Molecular dynamics analysis of a series of 22 potential farnesyltransferase substrates containing a CaaX-motif

Protein farnesyltransferase (FTase) is an important target in many research fields, more markedly so in cancer investigation since several proteins known to be involved in human cancer development are thought to serve as substrates for FTase and to require farnesylation for proper biological activity. Several FTase inhibitors (FTIs) have advanced into clinical testing. Nevertheless, despite the progress in the field several functional and mechanistic doubts on the FTase catalytic activity have persisted. This work provides some crucial information on this important enzyme by describing the application of molecular dynamics simulations using specifically designed molecular mechanical parameters for a variety of 22 CaaX peptides known to work as natural substrates or inhibitors for this enzyme. The study involves a comparative analysis of several important molecular aspects, at the mechanistic level, of the behavior of substrates and inhibitors at the dynamic level, including the behavior of the enzyme and peptides, as well as their interaction, together with the effect of the solvent. Properties evaluated include the radial distribution function of the water molecules around the catalytically important zinc metal atom and cysteine sulfur of CaaX, the conformations of the substrate and inhibitor and the corresponding RMSF values, critical hydrogen bonds, and several catalytically relevant distances. These results are discussed in light of recent experimental and computational evidence that provides new insights into the activity of this enzyme.

The basis for K-Ras4B binding specificity to protein farnesyltransferase revealed by 2 A resolution ternary complex structures

Background: The protein farnesyltransferase (FTase) catalyzes addition of the hydrophobic farnesyl isoprenoid to a cysteine residue fourth from the C terminus of several protein acceptors that are essential for cellular signal transduction such as Ras and Rho. This addition is necessary for the biological function of the modified proteins. The majority of Ras-related human cancers are associated with oncogenic variants of K-RasB, which is the highest affinity natural substrate of FTase. Inhibition of FTase causes regression of Ras-mediated tumors in animal models. Results: We present four ternary complexes of rat FTase co-crystallized with farnesyl diphosphate analogs and K-Ras4B peptide substrates. The Ca(1)a(2)X portion of the peptide substrate binds in an extended conformation in the hydrophobic cavity of FTase and coordinates the active site zinc ion. These complexes offer the first view of the polybasic region of the K-Ras4B peptide substrate, which confers the major enhancement of affinity of this substrate. The polybasic region forms a type I beta turn and binds along the rim of the hydrophobic cavity. Removal of the catalytically essential zinc ion results in a dramatically different peptide conformation in which the Ca(1)a(2)X motif adopts a beta turn. A manganese ion binds to the diphosphate mimic of the farnesyl diphosphate analog. Conclusions: These ternary complexes provide new insight into the molecular basis of peptide substrate specificity, and further define the roles of zinc and magnesium in the prenyltransferase reaction. Zinc is essential for productive Ca(1)a(2)X peptide binding, suggesting that the beta-turn conformation identified in previous nuclear magnetic resonance (NMR) studies reflects a state in which the cysteine is not coordinated to the zinc ion. The structural information presented here should facilitate structure-based design and optimization of inhibitors of Ca(1)a(2)X protein prenyltransferases.     

Protein farnesyl transferase target selectivity is dependent upon peptide stimulated product release

Protein farnesyl transferase (FTase) catalyzes transfer of a 15 carbon farnesyl lipid to cysteine in the C-terminal Ca1a2X sequence of numerous proteins including Ras. Previous studies have shown that product release is rate limiting and is dependent on binding of either a new peptide or isoprenoid diphosphate substrate. While considerable progress has been made in understanding how FTase distinguishes between related target proteins, the relative importance of the two pathways for product release on substrate selectivity is unclear. A detailed analysis of substrate stimulated product release has now been performed and provides new insights into the mechanism of FTase target selectivity. To clarify how FTase selects between different Ca1a2X sequences, we have examined the competition of various peptide substrates for modification with the isoprenoids farnesyl diphosphate (FPP) and anilinogeranyl diphosphate (AGPP). We find that reactivity of some competing peptides is correlated with apparent Kmpeptide, while the reactivity of others is predicted by the selectivity factor apparent kcat/Kmpeptide. The peptide target selectivity also depends on the structure of the isoprenoid donor. Additionally, we observe two peptide substrate concentration dependent maxima and substrate inhibition in the steady-state reaction which require a minimum of three peptide binding states for the steady-state FTase reaction mechanism. We propose a model for the FTase reaction mechanism that, in addition to FPP stimulated product release, incorporates peptide binding to the FTase-FPP complex and the formation of an FTase-product-peptide complex followed by product release leading to an inhibitory FTase-peptide complex as a natural consequence of catalysis to explain these results.     

