Characterization and kinetic analysis of the intracellular domain of human protein tyrosine phosphatase <i>β</i> (HPTP <i>β</i>) using synthetic phosphopeptides

Harder, Kenneth W.; Owen, Philip; Wong, Lily; Aebersold, Ruedi; Clark‐Lewis, Ian; Jirik, Frank R.

doi:10.1042/bj2980395

Cited by 198 publications

(175 citation statements)

References 51 publications

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“…Thus, APase matches those mentioned above in its formal proficiency [(k cat ͞K m )͞k non ] as a catalyst. Among enzymes of this latter type, a high value of k cat (1.5 ϫ 10 4 s Ϫ1 ) has been recorded for a human protein tyrosine phosphatase (PTP; EC 3.1.3.48), acting on a model peptide through a phosphorylated cysteine residue as an intermediate (21). Comparison with the present rate constant for spontaneous hydrolysis of the phenyl phosphate dianion indicates that PTP enhances the rate of hydrolysis of phosphorylated tyrosine residues by Ϸ17 orders of magnitude.…”

Section: Resultsmentioning

confidence: 99%

The rate of hydrolysis of phosphomonoester dianions and the exceptional catalytic proficiencies of protein and inositol phosphatases

Lad

Williams

Wolfenden

2003

Proc. Natl. Acad. Sci. U.S.A.

257

349

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To evaluate the proficiency of phosphatases as catalysts, the rate of the uncatalyzed hydrolysis of simple phosphate monoester dianions was estimated by extrapolating rates measured over a range of high temperatures. The rate of spontaneous hydrolysis of phenyl phosphate dianion indicates that a linear free energy relationship reported earlier is reliable for leaving groups whose conjugate acids have pKa values up to at least 10. Using Teflon reaction vessels, it proved possible to follow the hydrolysis of methyl phosphate and 3-(4-carboxy)-2,2-dimethylpropyl phosphate in strong alkali. Even in 1 M KOH, the reaction was found to be specific acid catalyzed. These results establish an upper limit for dianion reactivity, which had been overestimated earlier as a result of the leaching by alkali of silicic acid from quartz reaction vessels. The present findings indicate that the half-time for attack by water on alkyl phosphate dianions is 1.1 ؋ 10 12 years (k ‫؍‬ 2 ؋ 10 ؊20 s) at 25°C and that phosphatases involved in cell signaling and regulation produce the largest rate enhancements that have been identified thus far. Protein phosphatase-1 and inositol 1-phosphatase exceed all other known enzymes in their affinities for the altered substrates in the transition state.M ost enzyme reactions proceed with k cat ͞K m values in the neighborhood of 10 7 M Ϫ1 ⅐s Ϫ1 and appear to be similarly efficient according to that criterion. However, to assess the proficiency of an enzyme as a catalyst, and its corresponding affinity for the altered substrate in the transition state, it is necessary to compare k cat ͞K m with the rate constant of the corresponding reaction under the same conditions in the absence of a catalyst. In contrast to the relatively narrow range of values of k cat ͞K m observed for enzyme catalyzed reaction rates, the rates of these uncatalyzed reactions span a range of at least 19 orders of magnitude (1). Thus, differences in enzyme proficiency tend to reflect differences in the rates of the uncatalyzed reactions rather than the catalyzed reactions, and enzymes differ greatly from one another in their prowess as catalysts. Enzymes that catalyze the slowest reactions are of practical interest, in that they offer sensitive targets for inhibition by transition-state analogues. Their mechanisms of action are also particularly challenging to rationalize. Conspicuous among these slow reactions is the hydrolysis of monoesters of phosphoric acid.In biological systems, phosphate monoesters are cleaved by two groups of monoesterases that act by different mechanisms.One group of phosphomonoesterases, typified by bacterial alkaline phosphatase (2) and protein tyrosine phosphatases (3), uses an active-site nucleophile to displace the alcohol-leaving group from the substrate to form a phosphoryl-enzyme intermediate, which is subsequently hydrolyzed. Rate enhancement by enzymes that act through a double-displacement mechanism is not simple to analyze in terms of transition-state affinity or transition-state stabilization (...

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Section: Resultsmentioning

confidence: 99%

The rate of hydrolysis of phosphomonoester dianions and the exceptional catalytic proficiencies of protein and inositol phosphatases

Lad

Williams

Wolfenden

2003

Proc. Natl. Acad. Sci. U.S.A.

