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Carol A. Fierke - One of the best experts on this subject based on the ideXlab platform.
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Analogs of farnesyl diphosphate alter CaaX substrate specificity and reactions rates of Protein Farnesyltransferase.
Bioorganic & medicinal chemistry letters, 2015Co-Authors: Benjamin C. Jennings, Yen Chih Wang, Mark D Distefano, Richard A. Gibbs, Amy M. Danowitz, Carol A. FierkeAbstract:Attempts to identify the prenyl-proteome of cells or changes in prenylation following drug treatment have used 'clickable' alkyne-modified analogs of the lipid substrates farnesyl- and geranylgeranyl-diphosphate (FPP and GGPP). We characterized the reactivity of four alkyne-containing analogs of FPP with purified Protein Farnesyltransferase and a small library of dansylated peptides using an in vitro continuous spectrofluorimetric assay. These analogs alter prenylation specificity and reactivity suggesting that in vivo results obtained using these FPP analogs should be interpreted cautiously.
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Farnesyl Diphosphate Analogues with Aryl Moieties are Efficient Alternate Substrates for Protein Farnesyltransferase
Biochemistry, 2012Co-Authors: Thangaiah Subramanian, Carol A. Fierke, Douglas A. Andres, June E. Pais, Suxia Liu, Jerry M. Troutman, Yuta Suzuki, Karunai Leela Subramanian, H. Peter SpielmannAbstract:Farnesylation is an important post-translational modification essential for the proper localization and function of many Proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to Proteins is catalyzed by Protein Farnesyltransferase (FTase). We employed a library of FPP analogues with a range of aryl groups substituting for individual isoprene moieties to examine some of the structural and electronic properties of the transfer of an analogue to the peptide catalyzed by FTase. Analysis of steady-state kinetics for modification of peptide substrates revealed that the multiple-turnover activity depends on the analogue structure. Analogues in which the first isoprene is replaced with a benzyl group and an analogue in which each isoprene is replaced with an aryl group are good substrates. In sharp contrast with the steady-state reaction, the single-turnover rate constant for dansyl-GCVLS alkylation was found to be the same for all analogues, despite the increased chemical reactivity of the ben...
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Expansion of Protein Farnesyltransferase Specificity Using “Tunable” Active Site Interactions DEVELOPMENT OF BIOENGINEERED PRENYLATION PATHWAYS
The Journal of biological chemistry, 2012Co-Authors: James L. Hougland, Soumyashree A. Gangopadhyay, Carol A. FierkeAbstract:Post-translational modifications play essential roles in regulating Protein structure and function. Protein Farnesyltransferase (FTase) catalyzes the biologically relevant lipidation of up to several hundred cellular Proteins. Site-directed mutagenesis of FTase coupled with peptide selectivity measurements demonstrates that molecular recognition is determined by a combination of multiple interactions. Targeted randomization of these interactions yields FTase variants with altered and, in some cases, bio-orthogonal selectivity. We demonstrate that FTase specificity can be "tuned" using a small number of active site contacts that play essential roles in discriminating against non-substrates in the wild-type enzyme. This tunable selectivity extends in vivo, with FTase variants enabling the creation of bioengineered parallel prenylation pathways with altered substrate selectivity within a cell. Engineered FTase variants provide a novel avenue for probing both the selectivity of prenylation pathway enzymes and the effects of prenylation pathway modifications on the cellular function of a Protein.
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Insights into the mechanistic dichotomy of the Protein Farnesyltransferase peptide substrates CVIM and CVLS.
Journal of the American Chemical Society, 2012Co-Authors: Yue Yang, Carol A. Fierke, Bing Wang, Melek N. Ucisik, Guanglei Cui, Kenneth M. MerzAbstract:Protein Farnesyltransferase (FTase) catalyzes farnesylation of a variety of peptide substrates. 3H α-secondary kinetic isotope effect (α-SKIE) measurements of two peptide substrates, CVIM and CVLS, are significantly different and have been proposed to reflect a rate-limiting SN2-like transition state with dissociative characteristics for CVIM, while, due to the absence of an isotope effect, CVLS was proposed to have a rate-limiting peptide conformational change. Potential of mean force quantum mechanical/molecular mechanical studies coupled with umbrella sampling techniques were performed to further probe this mechanistic dichotomy. We observe the experimentally proposed transition state (TS) for CVIM but find that CVLS has a symmetric SN2 TS, which is also consistent with the absence of a 3H α-SKIE. These calculations demonstrate facile substrate-dependent alterations in the transition state structure catalyzed by FTase.