Mechanistic studies of rat protein farnesyltransferase indicate an associative transition state

Protein farnesyltransferase is a zinc metalloenzyme that catalyzes the transfer of a 15-carbon farnesyl group to a conserved cysteine residue of a protein substrate. Both electrophilic and nucleophilic mechanisms have been proposed for this enzyme. In this work, we investigate the detailed catalytic mechanism of mammalian protein farnesyltransferase by measuring the effect of metal substitution and/or substrate alterations on the rate constant of the chemical step. Substitution of cadmium for the active site zinc enhances peptide affinity approximately 5-fold and decreases the rate constant for the formation of the thioether product approximately 6-fold, indicating changes in the metal-thiolate coordination in the catalytic transition state. In addition, the observed rate constant for product formation decreases for C3 fluoromethyl farnesyl pyrophosphate substrates, paralleling the number of fluorines at the C3 methyl position and indicating that a rate-contributing transition state has carbocation character. Magnesium ions do not affect the affinity of either the peptide or the isoprenoid substrate but specifically enhance the observed rate constant for product formation 700-fold, suggesting that magnesium coordinates and activates the diphosphate leaving group. These data suggest that FTase catalyzes protein farnesylation by an associative mechanism with an "exploded" transition state where the metal-bound peptide/protein sulfur has a partial negative charge, the C1 of FPP has a partial positive charge, and the bridge oxygen between C1 and the alpha phosphate of FPP has a partial negative charge. This proposed transition state suggests that stabilization of the developing charge on the carbocation and pyrophosphate oxygens is an important catalytic feature.     

Farnesylation of nonpeptidic thiol compounds by protein farnesyltransferase

Protein farnesyltransferase catalyzes the modification of protein substrates containing specific carboxyl-terminal Ca(1)a(2)X motifs with a 15-carbon farnesyl group. The thioether linkage is formed between the cysteine of the Ca(1)a(2)X 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(k(max)) and the thiol pK(a); model nucleophilic reactions of thiols display a Br?nsted correlation of approximately 0.4. These data demonstrate that zinc-sulfur coordination is a primary criterion for classification as a FTase substrate, but other interactions between the peptide and the FTase.isoprenoid complex provide significant enhancement of binding and catalysis. Finally, these results suggest that the mechanism of FTase provides in vivo selectivity for the farnesylation of protein substrates even in the presence of high concentrations of intracellular thiols.     

Genetic evidence for in vivo cross-specificity of the CaaX-box protein prenyltransferases farnesyltransferase and geranylgeranyltransferase-I in Saccharomyces cerevisiae.

