Photoreactive Analogues of Prenyl Diphosphates as Inhibitors and Probes of Human Protein Farnesyltransferase and Geranylgeranyltransferase Type I

Protein farnesyltransferase (FTase) is a zinc metalloenzyme that performs a post-translational modification on many proteins that is critical for their function. The importance of cysteine residues in FTase activity was investigated using cysteine-specific reagents. Zincdepleted FTase (apo-FTase), but not the holoenzyme, was completely inactivated by treatment with N-ethylmaleimide (NEM). Similar effects were detected after treatment of the enzyme with iodoacetamide. The addition of zinc to apo-FTase protects it from inactivation by NEM. These findings indicated the presence of specific cysteine residue(s), potentially located at the zinc binding site, that are required for FTase activity. We performed a selective labeling strategy whereby the cysteine residues exposed upon removal of zinc from the enzyme were modified with [ 3 H]NEM. The enzyme so modified was digested with trypsin, and four labeled peptides were identified and sequenced, one peptide being the major site of labeling and the remaining three labeled to lesser extents. The major labeled peptide contained a radiolabeled cysteine residue, Cys 299 , that is in the ␤ subunit of FTase and is conserved in all known protein prenyltransferases. This cysteine residue was changed to both alanine and serine by site-directed mutagenesis, and the mutant proteins were produced in Escherichia coli and purified. While both mutant proteins retained the ability to bind farnesyl diphosphate, they were found to have lost essentially all catalytic activity and ability to bind zinc. These results indicate that the Cys 299 in the ␤ subunit of FTase plays a critical role in catalysis by the enzyme and is likely to be one of the residues that directly coordinate the zinc atom in this enzyme.

supporting

confidence: 52%

Identification of a Cysteine Residue Essential for Activity of Protein Farnesyltransferase

Moomaw

et al. 1996

“…In cross-linking studies, substrate proteins and photoactivatable analogs of FPP and CAAX peptides have been shown to bind to the ␤-subunit of FTase (18,22,23). Similarly, photoactivatable geranylgeranyl diphosphate analogs can be cross-linked to the ␤-subunit of GGTase-I (24,25). The recently published x-ray crystal structure of rat FTase (Ͼ93% amino acid sequence identity to human FTase) identified three conserved amino acids of the ␤-subunit as ligands for the essential Zn 2ϩ ion (26).…”

mentioning

confidence: 99%

Mutational Analysis of Conserved Residues of the ॆ-Subunit of Human Farnesyl:Protein Transferase

Kral

Diehl

Desolms

et al. 1997

Self Cite

The roles of 11 conserved amino acids of the ␤-subunit of human farnesyl:protein transferase (FTase) were examined by performing kinetic and biochemical analyses of site-directed mutants. This biochemical information along with the x-ray crystal structure of rat FTase indicates that residues His-248, Arg-291, Lys-294, and Trp-303 are involved with binding and utilization of the substrate farnesyl diphosphate. Our data confirm structural evidence that amino acids Cys-299, Asp-297, and His-362 are ligands for the essential Zn 2؉ ion and suggest that Asp-359 may also play a role in Zn 2؉ binding. Additionally, we demonstrate that Arg-202 is important for binding the essential C-terminal carboxylate of the protein substrate.Farnesyl:protein transferase (FTase) 1 catalyzes thioether bond formation between farnesyl, from farnesyl diphosphate (FPP), and the sulfur atom of a cysteine residue near the C terminus of its protein substrate (1, 2). The protein substrate cysteine residue is located within a C-terminal motif called a CAAX box in which C is the cysteine that is S-farnesylated, A is often an aliphatic amino acid, and X is methionine, serine, glutamine, cysteine, or alanine (1, 3, 4). S-Farnesylation is the first step by which a number of proteins, including all forms of the Ras proto-oncoprotein, are post-translationally lipid-modified, facilitating membrane association and in some cases protein-protein interaction (2). Membrane association is required for Ras function, thus inhibitors of FTase have been proposed as antitumor agents (5). FTase inhibitors (FTIs) have been shown to inhibit anchorage-independent growth of ras-transformed rodent fibroblasts and human tumor cell lines (6 -8). In addition, FTIs have shown antitumor effects in rodents (6, 9).FTase kinetically proceeds through an ordered, sequential mechanism in which FPP is the first substrate bound (10, 11). Subsequent protein substrate binding to FTase depends on an enzyme-bound Zn 2ϩ ion (12, 13). Recent spectral data indicate that upon formation of the FTase⅐FPP⅐CAAX ternary complex, the cysteine thiol of the CAAX sequence interacts directly with Zn 2ϩ

“…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)(16)(17), although there is also evidence that the ␣ subunit may participate (15,18). In addition to its bound Zn 2ϩ , FTase also requires Mg 2ϩ for activity.…”

mentioning

confidence: 99%

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

Tschantz

Furfine

Casey

1997

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...