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Evaluation of Protein Farnesyltransferase substrate specificity using synthetic peptide libraries.
Bioorganic & medicinal chemistry letters, 2007Co-Authors: Amanda J. Krzysiak, Carol A. Fierke, Sarah A. Scott, Katherine A. Hicks, Richard A. GibbsAbstract:Farnesylation, catalyzed by Protein Farnesyltransferase (FTase), is an important post-translational modification guiding cellular localization. Recently predictive models for identifying FTase substrates have been reported. Here we evaluate these models through screening of dansylated-GCaaS peptides, which also provides new insights into the Protein substrate selectivity of FTase.
Patrick J. Casey - One of the best experts on this subject based on the ideXlab platform.
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Conversion of Protein Farnesyltransferase to a geranylgeranyltransferase.
Biochemistry, 2006Co-Authors: K.l. Terry, Patrick J. Casey, Lorena S. BeeseAbstract:Posttranslational modifications are essential for the proper function of a number of Proteins in the cell. One such modification, the covalent attachment of a single isoprenoid lipid (prenylation), is carried out by the CaaX prenyltransferases, Protein Farnesyltransferase (FTase) and Protein geranylgeranyltransferase type-I (GGTase-I). Substrate Proteins of these two enzymes are involved in a variety of cellular functions but are largely associated with signal transduction. These modified Proteins include members of the Ras superfamily, heterotrimeric G-Proteins, centromeric Proteins, and a number of Proteins involved in nuclear integrity. Although FTase and GGTase-I are highly homologous, they are quite selective for their substrates, particularly for their isoprenoid diphosphate substrates, FPP and GGPP, respectively. Here, we present both crystallographic and kinetic analyses of mutants designed to explore this isoprenoid specificity and demonstrate that this specificity is dependent upon two enzyme residues in the subunits of the enzymes, W102{beta} and Y365{beta} in FTase (T49{beta} and F324{beta}, respectively, in GGTase-I).
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Protein Farnesyltransferase in embryogenesis, adult homeostasis, and tumor development.
Cancer cell, 2005Co-Authors: Nieves Mijimolle, Patrick J. Casey, Juan Velasco, Pierre Dubus, Carmen Guerra, Carolyn Weinbaum, Victoria Campuzano, Mariano BarbacidAbstract:Protein Farnesyltransferase (FTase) is an enzyme responsible for posttranslational modification of Proteins carrying a carboxy-terminal CaaX motif. Farnesylation allows substrates to interact with membranes and Protein targets. Using gene-targeted mice, we report that FTase is essential for embryonic development, but dispensable for adult homeostasis. Six-month-old FTase-deficient mice display delayed wound healing and maturation defects in erythroid cells. Embryonic fibroblasts lacking FTase have a flat morphology and reduced motility and proliferation rates. Ablation of FTase in two ras oncogene-dependent tumor models has no significant consequences for tumor initiation. However, elimination of FTase during tumor progression had a limited but significant inhibitory effect. These results should help to better understand the role of Protein farnesylation in normal tissues and in tumor development.
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Kinetic studies of Protein Farnesyltransferase mutants establish active substrate conformation.
Biochemistry, 2003Co-Authors: Jennifer S Pickett, Patrick J. Casey, Heather L Hartman, Katherine E. Bowers, Alan C. Embry, Carol A. FierkeAbstract:The zinc metalloenzyme Protein Farnesyltransferase (FTase) catalyzes the transfer of a 15-carbon farnesyl moiety from farnesyl diphosphate (FPP) to a cysteine residue near the C-terminus of a Protein substrate. Several crystal structures of inactive FTase·FPP·peptide complexes indicate that K164α interacts with the α-phosphate and that H248β and Y300β form hydrogen bonds with the β-phosphate of FPP [Strickland, C. L., et al. (1998) Biochemistry 37, 16601−16611]. Mutations K164Aα, H248Aβ, and Y300Fβ were prepared and analyzed by single turnover kinetics and ligand binding studies. These mutations do not significantly affect the enzyme affinity for FPP but do decrease the farnesylation rate constant by 30-, 10-, and 500-fold, respectively. These mutations have little effect on the pH and magnesium dependence of the farnesylation rate constant, demonstrating that the side chains of K164α, Y300β, and H248β do not function either as general acid−base catalysts or as magnesium ligands. Mutation of H248β and Y30...