Two protein prenyltransferase enzymes, farnesyltransferase (FTase) and geranylgeranyltransferase-I (GGTase-I), catalyze the covalent attachment of a farnesyl or geranylgeranyl lipid group to the cysteine of a CaaX sequence (cysteine [C], two aliphatic amino acids [aa], and any amino acid [X]. In vitro studies reported here confirm previous reports that CaaX proteins with a C-terminal serine are farnesylated by FTase and those with a C-terminal leucine are geranylgeranylated by GGTase-I. In addition, we found that FTase can farnesylate CaaX proteins with a C-terminal leucine and can transfer a geranylgeranyl group to some CaaX proteins. Genetic data indicate that FTase and GGTase-I have the same substrate preferences in vivo as in vitro and also show that each enzyme can prenylate some of the preferred substrates of the other enzyme in vivo. Specifically, the viability of yeast cells lacking FTase is due to prenylation of Ras proteins by GGTase-I. Although this GGTase-I dependent prenylation of Ras is sufficient for growth, it is not sufficient for mutationally activated Ras proteins to exert deleterious effects on growth. The dependence of the activated Ras phenotype on FTase can be bypassed by replacing the C-terminal serine with leucine. This altered form of Ras appears to be prenylated by both GGTase-I and FTase, since it produces an activated phenotype in a strain lacking either FTase or GGTase-I. Yeast cells can grow in the absence of GGTase-I as long as two essential substrates are overexpressed, but their growth is slow. Such strains are dependent on FTase for viability and are able to grow faster when FTase is overproduced, suggesting that FTase can prenylate the essential substrates of GGTase-I when they are overproduced.     

Computational studies of the farnesyltransferase ternary complex part I: substrate binding

Farnesyltransferase (FTase) catalyzes the transfer of farnesyl from farnesyl diphosphate (FPP) to cysteine residues at or near the C-terminus of protein acceptors with a CaaX motif (a, aliphatic; X, Met). Farnesylation is a critical modification to many switch proteins involved in the extracellular signal transduction pathway, which facilitates their fixation on the cell membrane where the extracellular signal is processed. The malfunction caused by mutations in these proteins often causes uncontrolled cell reproduction and leads to tumor formation. FTase inhibitors have been extensively studied as potential anticancer agents in recent years, several of which have advanced to different phases of clinical trials. However, the mechanism of this biologically important enzyme has not been firmly established. Understanding how FTase recruits the FPP substrate is the first and foremost step toward further mechanistic investigations and the design of effective FTase inhibitors. Molecular dynamic simulations were carried out on the ternary structure of FTase complexed with the FPP substrate and an acetyl-capped tetrapeptide (acetyl-CVIM), which revealed that the FPP substrate maintains an inactive conformation and the binding of the diphosphate group can be largely attributed to residues R291beta, K164alpha, K294beta, and H248beta. The FPP substrate assumes an extended conformation in the binding site with restricted rotation of the backbone dihedral angles; however, it does not have a well-defined conformation when unbound in solution. This is evident from multinanosecond MD simulations of the FPP substrate in a vacuum and solution. Our conclusion is further supported by theoretical J coupling calculations. Our results on the FPP binding are in good agreement with previous experimental kinetic studies on FTase mutants. The hypothetical conformational activation of the FPP substrate is currently under investigation.     

Upstream polybasic region in peptides enhances dual specificity for prenylation by both farnesyltransferase and geranylgeranyltransferase type I

Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase I) catalyze the attachment of a farnesyl or geranylgeranyl lipid, respectively, near the C-terminus of their protein substrates. FTase and GGTase I differ in both their substrate specificity and magnesium dependence, where the activity of FTase, but not GGTase I, is activated by magnesium. Many protein substrates of these enzymes contain an upstream polybasic region that is proposed to increase the affinity of the substrate and aid in plasma membrane association. Here, we demonstrate that the addition of an upstream polybasic region to a peptide substrate enhances the binding affinity of FTase approximately 4-fold for the peptide but diminishes the catalytic efficiency of the reaction, reflected by decreases in both the prenylation rate constant and kcat/KM. Specifically, the prenylation rate constant decreases 7-fold at 5 mM MgCl2 for the peptide KKKSKTKCVIM (C-terminal sequence of K-Ras4B) in comparison to TKCVIM. This decrease is accompanied by an alteration in the dependence on magnesium, as the K(Mg) increases from 2.2 +/- 0.1 mM for TKCVIM to 11.5 +/- 0.1 mM for KKKSKTKCVIM. The presence of an upstream polybasic region does not significantly affect GGTase I-catalyzed reactions, as only minimal changes are seen in Kd, kcat/KM, and k(chem) values. Thus, the presence of an upstream polybasic region enhances the dual prenylation of these substrates, by decreasing the catalytic efficiency of farnesylation catalyzed by FTase to a level comparable to that of geranylgeranylation catalyzed by GGTase I.     

