Mammalian Protein Geranylgeranyltransferase-I: Substrate Specificity, Kinetic Mechanism, Metal Requirements, and Affinity Labeling

Yokoyama, Kohei; McGeady, Paul; Gelb, Michael H.

doi:10.1021/bi00004a029

Cited by 121 publications

(143 citation statements)

References 31 publications

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“…Using a gel-filtration assay we showed that RabGGTase forms a stable complex with Rab7±REP-1 in the absence of the lipid substrate GGpp. This is consistent with the findings that GGTase I and farnesyl transferase can form a stable complex with both protein and lipid substrate independently [9,11]. Formation of the stable complex was dependent on the presence of both Rab7 and REP-1, supporting the notion that Rab7 could be recognized by RabGGTase only in its complex with REP-1.…”

Section: Discussionsupporting

confidence: 91%

“…The choice between farnesylation and geranylgeranylation is determined by the hydrophobicity of the X residue [7]. 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].…”

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confidence: 99%

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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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Section: Discussionsupporting

confidence: 91%

mentioning

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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“…Previous kinetic data suggest that the kinetic scheme for GGTase I is similar to that of FTase (34,35), including that the substrate binding is functionally ordered with GGPP binding before peptide and that the rate constant for geranylgeranylation is faster than product dissociation (Scheme 1). The kinetic and thermodynamic data that we have determined for GGTase I are consistent with this basic kinetic mechanism.…”

Section: Resultsmentioning

confidence: 98%

“…Interestingly, the Mg(II) dependence of GGTase I is not well understood. Initial studies indicated that, similar to FTase, GGTase I required Mg(II) for maximal activity (33,34). However, later studies demonstrated that metal-chelating reagents have little effect on the steadystate turnover rate of GGTase I, suggesting that Mg(II) is not required for maximal activity (35).…”

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confidence: 99%

Lysine β311 of Protein Geranylgeranyltransferase Type I Partially Replaces Magnesium

Hartman

Bowers

Fierke

2004

Journal of Biological Chemistry

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Protein geranylgeranyltransferase type I (GGTase I) catalyzes the attachment of a geranylgeranyl lipid group near the carboxyl terminus of protein substrates. Unlike protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type II, which require both Zn(II) and Mg(II) for maximal turnover, GGTase I turnover is dependent only on Zn(II). In FTase, the magnesium ion is coordinated by aspartate ␤352 and the diphosphate of farnesyl diphosphate to stabilize the developing charge in the transition state (Pickett, J. S., Bowers, K. E., and Fierke, C. A. Prenylation is a type of post-translational modification where a lipid group from either farnesyl diphosphate (FPP) 1 or geranylgeranyl diphosphate (GGPP) is covalently attached via a thioether linkage to a conserved cysteine residue near the carboxyl terminus of a protein (1-6; for review, see Refs. 7 and 8). The attached lipid mediates membrane association and specific protein-protein interactions, which are obligatory for proper functioning of the modified protein (9, 10). Protein farnesyltransferase (FTase), protein geranylgeranyltransferase type I (GGTase I), and protein geranylgeranyltransferase type II (GGTase II) comprise the class of zinc-catalyzed sulfur alkylation enzymes known as the protein prenyltransferases.All three protein prenyltransferases are ␣/␤ heterodimers; FTase and GGTase I share common ␣-subunits and possess ␤-subunits with 25% sequence identity (8,11,12). FTase and GGTase I prenylate proteins or peptides containing carboxylterminal CaaX (C refers to a conserved cysteine residue, a refers to an aliphatic amino acid, and X generally refers to leucine or phenylalanine for GGTase I, and methionine, serine, alanine, or glutamine for FTase) sequences (13-18). The proteins Ras, RhoB, CENP-E, and CENP-F are known substrates of FTase; GGTase I modifies the ␥-subunits of most heterotrimeric G proteins as well as many Ras-related G proteins, including members of the Rac, Rap, and Rho families (9, 19 -23). In a fashion distinct from FTase and GGTase I, GGTase II catalyzes the attachment of two geranylgeranyl groups to members of the Rab family of GTPases that contain carboxylterminal sequences of XXCC, XCXC, XCCX, and CCXX (C refers to a conserved cysteine residue, and X refers to any amino acid) (24).Recently, much of the work on prenyltransferases has focused on FTase and the protein substrate, Ras, a small GTPase in the receptor tyrosine kinase signaling pathway that is a key regulator of cell division (25). 30% of all human cancers can be linked to mutations in the ras gene, which lead to constitutively activated Ras signaling (25). The post-translational attachment of a farnesyl group by FTase is required for the transforming activity of Ras oncoproteins (25). These observations prompted a search for FTase inhibitors as novel anticancer agents (25). Subsequent research demonstrated that the form of Ras most often mutated in human cancers is K-Ras4B, a protein substrate that can be farnesylated by FTase as well as geranylgeranylated by...