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Reaction path of Protein Farnesyltransferase at atomic resolution
Nature, 2002Co-Authors: Stephen B. Long, Patrick J. Casey, Lorena S. BeeseAbstract:Protein Farnesyltransferase (FTase) catalyses the attachment of a farnesyl lipid group to numerous essential signal transduction Proteins, including members of the Ras superfamily1. The farnesylation of Ras oncoProteins, which are associated with 30% of human cancers, is essential for their transforming activity2. FTase inhibitors are currently in clinical trials for the treatment of cancer2,3,4. 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.
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Farnesylation of Nonpeptidic Thiol Compounds by Protein Farnesyltransferase
Biochemistry, 2001Co-Authors: Kendra E. Hightower, Patrick J. Casey, Carol A. FierkeAbstract:Protein Farnesyltransferase catalyzes the modification of Protein substrates containing specific carboxyl-terminal Ca1a2X motifs with a 15-carbon farnesyl group. The thioether linkage is formed between the cysteine of the Ca1a2X motif and C1 of the farnesyl group. Protein substrate specificity is essential to the function of the enzyme and has been exploited to find enzyme-specific inhibitors for antitumor therapies. In this work, we investigate the thiol substrate specificity of Protein Farnesyltransferase by demonstrating that a variety of nonpeptidic thiol compounds, including glutathione and dithiothreitol, are substrates. However, the binding energy of these thiols is decreased 4−6 kcal/mol compared to a peptide derived from the carboxyl terminus of H-Ras. Furthermore, for these thiol substrates, both the farnesylation rate constant and the apparent magnesium affinity decrease significantly. Surprisingly, no correlation is observed between the pH-independent log(kmax) and the thiol pKa; model nucleop...
C. D. Poulter - One of the best experts on this subject based on the ideXlab platform.
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Yeast Protein Farnesyltransferase. pKas of Peptide Substrates Bound as Zinc Thiolates
Biochemistry, 1999Co-Authors: David B. Rozema, C. D. PoulterAbstract: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.
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Yeast Protein Farnesyltransferase. Site-directed mutagenesis of conserved residues in the beta-subunit.
Biochemistry, 1997Co-Authors: Julia M. Dolence, David B. Rozema, C. D. PoulterAbstract: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.
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Yeast Protein Farnesyltransferase: a pre-steady-state kinetic analysis.
Biochemistry, 1997Co-Authors: Jeffery R. Mathis, C. D. PoulterAbstract: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.
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Synthesis of Protein Farnesyltransferase and Protein Geranylgeranyltransferase Inhibitors: Rapid Access to Chaetomellic Acid A and Its Analogues
The Journal of organic chemistry, 1996Co-Authors: Elaref Ratemi, J. M. Dolence, C. D. Poulter, John C. VederasAbstract:A facile two-step stereospecific synthesis of the Protein Farnesyltransferase inhibitor chaetomellic acid A (1) and its analogues was developed. Addition of organocuprates derived from Grignard reagents (e.g. tetradecylmagnesium chloride and CuBr.Me(2)S) to dimethyl acetylenedicarboxylate (DMAD) in tetrahydrofuran containing hexamethylphosphoramide was followed by capture of the resulting copper enolates with a variety of electrophiles (e.g. methyl iodide) to give dimethyl cis-butenedioate derivatives 4-11. Hydrolysis with lithium hydroxide generated the corresponding lithium carboxylates, which readily closed to 2,3-disubstituted maleic anhydrides 17-20 upon acid treatment. Compound 16, an analogue wherein the tetradecyl group of 1 is replaced by a farnesyl moiety, is 7-fold more potent than 1 as an inhibitor of Protein Farnesyltransferase from yeast and displays a 100:1 selectivity for this enzyme relative to yeast Protein geranylgeranyltransferase. In contrast, analogue 15, which contains a geranylgeranyl side chain, shows ca. 10:1 selectivity for the latter enzyme.
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A mechanism for posttranslational modifications of Proteins by yeast Protein Farnesyltransferase
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Julia M. Dolence, C. D. PoulterAbstract: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.
Lorena S. Beese - One of the best experts on this subject based on the ideXlab platform.