Interplay of isoprenoid and peptide substrate specificity in protein farnesyltransferase

Protein farnesyltransferase (FTase) catalyzes the post-translational modification of many important cellular proteins, and is a potential anticancer drug target. Crystal structures of the FTase ternary complex illustrate an unusual feature of this enzyme, the fact that the isoprenoid substrate farnesyl diphosphate (FPP) forms part of the binding site for the peptide substrate. This implies that changing the structure of FPP could alter the specificity of the FPP-FTase complex for peptide substrates. We have found that this is the case; a newly synthesized FPP analogue, 3-MeBFPP, is a substrate with three peptide cosubstrates, but is not an effective substrate with a fourth peptide (dansyl-GCKVL). Addition of this analogue also inhibits farnesylation of dansyl-GCKVL by FPP. Surprisingly, the differential substrate abilities of these four peptides with FPP-FTase and 3-MeBFPP-FTase complexes do not correlate with their binding affinities for these isoprenoid-enzyme complexes. The possible mechanistic rationales for this observation, along with its potential utility for the study of protein prenylation, are discussed.     

Sequence dependence of protein isoprenylation

Several proteins have been shown to be post-translationally modified on a specific C-terminal cysteine residue by either of two isoprenoid biosynthetic pathway metabolites, farnesyl diphosphate or geranylgeranyl diphosphate. Three enzymes responsible for protein isoprenylation were resolved chromatographically from the cytosolic fraction of bovine brain: a farnesyl-protein transferase (FTase), which modified the cell-transforming Ras protein, and two geranyl-geranyl-protein transferases, one (GGTase-I) which modified a chimeric Ras having the C-terminal amino acid sequence of the gamma-6 subunit of heterotrimeric GTP-binding proteins, and the other (GGTase-II) which modified the Saccharomyces cerevisiae secretory GTPase protein YPT1. In a S. cerevisiae strain lacking FTase activity (ram1), both GGTases were detected at wild-type levels. In a ram2 S. cerevisiae strain devoid of FTase activity, GGTase-I activity was reduced by 67%, suggesting that GGTase-I and FTase activities derive from different enzymes but may share a common genetic feature. For the FTase and the GGTase-I activities, the C-terminal amino acid sequence of the protein substrate, the CAAX box, appeared to contain all the critical determinants for interaction with the transferase. In fact, tetrapeptides with amino acid sequences identical to the C-terminal sequences of the protein substrates for FTase or GGTase-I competed for protein isoprenylation by acting as alternative substrates. Changes in the CAAX amino acid sequence of protein substrates markedly altered their ability to serve as substrates for both FTase and GGTase-I. In addition, it appeared that FTase and GGTase-I had complementary affinities for CAAX protein substrates; that is, CAAX proteins that were good substrates for FTase were, in general, poor substrates for GGTase-I, and vice versa. In particular, a leucine residue at the C terminus influenced whether a CAAX protein was either farnesylated or geranylgeranylated preferentially. The YPT1 C terminus peptide, TGGGCC, did not compete or serve as a substrate for GGTase-II, indicating that the interaction between GGTase-II and YPT1 appeared to depend on more than the 6 C-terminal residues of the protein substrate sequence. These results identify three different isoprenyl-protein transferases that are each selective for their isoprenoid and protein substrates.     

Crystal structures of the fungal pathogen Aspergillus fumigatus protein farnesyltransferase complexed with substrates and inhibitors reveal features for antifungal drug design