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“…Protein prenyltransferases preferentially operate via an ordered mechanism for binding two substrates with the prenyl pyrophosphate substrate binding first [2,5,6]. Mammalian PGGT-I and PFT form a tight complex selectively with their substrates, geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP), respectively [7,8].…”

Section: Introductionmentioning

confidence: 99%

Protein geranylgeranyltransferase-I of Trypanosoma cruzi

Yokoyama

Gillespie

Voorhis

et al. 2008

Molecular and Biochemical Parasitology

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Protein geranylgeranyltransferase type I (PGGT-I) and protein farnesyltransferase (PFT) occur in many eukaryotic cells. Both consist of two subunits, the common αsubunit and a distinct β subunit. In the gene database of protozoa Trypanosoma cruzi, the causative agent of Chagas' disease, a putative protein that consists of 401 amino acids with ∼20% amino acid sequence identity to the PGGT-I β of other species was identified, cloned, and characterized. Multiple sequence alignments show that the T. cruzi ortholog contains all three of the zinc-binding residues and several residues uniquely conserved in the β subunit of PGGT-I. Co-expression of this protein and the α subunit of T. cruzi PFT in Sf9 insect cells yielded a dimeric protein that forms a tight complex selectively with [ 3 H] geranylgeranyl pyrophosphate, indicating a key characteristic of a functional PGGT-I. Recombinant T. cruzi PGGT-I ortholog showed geranylgeranyltransferase activity with distinct specificity toward the C-terminal CaaX motif of protein substrates compared to that of the mammalian PGGT-I and T. cruzi PFT. Most of the CaaX-containing proteins with X=Leu are good substrates of T. cruzi PGGT-I, and those with X=Met are substrates for both T. cruzi PFT and PGGT-I, whereas unlike mammalian PGGT-I, those with X=Phe are poor substrates for T. cruzi PGGT-I. Several candidates for T. cruzi PGGT-I or PFT substrates containing the C-terminal CaaX motif are found in the T. cruzi gene database. Among five C-terminal peptides of those tested, a peptide of a Ras-like protein ending with CVLL was selectively geranylgeranylated by T. cruzi PGGT-I. Other peptides with CTQQ (Tcj2 DNAJ protein), CAVM (TcPRL-1 protein tyrosine phosphatase), CHFM (a small GTPase like protein), and CQLF (TcRho1 GTPase) were specific substrates for T. cruzi PFT but not for PGGT-I. The mRNA and protein of the T. cruzi PGGT-I β ortholog were detected in three life-cycle stages of T. cruzi. Cytosol fractions from trypomastigotes (infectious mammalian stage) and epimastigotes (insect stage) were shown to contain levels of PGGT-I activity that are ∼100-fold lower than PFT activity. The CaaX mimetics known as PGGT-I inhibitors show very low potency against T. cruzi PGGT-I compared to the mammalian enzyme, suggesting the potential to develop selective inhibitors against the parasite enzyme.

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Mammalian Protein Geranylgeranyltransferase-I: Substrate Specificity, Kinetic Mechanism, Metal Requirements, and Affinity Labeling

Cited by 121 publications

References 31 publications

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

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

Lysine β311 of Protein Geranylgeranyltransferase Type I Partially Replaces Magnesium

Protein geranylgeranyltransferase-I of Trypanosoma cruzi

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