Involvement of the α Subunit of Farnesyl-Protein Transferase in Substrate Recognition

Pellicena, Patricia; Jd, Scholten; Zimmerman, Karen K.; Creswell, Mark W.; Huang, Chieh-Chen; Wt, Miller

doi:10.1021/bi961336h

Cited by 23 publications

(11 citation statements)

References 41 publications

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“…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 (15,18). In addition to its bound Zn 2ϩ , FTase also requires Mg 2ϩ for activity.…”

Section: Protein Farnesyltransferase (Ftase)mentioning

confidence: 99%

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

Tschantz

Furfine

Casey

1997

Journal of Biological Chemistry

109

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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...

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Section: Protein Farnesyltransferase (Ftase)mentioning

confidence: 99%

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

Tschantz

Furfine

Casey

1997

Journal of Biological Chemistry

109

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“…Substratebased photoaffinity labels have been described that specifically target the active site of farnesyltransferase. The farnesyl diphosphate derivatives label the β subunit of FTase [21,23,69], while peptide derivatives label both the α and β subunits [70,71]. Additional information about the peptidebinding site has been provided by transfer NOE experiments on bound peptide inhibitors [72,73], which indicated that they bound to FTase in a β-turn conformation.…”

Section: Structural Studiesmentioning

confidence: 99%

Non-Peptidic Prenyltransferase Inhibitors: Diverse Structural Classes and Surprising Anti-Cancer Mechanisms

Gibbs

Zahn

Sebolt–Leopold

2001

CMC

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The development of farnesyltransferase inhibitors (FTIs) has been one of the most active areas of anticancer drug development for the past ten years. This review presents a general overview of the developments in this area, along with a critical appraisal of the anticancer activity of FTIs. A historical survey of the protein prenylation field is given, in particular to emphasize the key role played by the Ras oncoprotein in driving the discovery of prenyltransferase enzymes. The different classes of prenylated proteins will be described along with the biochemical characteristics of the key drug target--farnesyltransferase (FTase). Numerous potent farnesyltransferase inhibitors have been developed. The FTIs developed can be separated into three different categories, based on their origin and/or mechanism of action: a) natural products; b) peptidomimetics and other CAAX-competitive inhibitors; c) farnesyl pyrophosphate (FPP) mimetics or analogs and other FPP-competitive inhibitors. Along with a survey of newer FTIs in each class, the development of several representative, potent compounds will be discussed in depth as we discuss the potential advantages and liabilities of each class. Particular emphasis is given to the discovery of new, more potent FPP-competitive FTIs of several diverse structural classes. Testing of different FTIs for their ability to block the growth of various cancer cell types in animal models will be discussed. There are a number of key differences between these compounds and traditional cytotoxic cancer chemotherapeutic agents, with surprising exceptions to their expected modes of action. As some FTIs have entered human clinical trials, answers may soon become available to key mechanistic questions concerning the extent and nature of their antitumor growth properties.

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“…Mutagenesis of a Zn 2ϩ -coordinating Cys residue in the ␤ subunit of FTase indicates a role for this subunit in isoprenoid binding and a direct role in enzymatic catalysis (26). Photoaffinity cross-linking of peptides to FTase indicates the presence of an intersubunit location for the catalytic active site (27,28). Further studies of the FTase ␤ subunit indicate that distinct NH 2 -terminal and COOH-terminal regions of this subunit are involved in determining the -CAAX specificity of this enzyme and that single site mutations are sufficient to produce a mutant FTase that has an increased affinity for -CAAL substrates (29).…”

Section: Ddlf Ftase Farnesylated Ras-cii(smentioning

confidence: 99%

Amino Acid Residues That Define Both the Isoprenoid and CAAX Preferences of the Saccharomyces cerevisiae Protein Farnesyltransferase

Caplin

Ohya

Marshall

1998

Journal of Biological Chemistry

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Studies of the yeast protein farnesyltransferase (FTase) have shown that the enzyme preferentially farnesylates proteins ending in CAAX (C ‫؍‬ cysteine, A ‫؍‬ aliphatic residue, X ‫؍‬ cysteine, serine, methionine, alanine) and to a lesser degree CAAL. Furthermore, like the type I protein geranylgeranyltransferase (GGTase-I), FTase can also geranylgeranylate methionine-and leucine-ending substrates both in vitro and in vivo. Substrate overlap of FTase and GGTase I has not been determined to be biologically significant. In this study, specific residues that influence the substrate preferences of FTase have been identified using site-directed mutagenesis. Three of the mutations altered the substrate preferences of the wild type enzyme significantly.

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Involvement of the α Subunit of Farnesyl-Protein Transferase in Substrate Recognition

Cited by 23 publications

References 41 publications

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

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

Non-Peptidic Prenyltransferase Inhibitors: Diverse Structural Classes and Surprising Anti-Cancer Mechanisms

Amino Acid Residues That Define Both the Isoprenoid and CAAX Preferences of the Saccharomyces cerevisiae Protein Farnesyltransferase

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