A Fluorescence Assay for Geranylgeranyl Transferase Type I

Pickett, Walter C.; Zhang, F.L.; Silverstrim, Christine; Schow, Steven R.; Wick, Michael M.; Kerwar, S.S.

doi:10.1006/abio.1995.1108

Cited by 27 publications

(19 citation statements)

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“…Effect of Mutagenesis on Steady-state Turnover-The steadystate parameter, k cat , was measured using a continuous fluorescence assay with a dansylated peptide substrate (43,44,48). …”

Section: Resultsmentioning

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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“…Effect of Mutagenesis on Steady-state Turnover-The steadystate parameter, k cat , was measured using a continuous fluorescence assay with a dansylated peptide substrate (43,44,48). …”

Section: Resultsmentioning

confidence: 99%

Lysine β311 of Protein Geranylgeranyltransferase Type I Partially Replaces Magnesium

Hartman

Bowers

Fierke

2004

Journal of Biological Chemistry

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“…The reaction was reported to follow a random Bi Bi pathway with steady-state kinetics, although GGPP binding is the kinetically preferred first step. The order of addition of substrates is reflected in their relative K m values of low nanomolar for GGPP and micromolar for peptide substrate, although the absolute numbers vary dramatically with the particular CAAX sequence and length of the peptide substrate (7,13,15,16). The reaction mechanism thus appears identical to that of FPTase (17).…”

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confidence: 96%

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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“…Several methods to use labeled or unlabeled CAAX peptides to assay FPTase and GGPTase I activities have been developed [7,[12][13][14][15]. To date, no method has been reported to use a labeled peptide for the quantitative determination of FPP or GGPP in biological samples.…”

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confidence: 99%