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Conversion of Protein Farnesyltransferase to a geranylgeranyltransferase.
Biochemistry, 2006Co-Authors: K.l. Terry, Patrick J. Casey, Lorena S. BeeseAbstract:Posttranslational modifications are essential for the proper function of a number of Proteins in the cell. One such modification, the covalent attachment of a single isoprenoid lipid (prenylation), is carried out by the CaaX prenyltransferases, Protein Farnesyltransferase (FTase) and Protein geranylgeranyltransferase type-I (GGTase-I). Substrate Proteins of these two enzymes are involved in a variety of cellular functions but are largely associated with signal transduction. These modified Proteins include members of the Ras superfamily, heterotrimeric G-Proteins, centromeric Proteins, and a number of Proteins involved in nuclear integrity. Although FTase and GGTase-I are highly homologous, they are quite selective for their substrates, particularly for their isoprenoid diphosphate substrates, FPP and GGPP, respectively. Here, we present both crystallographic and kinetic analyses of mutants designed to explore this isoprenoid specificity and demonstrate that this specificity is dependent upon two enzyme residues in the subunits of the enzymes, W102{beta} and Y365{beta} in FTase (T49{beta} and F324{beta}, respectively, in GGTase-I).
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Thematic review series: lipid posttranslational modifications. Structural biology of Protein Farnesyltransferase and geranylgeranyltransferase type I.
Journal of lipid research, 2006Co-Authors: Kimberly T. Lane, Lorena S. BeeseAbstract:More than 100 Proteins necessary for eukaryotic cell growth, differentiation, and morphology require posttranslational modification by the covalent attachment of an isoprenoid lipid (prenylation). Prenylated Proteins include members of the Ras, Rab, and Rho families, lamins, CENPE and CENPF, and the γ subunit of many small heterotrimeric G Proteins. This modification is catalyzed by the Protein prenyltransferases: Protein Farnesyltransferase (FTase), Protein geranylgeranyltransferase type I (GGTase-I), and GGTase-II (or RabGGTase). In this review, we examine the structural biology of FTase and GGTase-I (the CaaX prenyltransferases) to establish a framework for understanding the molecular basis of substrate specificity and mechanism. These enzymes have been identified in a number of species, including mammals, fungi, plants, and protists. Prenyltransferase structures include complexes that represent the major steps along the reaction path, as well as a number of complexes with clinically relevant inhibitors. Such complexes may assist in the design of inhibitors that could lead to treatments for cancer, viral infection, and a number of deadly parasitic diseases.
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Reaction path of Protein Farnesyltransferase at atomic resolution
Nature, 2002Co-Authors: Stephen B. Long, Patrick J. Casey, Lorena S. BeeseAbstract:Protein Farnesyltransferase (FTase) catalyses the attachment of a farnesyl lipid group to numerous essential signal transduction Proteins, including members of the Ras superfamily1. The farnesylation of Ras oncoProteins, which are associated with 30% of human cancers, is essential for their transforming activity2. FTase inhibitors are currently in clinical trials for the treatment of cancer2,3,4. 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.
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Structures of Protein Farnesyltransferase
The Enzymes, 2001Co-Authors: Stephen B. Long, Lorena S. BeeseAbstract:High resolution three-dimensional crystal structures of Protein Farnesyltransferase (FTase) complexed with substrates and inhibitors provide a framework for understanding the molecular basis of substrate specificity and mechanism and may facilitate the development of improved chemotherapeutics. The 2.25A resolution crystal structure of rat FTase provided the first structural information on any Protein prenyltransferase enzyme (1). Rat FTase shares 93% sequence identity with the human enzyme and is predicted to be indistinguishable from human FTase in the active site region. Subsequently, a co-crystal structure of rat FTase with bound farnesyl diphosphate (FPP) revealed the location of the isoprenoid binding and gave insight into the molecular basis of isoprenoid substrate specificity (2). Recently, two co-crystal structures of rat FTase with a bound peptide substrate and a nonreactive isoprenoid diphosphate analog have identified the location of both the peptide and isoprenoid binding sites in a ternary enzyme complex (3,4). In this chapter we describe the recent crystal structures of rat FTase, and discuss their implications on understanding substrate specificity, mechanism, and inhibitor design.
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Conversion of Tyr361 beta to Leu in mammalian Protein Farnesyltransferase impairs product release but not substrate recognition.