Species of the fungal genus Aspergillus are significant human and agricultural pathogens that are often refractory to existing antifungal treatments. Protein farnesyltransferase (FTase), a critical enzyme in eukaryotes, is an attractive potential target for antifungal drug discovery. We report high‐resolution structures of A. fumigatus FTase (AfFTase) in complex with substrates and inhibitors. Comparison of structures with farnesyldiphosphate (FPP) bound in the absence or presence of peptide substrate, corresponding to successive steps in ordered substrate binding, revealed that the second substrate‐binding step is accompanied by motions of a loop in the catalytic site. Re‐examination of other FTase structures showed that this motion is conserved. The substrate‐ and product‐binding clefts in the AfFTase active site are wider than in human FTase (hFTase). Widening is a consequence of small shifts in the α‐helices that comprise the majority of the FTase structure, which in turn arise from sequence variation in the hydrophobic core of the protein. These structural effects are key features that distinguish fungal FTases from hFTase. Their variation results in differences in steady‐state enzyme kinetics and inhibitor interactions and presents opportunities for developing selective anti‐fungal drugs by exploiting size differences in the active sites. We illustrate the latter by comparing the interaction of ED5 and Tipifarnib with hFTase and AfFTase. In AfFTase, the wider groove enables ED5 to bind in the presence of FPP, whereas in hFTase it binds only in the absence of substrate. Tipifarnib binds similarly to both enzymes but makes less extensive contacts in AfFTase with consequently weaker binding.     

Kinetic studies of protein farnesyltransferase mutants establish active substrate conformation

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 K164alpha interacts with the alpha-phosphate and that H248beta and Y300beta form hydrogen bonds with the beta-phosphate of FPP [Strickland, C. L., et al. (1998) Biochemistry 37, 16601-16611]. Mutations K164Aalpha, H248Abeta, and Y300Fbeta 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 K164alpha, Y300beta, and H248beta do not function either as general acid-base catalysts or as magnesium ligands. Mutation of H248beta and Y300beta, but not K164alpha, decreases the farnesylation rate constant using farnesyl monophosphate (FMP). These data suggest that, contrary to the conclusions derived from analysis of the static crystal structures, the transition state for farnesylation is stabilized by interactions between the alpha-phosphate of the isoprenoid substrate and the side chains of Y300beta and H248beta. These results suggest an active substrate conformation for FTase wherein the C1 carbon of the FPP substrate moves toward the zinc-bound thiolate of the protein substrate to react, resulting in a rearrangement of the diphosphate group relative to its ground state position in the binding pocket.     

Steady-state kinetic mechanism of Ras farnesyl:protein transferase.

The steady-state kinetic mechanism of bovine brain farnesyl:protein transferase (FPTase) has been determined using a series of initial velocity studies, including both dead-end substrate and product inhibitor experiments. Reciprocal plots of the initial velocity data intersected on the 1/[s] axis, indicating that a ternary complex forms (sequential mechanism) and suggesting that the binding of one substrate does not affect the binding of the other. The order of substrate addition was probed by determining the patterns of dead-end substrate and product inhibition. Two nonhydrolyzable analogues of farnesyl diphosphate, (alpha-hydroxyfarnesyl)phosphonic acid (1) and [[(farnesylmethyl)hydroxyphosphinyl]methyl]phosphonic acid (2), were both shown to be competitive inhibitors of farnesyl diphosphate and noncompetitive inhibitors of Ras-CVLS. Four nonsubstrate tetrapeptides, CV[D-L]S, CVLS-NH2, N-acetyl-L-penicillamine-VIM, and CIFM, were all shown to be noncompetitive inhibitors of farnesyl diphosphate and competitive inhibitors of Ras-CVLS. These data are consistent with random order of substrate addition. Product inhibition patterns corroborated the results found with the dead-end substrate inhibitors. We conclude that bovine brain FPTase proceeds through a random order sequential mechanism. Determination of steady-state parameters for several physiological Ras-CaaX variants showed that amino acid changes affected the values of KM, but not those of kcat, suggesting that the catalytic efficiencies (kcat/KM) of Ras-CaaX substrates depend largely upon their relative binding affinity for FPTase.     

