1995
DOI: 10.1021/bi00046a044
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Catalytic Activity of the SH2 Domain of Human pp60c-src; Evidence from NMR, Mass Spectrometry, Site-Directed Mutagenesis and Kinetic Studies for an Inherent Phosphatase Activity

Abstract: During solution structural studies it was apparent that the human recombinant pp60c-src SH2 domain (srcSH2, residues 144-249) possessed an inherent phosphatase (Pase) activity. Complexes of U[13C,15N]srcSH2 with unlabeled Ac-pYEEIE (I) were examined using 31P and 1H-detected isotope filtered NMR methods. The presence of a high-affinity complex in equimolar solutions of I and U[13C, 15N]-srcSH2 was demonstrated by chemical shift perturbations, line broadening, and the observation of intermolecular nuclear Overh… Show more

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Cited by 11 publications
(7 citation statements)
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“…In addition, the Cys42 side chain has been associated with a weak phosphoesterase activity that may serve to regulate the binding affinity or timescale of interaction between phosphopeptides and the Src SH2 domain 62. The increased preference for the thiol to be deprotonated in the context of the pY‐binding site, manifested in the ∼0.5–0.8 pH unit decrease in p K a compared to solvent‐exposed cysteine (p K a ∼ 9.3), might explain the reported increased catalytic activity of this thiol at physiological pH.…”
Section: Resultsmentioning
confidence: 99%
“…In addition, the Cys42 side chain has been associated with a weak phosphoesterase activity that may serve to regulate the binding affinity or timescale of interaction between phosphopeptides and the Src SH2 domain 62. The increased preference for the thiol to be deprotonated in the context of the pY‐binding site, manifested in the ∼0.5–0.8 pH unit decrease in p K a compared to solvent‐exposed cysteine (p K a ∼ 9.3), might explain the reported increased catalytic activity of this thiol at physiological pH.…”
Section: Resultsmentioning
confidence: 99%
“…Peptides were immobilized on a sensor chip by covalent coupling to a carboxylated dextran matrix on its surface as described (34). Purified recombinant Src SH2 and SH3/SH2 domains were expressed and purified as described (35,36) and injected over the sensor chips in 10 mM HEPES pH 7.4, 150 mM NaCl, and 0.05% (v/v) of a 10% P-20 surfactant solution. As described (34) data points were collected when the responses reached steady state and the equilibrium dissociation constants calculated using the Hill equation,…”
Section: Methodsmentioning
confidence: 99%
“…Off-line sampling enables the inclusion of any suitable treatment or separation technique necessary to eliminate the possible sources of interference, or to prepare sample solutions for analysis by any available ionization method. For this reason, off-line applications have been accomplished by using a variety of techniques, including FAB/CF-FAB (Caprioli, 1985(Caprioli, , 1988aShao et al, 1987;Langridge et al, 1993;Newton et al, 1997); ESI-MS (Wilson, Perez, & Pasternak, 1993;Aliprandis & Canary, 1994;Gonnet et al, 1996;Charbonniere et al, 1997;Takayama et al, 1997;Wu et al, 1997;Esperson et al, 1998;Kelleher, Hendrickson, & Walsh, 1999;Bothner et al, 2000;Fabris, 2000;Ge et al, 2001;Norris et al, 2001a,b;Pi et al, 2002); LC-MS (Boerner et al, 1995;Hsieh et al, 1995;Li et al, 2000); and MALDI (Craig et al, 1994;Matsumoto, Kahn, & Komori, 1998).…”
Section: Off-line Samplingmentioning
confidence: 99%
“…These advances have enabled the direct characterization of stable covalent adducts formed by many classes of enzymes with substrates, intermediates, products, or inhibitors (Aplin et al, 1990;Stevenson, Feng, & Storer, 1990;Ashton et al, 1991;Menard et al, 1991;Shneier et al, 1991;Orning et al, 1992;Knight et al, 1993;Miao et al, 1994;Boerner et al, 1995;Nairn et al, 1995;Saves et al, 1995;Boerner et al, 1996;Brown, Aplin, & Schofield, 1996;Thiruvikraman et al, 1996;Branchini et al, 1997;Febbraio et al, 1997;Gigant et al, 1997;Ramjee et al, 1997;Skorey et al, 1997;Wandall et al, 1997;Srivastava et al, 1999;Wu et al, 1999;Ichiyama et al, 2000;Regal et al, 2000;Vocadlo et al, 2001). Furthermore, since the initial work reporting the observation of an intact Michaelis complex between hen egg white lysozyme and the hexasaccharide substrate N-acetylglucosamine (NAG 6 ) (Ganem, Li, & Henion, 1991), ESI-MS has been widely used to obtain the stoichiometry, composition, and binding strength of non-covalent complexes formed by the interaction of proteins with different ligands (substrates, products, inhibitors, cofactors, etc.).…”
Section: Off-line Investigation Of Catalysis In Biological Systemsmentioning
confidence: 99%