Biochemistry, 2000Co-Authors: Rebecca A. Spence, Kendra E. Hightower, Carol A. Fierke, Lorena S. Beese, K.l. Terry, Patrick J. CaseyAbstract:Protein Farnesyltransferase catalyzes the lipid modification of Protein substrates containing Met, Ser, Gln, or Ala at their C-terminus. A closely related enzyme, Protein geranylgeranyltransferase type I, carries out a similar modification of Protein substrates containing a C-terminal Leu residue. Analysis of a mutant of Protein Farnesyltransferase containing a Tyr-to-Leu substitution at position 361 in the beta subunit led to the conclusion that the side chain of this Tyr residue played a major role in recognition of the Protein substrates. However, no interactions have been observed between this Tyr residue and peptide substrates in the crystal structures of Protein Farnesyltransferase. In an attempt to reconcile these apparently conflicting data, a thorough kinetic characterization of the Y361L variant of mammalian Protein Farnesyltransferase was performed. Direct binding measurements for the Y361L variant yielded peptide substrate binding that was actually some 40-fold tighter than that with the wild-type enzyme. In contrast, binding of the peptide substrate for Protein geranylgeranyltransferase type I was very weak. The basis for the discrepancy was uncovered in a pre-steady-state kinetic analysis, which revealed that the Y361L variant catalyzed farnesylation of a normal peptide substrate at a rate similar to that of the wild-type enzyme in a single turnover, but that subsequent turnover was prevented. These and additional studies revealed that the Y361L variant does not "switch" Protein substrate specificity as concluded from steady-state parameters; rather, this variant exhibits severely impaired product dissociation with its normal substrate, a situation resulting in a greatly compromised steady-state activity.
Joseph L. Goldstein - One of the best experts on this subject based on the ideXlab platform.
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Tetrapeptide inhibitors of Protein Farnesyltransferase : amino-terminal substitution in phenylalanine-containing tetrapeptides restores farnesylation
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Michael S. Brown, Joseph L. Goldstein, Kenneth J. Paris, John Burnier, James C. MarstersAbstract:Abstract Protein Farnesyltransferase from rat brain transfers farnesyl residues to cysteine residues in tetrapeptides that conform to the sequence CA1A2X, where C is cysteine, A1 and A2 are aliphatic amino acids, and X is methionine or serine. When the A2 residue is aromatic [e.g., phenylalanine as in Cys-Val-Phe-Met (CVFM)], the tetrapeptide continues to bind to the enzyme, but it can no longer accept a farnesyl group, and it becomes a pure inhibitor. The current studies show that this resistance to farnesylation also requires a positive charge on the cysteine amino group. Derivatization of this group with acetyl, octanoyl, or cholic acid residues or extension of the peptide with an additional amino acid restores the ability of phenylalanine-containing peptides to accept a farnesyl residue. The same result was obtained when the amino group of cysteine was deleted (mercaptopropionyl-VFM). These data suggest that the positive change on the cysteine amino group acts in concert with an aromatic residue in the A2 position to render peptides resistant to farnesylation by the rat brain enzyme.
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Divalent cation and prenyl pyrophosphate specificities of the Protein Farnesyltransferase from rat brain, a zinc metalloenzyme.
The Journal of biological chemistry, 1992Co-Authors: Yuval Reiss, Michael S. Brown, Joseph L. GoldsteinAbstract:The separate catalytic roles of Zn2+ and Mg2+ and the specificity of the prenyl pyrophosphate-binding site of the rat brain Protein Farnesyltransferase were explored using a purified enzyme preparation. The binding of p21Hras to the enzyme was abolished by dialysis against EDTA and restored by addition of ZnCl2, as demonstrated by chemical cross-linking. The binding of the other substrate, farnesyl pyrophosphate, was independent of divalent cations, as demonstrated by gel filtration. Transfer of the enzyme-bound farnesyl group to the bound p21Hras required Mg2+. Geranylgeranyl pyrophosphate bound to the prenyl pyrophosphate-binding site with an affinity equal to that of farnesyl pyrophosphate, but the geranylgeranyl group was not transferred efficiently to p21Hras. It also was not transferred to a modified p21Hras containing COOH-terminal leucine, a Protein that was shown previously to be a good substrate for a rat brain geranylgeranyltransferase. We conclude that the Protein Farnesyltransferase is a metalloenzyme that most likely contains Zn2+ at the peptide-binding site. It thus resembles certain metallopeptidases, including carboxypeptidase A and the angiotensin-converting enzyme. Strategies previously developed to screen for inhibitors of those enzymes may aid in the search for inhibitors of the Protein Farnesyltransferase.