Novel limonene phosphonate and farnesyl diphosphate analogues: design, synthesis, and evaluation as potential protein-farnesyl transferase inhibitors

Limonene and its metabolite perillyl alcohol are naturally-occurring isoprenoids that block the growth of cancer cells both in vitro and in vivo. This cytostatic effect appears to be due, at least in part, to the fact that these compounds are weak yet selective and non-toxic inhibitors of protein prenylation. Protein-farnesyl transferase (FTase), the enzyme responsible for protein farnesylation, has become a key target for the rational design of cancer chemotherapeutic agents. Therefore, several alpha-hydroxyphosphonate derivatives of limonene were designed and synthesized as potentially more potent FTase inhibitors. A noteworthy feature of the synthesis was the use of trimethylsilyl triflate as a mild, neutral deprotection method for the preparation of sensitive phosphonates from the corresponding tert-butyl phosphonate esters. Evaluation of these compounds demonstrates that they are exceptionally poor FTase inhibitors in vitro (IC50 > or = 3 mM) and they have no effect on protein farnesylation in cells. In contrast, farnesyl phosphonyl(methyl)phosphinate, a diphosphate-modified derivative of the natural substrate farnesyl diphosphate, is a very potent FTase inhibitor in vitro (Ki=23 nM).     

Synthesis of imidazole-containing analogues of farnesyl pyrophosphate and evaluation of their biological activity on protein farnesyltransferase

With the aim of creating new bisubstrate inhibitors of protein farnesyltransferase (FTase), new carboxylic farnesyl pyrophosphate analogues have been designed and synthesized. The original structures are built around three elements: a prenyl moiety, a 1,4-diacid motif and an imidazole ring. All the compounds were evaluated for their ability to inhibit FTase and compared with the corresponding derivatives lacking the imidazole ring, synthesized for that purpose. These new compounds are not bisubstrate inhibitors probably because the imidazole ring is not in the right position to interact with the zinc atom. However these derivatives display FPP competitive inhibition with a good activity in the carboxylic farnesyl pyrophosphate analogues series.     

Effect of metal substitution in copper amine oxidase from lentil seedlings

 The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the k _c value is different from that of native or Cu-fully-reconstituted enzyme, while K _m is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism. Received: 5 March 1999 / Accepted: 22 July 1999     

A mutant form of human protein farnesyltransferase exhibits increased resistance to farnesyltransferase inhibitors.

Protein farnesyltransferase (FTase) is a key enzyme responsible for the lipid modification of a large and important number of proteins including Ras. Recent demonstrations that inhibitors of this enzyme block the growth of a variety of human tumors point to the importance of this enzyme in human tumor formation. In this paper, we report that a mutant form of human FTase, Y361L, exhibits increased resistance to farnesyltransferase inhibitors, particularly a tricyclic compound, SCH56582, which is a competitive inhibitor of FTase with respect to the CAAX (where C is cysteine, A is an aliphatic amino acid, and X is the C-terminal residue that is preferentially serine, cysteine, methionine, glutamine or alanine) substrates. The Y361L mutant maintains FTase activity toward substrates ending with CIIS. However, the mutant also exhibits an increased affinity for peptides terminating with CIIL, a motif that is recognized by geranylgeranyltransferase I (GGTase I). The Y361L mutant also demonstrates activity with Ha-Ras and Cdc42Hs proteins, substrates of FTase and GGTase I, respectively. In addition, the Y361L mutant shows a marked sensitivity to a zinc chelator HPH-5 suggesting that the mutant has altered zinc coordination. These results demonstrate that a single amino acid change at a residue at the active site can lead to the generation of a mutant resistant to FTase inhibitors. Such a mutant may be valuable for the study of the effects of FTase inhibitors on tumor cells.     

Analysis of farnesyl transferase activity during hormone-induced maturation of Xenopus laevis oocytes

