Evidence for a Catalytic Role of Zinc in Protein Farnesyltransferase

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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...

Section: Discussionmentioning

confidence: 99%

Lysine β311 of Protein Geranylgeranyltransferase Type I Partially Replaces Magnesium

Hartman

Bowers

Fierke

2004

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“…Because the zinc ion participates in FTase catalysis (5)(6)(7)(8)(9), such an approach may be effective. In this regard, it is intriguing that HPH-5 may form a complex with the enzyme rather than simply chelating a zinc ion away from the enzyme.…”

Section: Discussionmentioning

confidence: 99%

A Mutant Form of Human Protein Farnesyltransferase Exhibits Increased Resistance to Farnesyltransferase Inhibitors

Villar¹,

Urano²,

Guo³

et al. 1999

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.Protein farnesyltransferase (FTase) 1 catalyzes the transfer of a farnesyl group onto a conserved cysteine residue four amino acids from the C terminus of a number of proteins, such as Ras, that are involved in cell growth and morphogenesis (1-4). This modification is critical for membrane association and subsequent protein-protein interactions of these proteins. FTase is a heterodimeric enzyme consisting of ␣-and ␤-subunits. The ␣-subunit of FTase is shared with the related prenyltransferase, protein geranylgeranyltransferase type I (GGTase I), whereas the ␤-subunits of FTase and GGTase I are approximately 30% homologous. The FTase enzyme recognizes the CAAX motif (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) that is found at the C termini of the substrate proteins. GGTase I enzyme also recognizes a CAAX motif; however, the terminal X amino acid is predominantly leucine or phenylalanine. This motif is referred to as the CAAL motif.The three-dimensional structures of the rat FTase enzyme have recently been resolved without substrate bound (5, 6), with bound substrate farnesyl pyrophosphate (FPP) (7), and with bound FPP and CAAX peptide analogs (8). In the structure without bound substrate, a non-cognate peptide provided by an adjacent ␤-subunit was modeled into the active site of the enzyme. The structure showed that the ␣-and ␤-subunits are largely composed of ␣-helices. The ␤-subunit forms a barrel-like structure, and one side of this barrel i...

“…The overall k cat under steady-state conditions is a relatively sluggish 1-3 min Ϫ1 for the mammalian enzymes (8,23), with product dissociation being rate determining in catalysis (22). The rate of the chemical step has been directly determined to be 17 s Ϫ1 through spectroscopic studies using enzyme containing a Co 2ϩ -for-Zn 2ϩ substitution (19). This spectroscopic study also revealed that the sulfur atom of the product thioether remains coordinated to the metal atom, an observation that may in part explain the slow release of product in steady-state turnover.…”

mentioning

confidence: 99%

“…In addition to its bound Zn 2ϩ , FTase also requires Mg 2ϩ for activity. The Zn 2ϩ is involved coordinating the thiol of the peptide substrate in the ternary complex of enzyme-isoprenoid-peptide substrate (19) and thus is presumed to play a direct role in catalysis. The role of Mg 2ϩ…”

mentioning

confidence: 99%

Substrate Binding Is Required for Release of Product from Mammalian Protein Farnesyltransferase

Tschantz

Furfine

Casey

1997

Self Cite

109

Protein farnesyltransferase (FTase) catalyzes the modification by a farnesyl lipid of Ras and several other key proteins involved in cellular regulation. Previous studies on this important enzyme have indicated that product dissociation is the rate-limiting step in catalysis. A detailed examination of this has now been performed, and the results provide surprising insights into the mechanism of the enzyme. Examination of the binding of a farnesylated peptide product to free enzyme revealed a binding affinity of ϳ1 M. However, analysis of the product release step under single turnover conditions led to the surprising observation that the peptide product did not dissociate from the enzyme unless additional substrate was provided. Once additional substrate was provided, the enzyme released the farnesylated peptide product with rates comparable with that of overall catalysis by FTase. Additionally, stable FTasefarnesylated product complexes were formed using Ras proteins as substrates, and these complexes also require additional substrate for product release. These data have major implications in both our understanding of overall mechanism of this enzyme and in design of inhibitors against this therapeutic target. Protein farnesyltransferase (FTase)1 catalyzes the S-farnesylation of a number of key cellular regulatory proteins. Farnesylation is directed by a C-terminal CAAX motif, where C is cysteine, A is usually an aliphatic residue, and X is typically methionine, serine, glutamine, or alanine (1, 2). The farnesyl lipid is attached to the substrate protein via a thioether linkage to the cysteine residue using farnesyl diphosphate (FPP) as the prenyl donor. Among the substrates for FTase are the Ras family of proto-oncogenes, several ␥ subunits of heterotrimeric G proteins, and nuclear lamins (1, 3). Farnesylation of these proteins is required for their proper membrane localization and activity. In the case of oncogenic forms of Ras proteins, the finding that farnesylation is required for expression of their transforming activity has led to FTase becoming an important target for anticancer drug design (4). Both in cell culture (5, 6) and in animal models (7), specific inhibitors of FTase have been shown to reverse the oncogenic phenotype induced by mutationally activated Ras.FTase has been purified to homogeneity from both rat and bovine brain by affinity purification on immobilized CAAX peptide substrates (8, 9). The enzyme is a Zn 2ϩ metalloenzyme that consists of ␣ and ␤ subunits that migrate on SDS-PAGE with apparent molecular masses of 48 and 46 kDa, respectively (8). Both subunits of the enzyme have been cloned (10 -12), and their co-expression in either Sf9 (13) or E. coli (14) results in production of quantities of the enzyme required for detailed biochemical and structural analyses. Cross-linking experiments have provided strong evidence that the ␤ subunit is involved in recognition of both the isoprenoid and protein substrates (15-17), although there is also evidence that the ␣ subunit may participate (15...