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

Dolence, Julia M.; Cassidy, Pamela B.; Mathis, Jeffery R.; Poulter, C. Dale

doi:10.1021/bi00051a017

Cited by 62 publications

(63 citation statements)

References 30 publications

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“…The reaction mechanism thus appears identical to that of FPTase (17). On the other hand, the yeast enzymes yGGPTase I and yFPTase have been reported to proceed through an ordered Bi Bi mechanism (18,19). Interestingly, and in contrast to FPTase, GGPTase I does not require Mg 2ϩ for GGPP binding or catalysis.…”

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confidence: 76%

Anions Modulate the Potency of Geranylgeranyl-Protein Transferase I Inhibitors

Huber

Robinson

Watkins

et al. 2001

Journal of Biological Chemistry

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We have identified and characterized potent and specific inhibitors of geranylgeranyl-protein transferase type I (GGPTase I), as well as dual inhibitors of GGPTase I and farnesyl-protein transferase. Many of these inhibitors require the presence of phosphate anions for maximum activity against GGPTase I in vitro. Inhibitors with a strong anion dependence were competitive with geranylgeranyl pyrophosphate (GGPP), rather than with the peptide substrate, which had served as the original template for inhibitor design. One of the most effective anions was ATP, which at low millimolar concentrations increased the potency of GGPTase I inhibitors up to several hundred-fold. In the case of clinical candidate L-778,123, this increase in potency was shown to result from two major interactions: competitive binding of inhibitor and GGPP, and competitive binding of ATP and GGPP. At 5 mM, ATP caused an increase in the apparent K d for the GGPP-GGPTase I interaction from 20 pM to 4 nM, resulting in correspondingly tighter inhibitor binding. A subset of very potent GGPP-competitive inhibitors displayed slow tight binding to GGPTase I with apparent on and off rates on the order of 10 6 M ؊1 s ؊1 and 10 ؊3 s ؊1 , respectively. Slow binding and the anion requirement suggest that these inhibitors may act as transition state analogs. After accounting for anion requirement, slow binding, and mechanism of competition, the structure-activity relationship determined in vitro correlated well with the inhibition of processing of GGPTase I substrate Rap1a in vivo.Inhibition of prenylation of Ras oncoproteins has long been considered an attractive approach to anti-cancer therapy of Ras-dependent tumors. Four isoforms of Ras are found in mammalian cells, Kirsten-Ras (Ki-Ras) 4A and 4B, Harvey-Ras (Ha-Ras), and N-Ras. Ki4B-Ras is the most frequently mutated isozyme in human cancer, ranging from 20% up to 90% in pancreatic cancers, whereas Ha-Ras mutations are found in a small percentage of bladder cancers. Most oncogenic mutations in Ras result in the elimination of the GTPase activity, locking Ras in its active, GTP-bound conformation. All Ras proteins undergo multiple post-translational modifications on their carboxyl termini that are required for membrane localization and biological activity. The COOH-terminal CAAX motif (where C is cysteine, A stands for aliphatic, and X for any amino acid) is recognized by farnesyl-protein transferase (FPTase), 1 which transfers a farnesyl group from farnesyl pyrophosphate (FPP) to the cysteine thiol. This prenylation step is followed by proteolytic clipping of the three terminal amino acids by a farnesyl-CAAX specific protease and subsequent methylation of the cysteine residue by a carboxymethyl transferase. Ha-Ras and N-Ras are further modified by palmitoylation which provides additional membrane interactions. A polylysine stretch in KiRas near the COOH terminus serves the same purpose. However, the prenylation step has been shown to be the single most critical modification for function of all Ras prote...

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confidence: 76%

Anions Modulate the Potency of Geranylgeranyl-Protein Transferase I Inhibitors

Huber

Robinson

Watkins

et al. 2001

Journal of Biological Chemistry

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“…Original studies suggested that the prenylation reaction catalyzed by farnesyl transferase or GGTase I proceeds via a random sequential mechanism by which the enzyme can bind the protein and lipid substrates independently [8,9]. However, recent studies with human and yeast farnesyl transferase supported an ordered addition of substrates [10,11].In contrast with other known prenyl transferases, RabGGTase does not recognize its substrate directly but exerts its function in concert with another protein termed REP (Rab escort protein) [12,13]. Two known mammalian proteins, Eur.…”

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confidence: 99%

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confidence: 99%

Characterization of the ternary complex between Rab7, REP‐1 and Rab geranylgeranyl transferase