Preincubation of Xenopus laevis oocytes with insulin or insulin-like growth factor 1 (IGF-1) resulted in inhibition of farnesyl transferase (FTase) activity measured both in vivo (after microinjection of tritiated farnesyl pyrophosphate and Ras-CVIM into oocytes) and in extracts using a filtration assay. FTase activity measured in oocyte extracts was inhibited 55% after a 20 min treatment of oocytes with 1 microM insulin or 10 nM IGF-1. The apparent IC(50) for inhibition of oocyte FTase by IGF-1 is 0.3 nM. The observed decrease in FTase activity was apparently not due to translocation of enzyme from cytosol to membrane, since activities measured both in soluble extracts and resuspended crude pellets displayed comparable levels of inhibition following hormone treatment. Using a hexapeptide (TKCVIM) as substrate, FTase activity was also inhibited 65% when oocytes were pretreated with 10 nM IGF-1. Two FTase inhibitors [(alpha-hydroxyfarnesyl) phosphonic acid (HFPA) and chaetomellic acid A (CA)] effectively inhibited Xenopus oocyte FTase by 80-90% when added to assay mixtures (IC(50) values of 338 +/- 96 nM HFPA and 232 +/- 80 nM CA) or after incubation of oocytes in drug before preparation of soluble extracts for assay (IC(50) values of 7 +/- 6 nM HFPA and 328 +/- 128 nM CA). The farnesyl transferase inhibitors were observed to slow the time course of oocyte maturation but did not block the IGF-1-induced maturation response. J. Exp. Zool. 286:193-203, 2000.Copyright 2000 Wiley-Liss, Inc.     

Identification of Novel Peptide Substrates for Protein Farnesyltransferase Reveals Two Substrate Classes with Distinct Sequence Selectivities

Prenylation is a posttranslational modification essential for the proper localization and function of many proteins. Farnesylation, the attachment of a 15-carbon farnesyl group near the C-terminus of protein substrates, is catalyzed by protein farnesyltransferase (FTase). Farnesylation has received significant interest as a target for pharmaceutical development, and farnesyltransferase inhibitors are in clinical trials as cancer therapeutics. However, as the total complement of prenylated proteins is unknown, the FTase substrates responsible for farnesyltransferase inhibitor efficacy are not yet understood. Identifying novel prenylated proteins within the human proteome constitutes an important step towards understanding prenylation-dependent cellular processes. Based on sequence preferences for FTase derived from analysis of known farnesylated proteins, we selected and screened a library of small peptides representing the C-termini of 213 human proteins for activity with FTase. We identified 77 novel FTase substrates that exhibit multiple-turnover (MTO) reactivity within this library; our library also contained 85 peptides that can be farnesylated by FTase only under single-turnover (STO) conditions. Based on these results, a second library was designed that yielded an additional 29 novel MTO FTase substrates and 45 STO substrates. The two classes of substrates exhibit different specificity requirements. Efficient MTO reactivity correlates with the presence of a nonpolar amino acid at the a₂ position and a Phe, Met, or Gln at the terminal X residue, consistent with the proposed Ca₁a₂X sequence model. In contrast, the sequences of the STO substrates vary significantly more at both the a₂ and the X residues and are not well described by current farnesylation algorithms. These results improve the definition of prenyltransferase substrate specificity, test the efficacy of substrate algorithms, and provide valuable information about therapeutic targets. Finally, these data illuminate the potential for in vivo regulation of prenylation through modulation of STO versus MTO peptide reactivity with FTase.     

19F nuclear magnetic resonance observations of aldehyde dismutation catalyzed by horse liver alcohol dehydrogenase

We have examined aspects of the second catalytic activity of alcohol dehydrogenase from horse liver (LADH), which involves an apparent dismutation of an aldehyde substrate into alcohol and acid in the presence of LADH and NAD. Using the substrate p-trifluoromethylbenzaldehyde, we have observed various bound complexes by ¹⁹F NMR in an effort to further characterize the mechanism of the reaction. The mechanism appears to involve the catalytic activity of LADH · NAD · aldehyde complex which reacts to form an enzyme · NADH · acid complex. The affinity of the acid product for LADH · NADH is weak and the acid product readily desorbs from the ternary complex. The resulting LADH · NADH can then react with a second molecule of aldehyde to form NAD and the corresponding alcohol. The result is the conversion of two molecules of aldehyde to one each of acid and alcohol, with LADH and NAD acting catalytically. This sequence of reactions can also explain the slow formation of acid product observed when alcohol and NAD are incubated with the enzyme.