Mutagenesis Studies of Protein Farnesyltransferase Implicate Aspartate β352 as a Magnesium Ligand

Pickett, Jennifer S.; Bowers, Katherine; Fierke, Carol A.

doi:10.1074/jbc.m309226200

Cited by 45 publications

(74 citation statements)

References 50 publications

(70 reference statements)

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“…The affinities of K␤311A and K␤311D GGTase I for dansylated GCVLL were decreased ϳ2-fold (30 Ϯ 3 nM and 20 Ϯ 2 nM, respectively, Table I) compared with wild type GGTase I. These alterations in the binding affinity are modest and are likely explained by changes in the van der Waals volume of the altered side chain (32,47).…”

Section: Resultsmentioning

confidence: 95%

“…Mutagenesis of Lys-␤311-Previous mutagenesis studies of rat FTase suggest that the side chain of Asp-␤352 coordinates a catalytic Mg(II) ion (32). An aspartate is conserved in the homologous position in GGTase II; however, sequence alignments and crystallographic analysis of GGTase I indicate that this aspartate is replaced by lysine (Lys-␤311) in GGTase I (12,32,36).…”

Section: Resultsmentioning

confidence: 99%

“…An aspartate is conserved in the homologous position in GGTase II; however, sequence alignments and crystallographic analysis of GGTase I indicate that this aspartate is replaced by lysine (Lys-␤311) in GGTase I (12,32,36). Therefore, the positive charge of the side chain of Lys-␤311 has been proposed to replace the catalytic function of Mg(II) in GGTase I (12,36).…”

Section: Resultsmentioning

confidence: 99%

“…apparent magnesium affinity of these mutants is only slightly weaker than FTase with the comparable side chain at this position, K Mg ϭ 4.0 Ϯ 0.3 mM and K Mg ϭ 110 Ϯ 30 mM for wild type and D␤352A FTase, respectively (31,32). For K␤311D GGTase I, like wild type FTase (31,32), the apparent magnesium affinity, K Mg , and the chemical rate constant, k max , vary little as the pH varies from 7.8 to 8.3 (data not shown). However, the value of K Mg increases as pH decreases, as is also observed for wild type FTase (data not shown; 31, 32).…”

Section: Resultsmentioning

confidence: 99%

“…In FTase, Mg(II) accelerates the chemical step 700-fold by coordinating the diphosphate group of the isoprenoid substrate, thereby stabilizing the negative charge buildup on the diphosphate leaving group (29 -31). Mutagenesis studies provide evidence that Mg(II) is also coordinated by an aspartate residue in FTase (32). Interestingly, the Mg(II) dependence of GGTase I is not well understood.…”

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confidence: 99%

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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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Section: Resultsmentioning

confidence: 95%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Lysine β311 of Protein Geranylgeranyltransferase Type I Partially Replaces Magnesium

Hartman

Bowers

Fierke

2004

Journal of Biological Chemistry

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The Search for the Mechanism of the Reaction Catalyzed by Farnesyltransferase

Sousa¹,

Fernandes²,

Ramos³

2009

Chemistry A European J

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Atomic-level portrait: The mechanism of the reaction catalyzed by the puzzling enzyme farnesyltransferase is elucidated by using computational methods, allowing the obtainment of the first real detailed atomistic quantum-chemical transition-state structure (see figure) for the reaction catalyzed by this enzyme. The results obtained provide an atomic-level framework for the design of more potent and specific inhibitors for this important enzyme.

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

et al. 2014

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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 a-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.

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Mutagenesis Studies of Protein Farnesyltransferase Implicate Aspartate β352 as a Magnesium Ligand

Cited by 45 publications

References 50 publications

Lysine β311 of Protein Geranylgeranyltransferase Type I Partially Replaces Magnesium

Lysine β311 of Protein Geranylgeranyltransferase Type I Partially Replaces Magnesium

The Search for the Mechanism of the Reaction Catalyzed by Farnesyltransferase

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

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