Alexandrov

Simón²,

Yurchenko³

et al. 1999

European Journal of Biochemistry

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Geranylgeranylation is a post-translational modification of Rab GTPases that enables them to associate reversibly with intracellular membranes. Geranylgeranylation of Rab proteins is critical for their activity in controlling intracellular membrane transport. According to the currently accepted model for their action, newly synthesized Rab proteins are recruited by Rab escort protein (REP) and are presented to the Rab geranylgeranyl transferase (RabGGTase) which covalentely modifies the Rab protein with two geranylgeranyl moieties. After prenylation, the Rab protein remains in complex with REP and is delivered to the target membrane by the latter. In this work, we show that RabGGTase can form a stable complex with Rab7±REP in the absence of its lipid substrate geranylgeranyl pyrophosphate. In order to characterize this interaction, we developed three fluorescence assays reporting on the interaction of RabGGTase with the Rab7±REP complex. For this interaction we determined a K d value of about 120 nm.Association of RabGGTase with the Rab7±REP complex occurs with a rate constant of < 10 8 m 21´s21 . We demonstrate that the state of the nucleotide bound to Rab7 does not influence the affinity of RabGGTase for the Rab7±REP-1 complex. Finally, we address the issue of substrate specificity of RabGGTase. Titration experiments demonstrate that, in contrast with farnesyl transferase, RabGGTase does not recognize a defined C-terminal sequence motif. Experiments using Rab7 mutants in which the last 16 amino acids were either mutated or truncated revealed that the distal part of the C-terminus makes only a limited contribution to the binding affinity between RabGGTase and the Rab7±REP-1 complex. This demonstrates the functional dissimilarity between RabGGTase and geranylgeranyl transferase I and farnesyl transferase, which interact specifically with the C-terminus of their substrates. Based on these experiments, we propose that RabGGTase recognizes the overall structure arising from the association of Rab and REP and then`scans' the flexible C-terminus to position the proximal cysteines into the active site.Keywords: geranylgeranylation; prenyltransferase; Rab7; Rab geranylgeranyl transferase; REP.Rab proteins are members of the Ras superfamily of small GTP-binding proteins. Many of them were shown to be implicated in control of intracellular vesicular traffic [1,2]. For their function Rab proteins require modification by geranylgeranyl isoprenoids. This allows them to associate reversibly with a membrane in a tightly controlled fashion [3,4]. Covalent attachment of geranylgeranyl groups to two C-terminal cysteines is catalyzed by Rab geranylgeranyl transferase (RabGGTase).Mammalian RabGGTase (GGTase II) is a heterodimer composed of tightly associated a and b subunits of 60 and 38 kDa, respectively [5,6], and belongs to the family of protein prenyl transferases together with farnesyl transferase and geranylgeranyl transferase I (GGTase I) [7]. Substrates of farnesyl transferase include members of the Ras family, lamin b and...

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“…Steady-state kinetic analyses of PFT and PGGT-I show that both enzymes can operate by a random sequential mechanism in which either prenyl donor or acceptor binds first to the enzyme followed by binding of the other substrate to form a ternary complex that goes on to products. However, operationally, both enzymes show a strong tendency to bind prenyl pyrophosphate first (25)(26)(27)(28). Both PFT and PGGT-I have been shown to tightly bind both FPP and GGPP (12,29,30).…”

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confidence: 99%

Differential Prenyl Pyrophosphate Binding to Mammalian Protein Geranylgeranyltransferase-I and Protein Farnesyltransferase and Its Consequence on the Specificity of Protein Prenylation

Yokoyama

Zimmerman²,

Scholten³

et al. 1997

Journal of Biological Chemistry

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Protein geranylgeranyltransferase-I (PGGT-I) and protein farnesyltransferase (PFT) attach geranylgeranyl and farnesyl groups, respectively, to the C termini of eukaryotic cell proteins. In vitro, PGGT-I and PFT can transfer both geranylgeranyl and farnesyl groups from geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP) to their protein or peptide prenyl acceptor substrates. In the present study it is shown that PGGT-I binds GGPP 330-fold tighter than FPP and that PFT binds FPP 15-fold tighter than GGPP. Therefore, in vivo, where both GGPP and FPP compete for the binding to prenyltransferases, PGGT-I and PFT will likely be bound predominantly to GGPP and FPP, respectively. Previous studies have shown that K-Ras4B and the Ras-related GTPase TC21 are substrates for both PGGT-I and PFT in vitro. It is shown that TC21 can compete with the C-terminal peptide of the ␥ subunit of heterotrimeric G proteins and with the C-terminal peptide of lamin B for geranylgeranylation by PGGT-I and for farnesylation by PFT, respectively. K-Ras4B competes in both cases but is almost exclusively farnesylated by PFT in the presence of the lamin B peptide competitor. Rapid and single turnover kinetic studies indicate that the rate constant for the PGGT-I-catalyzed geranylgeranyl transfer step of the reaction cycle is 14-fold larger than the steady-state turnover number, which indicates that the rate of the overall reaction is limited by a step subsequent to prenyl transfer such as release of products from the enzyme. PGGT-I-catalyzed farnesylation is 37-fold slower than geranylgeranylation and is limited by the farnesyl transfer step. These results together with earlier studies provide a paradigm for the substrate specificity of PGGT-I and PFT and provide information that is critical for the design of prenyltransferase inhibitors as anti-cancer agents.Modification of the C termini of specific eukaryotic proteins by attachment of either 15-carbon farnesyl or 20-carbon geranylgeranyl groups is required for their proper membrane targeting and functional activation (1-9). Two closely related enzymes protein farnesyltransferase (PFT) 1 and PGGT-I transfer prenyl groups from prenyl pyrophosphates to proteins that contain a C-terminal CaaX motif (C is cysteine, a is usually an aliphatic amino acid, and X is a variety of amino acids) (10 -13). The X residue of this motif plays a major role in recognition by these two enzymes (14). PFT preferentially transfers a farnesyl group to the cysteine residue of the CaaX motif when X is serine, methionine, glutamine, or cysteine, and possibly other residues. PGGT-I preferentially geranylgeranylates proteins having a C-terminal leucine or phenylalanine. PFT and PGGT-I consist of a common ␣ subunit and distinct ␤ subunits (15-18). A third enzyme, protein geranylgeranyltransferase-II, also known as Rab geranylgeranyltransferase, transfers the 20-carbon prenyl group to both cysteines of Rab proteins that have C-terminal sequences CXC, CC, or possibly CCXX (19,20). Although the C-terminal CaaX...

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

Cited by 62 publications

References 30 publications

Anions Modulate the Potency of Geranylgeranyl-Protein Transferase I Inhibitors

Anions Modulate the Potency of Geranylgeranyl-Protein Transferase I Inhibitors

Characterization of the ternary complex between Rab7, REP‐1 and Rab geranylgeranyl transferase

Differential Prenyl Pyrophosphate Binding to Mammalian Protein Geranylgeranyltransferase-I and Protein Farnesyltransferase and Its Consequence on the Specificity of Protein Prenylation

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