Compounds structurally related to complexone I. Tris(cabroxyethyl)phosphine

Podlaha, Jaroslav; Podlahová, J.

doi:10.1135/cccc19731730

Cited by 29 publications

(18 citation statements)

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“…Protein concentrations were determined by the Coomassie Blue binding method using a commercial kit (Bio-Rad). (22,23).) Using these conditions, the activity of Co 2ϩ -FTase is comparable with that of Zn 2ϩ -FTase (Fig.…”

Section: Methodsmentioning

confidence: 85%

Evidence for a Catalytic Role of Zinc in Protein Farnesyltransferase

Huang

Casey

Fierke

1997

Journal of Biological Chemistry

155

124

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. These data provide evidence that the zinc ion plays an important catalytic role in FTase, most likely by activation of the cysteine thiol of the protein substrate for nucleophilic attack on the isoprenoid.Protein farnesyltransferase (FTase) 1 catalyzes the transfer of the farnesyl group of farnesyl pyrophosphate (FPP) to a conserved cysteine residue of a protein substrate, including Ras proteins, nuclear lamins, and several proteins involved in cell signaling (1-4). Protein farnesylation mediates membrane association and, possibly, interactions with other proteins essential for the localization and function of these proteins (3, 4). One example in this regard is the requirement of farnesylation for the cell transforming ability of oncogenic Ras proteins (5); this result has stimulated an intense search for FTase inhibitors as potential anticancer drugs (6, 7). An increased understanding of the molecular mechanism of FTase should enhance the rational design of such FTase inhibitors.FTase is a metalloenzyme that contains one zinc ion per ␣/␤ heterodimer that is essential for optimal activity (8, 9). Crosslinking and direct binding studies indicate that the zinc ion is required for the binding of protein but not isoprenoid substrates (8). Additionally, FTase containing Cd 2ϩ substituted for Zn 2ϩ has essentially normal catalytic activity, demonstrating that other metal ions can functionally substitute for the zinc (10). Chemical modification and site-directed mutagenesis studies have identified a conserved cysteine residue of FTase, Cys 299 , in the ␤ subunit, as important for catalytic activity and zinc binding, suggesting that the thiolate of this residue may directly coordinate the zinc ion (11).Although it is clear that the zinc ion in FTase is critical for activity, the precise function of the metal, particularly the question of whether the primary role of the zinc ion is structural or catalytic, is not yet known. Proposed catalytic functions for the zinc ion in FTase include increasing the nucleophilicity of the cysteine residue of the protein substrate (3,8,11,12) and/or activating the diphosphate leaving group (11,13,14). Here we investigate the metal coordination polyhedron in FTase by substituting Co 2ϩ for Zn 2ϩ , which does not change the catalytic activity of the enzyme. This substitution provides a useful spectroscopic probe of the composition and geometric arrangement of the ligands around the metal ion (15,16), and the spectral data obtained indicate that the metal ion coordinates the thiolate of the peptide substrate in the presence of bound FPP. EXPERIMENTAL PROCEDURESPreparation of Apo-FTase-Recombinant Rat FTase was produced and purified as described (9). Apo-FTase was prepared by dialyzing the holo-enzyme (Ͼ1 mg/ml) for 24 h against 50 mM Tris-Cl, pH 7.8, 1 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Buffer 1) and 5 mM EDTA at 4°C followed by an additional 24 h dialysis against Buffer 1 containing 50 M EDTA. After dialysis, the apo-FTase was concentrated to ϳ100 M, flash-frozen, ...

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Section: Methodsmentioning

confidence: 85%

Evidence for a Catalytic Role of Zinc in Protein Farnesyltransferase

Huang

Casey

Fierke

1997

Journal of Biological Chemistry

155

124

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“…Throughout this range TCEP should exist predominantly as the trialkylphosphonium (X3PH+) rather than the phosphine (X,P), because its pK, is 7.66 (Podlaha & Podlahova, 1973). The results suggest that a group with pK, of approx.…”

Section: Reduction Of Disulfides By Trialkylphosphinesmentioning

confidence: 91%

“…Reduction of endothelin at lower pH produced the rather surprising result (Table 2) that there was only a slow decline in the rate of reduction between pH 5 and 3, but a sharp decrease between pH 3 and 2. Throughout this range TCEP should exist predominantly as the trialkylphosphonium (X3PH+) rather than the phosphine (X,P), because its pK, is 7.66 (Podlaha & Podlahova, 1973). The results suggest that a group with pK, of approx.…”

Section: Reduction Of Disulfides By Trialkylphosphinesmentioning

confidence: 91%

Disulfide structures of highly bridged peptides: A new strategy for analysis

1993

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A new approach is described for analyzing disulfide linkage patterns in peptides containing tightly clustered cystines. Such peptides are very difficult to analyze with traditional strategies, which require that the peptide Chain be split between close or adjacent Cys residues. The water-soluble tris-(2-~arboxyethyl)-phosphine (TCEP) reduced disulfides at pH 3, and partially reduced peptides were purified by high performance liquid chromatography with minimal thiol-disulfide exchange. Alkylation of free thiols, followed by sequencer analysis, provided explicit assignment of disulfides that had been reduced. Thiol-disulfide exchange occurred during alkylation of some peptides, but correct deductions were still possible. Alkylation competed best with exchange when peptide solution was added with rapid mixing to 2.2 M iodoacetamide. Variants were developed in which up to three alkylating agents were used to label different pairs of thiols, allowing a full assignment in one sequencer analysis. Model peptides used included insulin (three bridges, intra-and interchain disulfides; -Cys.Cys-pair), endothelin and apamin (two disulfides; -Cys.x.Cys-pair), conotoxin GI and isomers (two disulfides; -Cys.Cys-pair), and bacterial enterotoxin (three bridges within 13 residues; two -Cys.Cys-pairs). With insulin, all intermediates in the reduction pathway were identified; with conotoxin GI, analysis was carried out successfully for all three disulfide isomers. In addition to these known structures, the method has been applied successfully to the analysis of several previously unsolved structures of similar complexity. Rates of reduction of disulfide bonds varied widely, but most peptides did not show a strongly preferred route for reduction.

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“…5). Of the remaining nucleophiles that were tested only the phosphine (TCEP, p K a 7.620) (used as a disulfide reducing agent) (entry n) and azide (a preservative) (entry o) gave adducts at pH 11 that were detectable under these ESI‐MS conditions.…”

Section: Resultsmentioning

confidence: 99%