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Cloning and expression of a cDNA encoding the alpha subunit of rat p21ras Protein Farnesyltransferase.
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Wenji Chen, Douglas A. Andres, Joseph L. Goldstein, Michael S. BrownAbstract:The complete amino acid sequence of the alpha subunit of heterodimeric p21ras Protein Farnesyltransferase from rat has been deduced from the sequence of a cloned cDNA. The cDNA encodes a 377-amino acid Protein that migrates on NaDodSO4/polyacrylamide gels identically to the alpha subunit purified from rat brain. When introduced into mammalian cells by transfection, the cDNA for the alpha subunit produced no immunodetectable Protein or Farnesyltransferase activity unless the cells were simultaneously transfected with a cDNA encoding beta subunit. In light of previous evidence that alpha subunit forms a heterodimer with at least two different beta subunits, current data suggest a mechanism for coordinating amounts of alpha and beta subunits. If an alpha subunit were stable only as a complex with a beta subunit, the number of alpha subunits would be automatically maintained at a level just sufficient to balance all beta subunits, thereby avoiding the potentially toxic overaccumulation of free alpha subunits.
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Nonfarnesylated tetrapeptide inhibitors of Protein Farnesyltransferase.
The Journal of biological chemistry, 1991Co-Authors: Joseph L. Goldstein, Sarah J. Stradley, Yuval Reiss, Michael S. Brown, Lila M. GieraschAbstract:The Protein Farnesyltransferase from rat brain was previously shown to be inhibited competitively by tetrapeptides that conform to the consensus Cys-A1-A2-X, where A1 and A2 are aliphatic amino acids and X is methionine, serine, or phenylalanine. In the current studies we use a thin layer chromatography assay to show that most of these tetrapeptides are themselves farnesylated by the purified enzyme. Two classes of tetrapeptides are not farnesylated and therefore act as true inhibitors: 1) those that contain an aromatic residue at the A2 position and 2) those that contain penicillamine (beta,beta-dimethylcysteine) in place of cysteine. The most potent of these pure inhibitors was Cys-Val-Phe-Met, which inhibited Farnesyltransferase activity by 50% at less than 0.1 microM. These data indicate that the inclusion of bulky aromatic or methyl residues in a tetrapeptide can abolish prenyl group transfer without blocking binding to the enzyme. This information should be useful in the design of peptides or peptidomimetics that inhibit farnesylation and thus block the action of p21ras Proteins in animal cells.
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Sequence requirement for peptide recognition by rat brain p21ras Protein Farnesyltransferase
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Yuval Reiss, Sarah J. Stradley, Lila M. Gierasch, Michael S. Brown, Joseph L. GoldsteinAbstract:Abstract We tested 42 tetrapeptides for their ability to bind to the rat brain p21ras Protein Farnesyltransferase as estimated by their ability to compete with p21Ha-ras in a farnesyltransfer assay. Peptides with the highest affinity had the structure Cys-A1-A2-X, where positions A1 and A2 are occupied by aliphatic amino acids and position X is occupied by a COOH-terminal methionine, serine, or phenylalanine. Charged residues reduced affinity slightly at the A1 position and much more drastically at the A2 and X positions. Effective inhibitors included tetrapeptides corresponding to the COOH termini of all animal cell Proteins known to be farnesylated. In contrast, the tetrapeptide Cys-Ala-Ile-Leu (CAIL), which corresponds to the COOH termini of several neural guanine nucleotide binding (G) Protein gamma subunits, did not compete in the farnesyl-transfer assay. Inasmuch as several of these Proteins are geranylgeranylated, the data suggest that the two isoprenes (farnesyl and geranylgeranyl) are transferred by different enzymes. A biotinylated heptapeptide corresponding to the COOH terminus of p21Ki-rasB was farnesylated, suggesting that at least some of the peptides serve as substrates for the transferase. The data are consistent with a model in which a hydrophobic pocket in the Protein Farnesyltransferase recognizes tetrapeptides through interactions with the cysteine and the last two amino acids.
