapeutic agents against advanced rat mammary cancer. Limonene is currently undergoing clinical testing in cancer patients. These monoterpenes and their oxygenated metabolites have been previously shown to inhibit protein prenylation in cultured cells. Since farnesylation of ras protein is critical for its ability to cause oncogenic transformation, inhibition of protein prenylation may be the basis of the anti-tumor effects of limonene and perillyl alcohol. In this study we test the ability of limonene and its oxygenated analogs to inhibit protein prenylation enzymes in vitro. Limonene and perillyl alcohol and their major in vivo metabolite, perillic acid, are weak inhibitors of both mammalian and yeast protein farnesyl transferase (PFT) and protein geranylgeranyl transferase (PGGT). In contrast, a minor metabolite of both limonene and perillyl alcohol, perillic acid methyl ester, is a potent inhibitor of both enzymes. Perillic acid methyl ester is a competitive inhibitor of yeast PFT with respect to farnesyl pyrophosphate. These studies suggest that if the inhibition of protein prenylation is a mechanism for limonene's and perillyl alcohol's anti-cancer activities, these monoterpenes may be prodrugs that are converted into pharmacologically-active substances by metabolic modification.

Purified yeast protein farnesyltransferase is structurally and functionally similar to its mammalian counterpart

Protein farnesyltransferase (FTase) catalyses the addition of a farnesyl group to a cysteine within the so-called 'CAAX box' at the C-terminus of various proteins. In the present paper we report purification of Saccharomyces cerevisiae FTase to near-homogeneity. This was accomplished by constructing a yeast strain overproducing FTase approx. 100-fold. The purified enzyme was a heterodimer of approx. 90 kDa and consisted of 43 kDa and 34 kDa subunits. The 43 kDa subunit was shown to be the product of the DPR1 gene by using antibody raised against baculovirus-produced DPR1 polypeptide. The purified enzyme required Mg2+, showed a pH optimum of 7.8 and was most active at 50 degrees C. The Km values for farnesyl pyrophosphate and GST-CIIS (glutathione S-transferase fused to the C-terminal 12 amino acids of yeast RAS2 protein), KmFpp and KmGST CIIS, were 8.1 and 5.1 microM respectively. The enzyme was capable of farnesylating GST-CIIL (the same as GST-CIIS, except that the C-terminal serine is changed to leucine), a substrate protein for the enzyme geranylgeranyltransferase, although with a higher apparent Km than for GST-CIIS. Like its mammalian counterpart, yeast FTase activity was inhibited by peptides containing the C-terminal CAAX sequence (that is, one where C = cysteine, A = aliphatic amino acid and X = any amino acid). These results provide direct evidence for the idea that the yeast and mammalian FTases are structurally and functionally very similar.