257

349

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“…PTP1B activity was assayed using a modification of the technique described by Harder et al 33 Briefly, the enzyme reactions were carried out in a final volume of 30 µL on Costar 384 well black plates with clear bottoms (part number 3711, Corning Inc., Corning, NY). To each well were added 50 µM of phosphorylated peptide and 0.9 nM (1.6 ng) of PTP1B diluted in 25 mM HEPES (pH 7.0), 0.3 mM EDTA, 3.3 mM DTT, 3.3% glycerol, and 0.03% bovine serum albumin, with or without inhibitors.…”

Section: Methodsmentioning

confidence: 99%

“…Compounds selected by the docking program were screened for their ability to inhibit the PTP1B dephosphorylation of the insulin receptor peptide. The assay used the malachite green-ammonium molybdate method 33 to detect phosphate liberated from the 1142-1153 insulin receptor phosphotyrosyl dodecapeptide, TRDI(P)YETDYYPRK. Peptide substrate (20 µL) was added to a 96 well plate at a final concentration of 50 µM.…”

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confidence: 99%

Molecular Docking and High-Throughput Screening for Novel Inhibitors of Protein Tyrosine Phosphatase-1B

et al. 2002

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High-throughput screening (HTS) of compound libraries is used to discover novel leads for drug development. When a structure is available for the target, computer-based screening using molecular docking may also be considered. The two techniques have rarely been used together on the same target. The opportunity to do so presented itself in a project to discover novel inhibitors for the enzyme protein tyrosine phosphatase-1B (PTP1B), a tyrosine phosphatase that has been implicated as a key target for type II diabetes. A corporate library of approximately 400 000 compounds was screened using high-throughput experimental techniques for compounds that inhibited PTP1B. Concurrently, molecular docking was used to screen approximately 235 000 commercially available compounds against the X-ray crystallographic structure of PTP1B, and 365 high-scoring molecules were tested as inhibitors of the enzyme.Of approximately 400 000 molecules tested in the high-throughput experimental assay, 85 (0.021%) inhibited the enzyme with IC 50 values less than 100 µM; the most active had an IC 50 value of 4.2 µM. Of the 365 molecules suggested by molecular docking, 127 (34.8%) inhibited PTP1B with IC 50 values less than 100 µM; the most active of these had an IC 50 of 1.7 µM. Structure-based docking therefore enriched the hit rate by 1700-fold over random screening. The hits from both the high-throughput and docking screens were dissimilar from phosphotyrosine, the canonical substrate group for PTP1B; the two hit lists were also very different from each other. Surprisingly, the docking hits were judged to be more druglike than the HTS hits. The diversity of both hit lists and their dissimilarity from each other suggest that docking and HTS may be complementary techniques for lead discovery.

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“…Phosphoinositide phosphatase activity was measured using a chromogenic assay based on the malachite green method, previously used for the analysis of protein phosphatases (Harder et al, 1994;Marcus et al, 2001). Briefly, recombinant purified protein was incubated with a reaction mixture consisting of 100 mM Tris/HCl pH 8, 10 mM dithiothreitol, 0?5 mM diC 16 -phosphatidylserine (Sigma P-1185) and 25 mM phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns 3,4,5-P 3 ) (diC 16 , Biomol PH-107) (temperature 37 uC, reaction volume 50 ml).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Cdc42-dependent signalling in Saccharomyces cerevisiae by phosphatase-dead SigD/SopB from Salmonella typhimurium

et al. 2006

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Heterologous expression of bacterial virulence factors in Saccharomyces cerevisiae is a feasible approach to study their molecular function. The authors have previously reported that the Salmonella typhimurium SigD protein, a phosphatidylinositol phosphatase involved in invasion of the host cell, inhibits yeast growth, presumably by depleting an essential pool of phosphatidylinositol 4,5-bisphosphate, and also that a catalytically inactive version, SigDR468A, was able to arrest growth by a different mechanism that involved disruption of the actin cytoskeleton. This paper describes marked differences between the phenotypes elicited by expression of SigD and SigDR468A in yeast. First, expression of SigDR468A caused accumulation of large unbudded cells and loss of septin organization, while SigD expression caused none of these effects. Second, growth inhibition by SigDR468A was mediated by a cell cycle arrest in G2 dependent on the Swe1 morphogenetic checkpoint, but SigD-induced growth inhibition was cell cycle independent. And third, SigD caused strong activation of the yeast MAP kinase Slt2, whereas SigDR468A rather inactivated another MAP kinase, Kss1. In a screen for suppressors of SigDR468A-induced growth arrest by overexpression of a yeast cDNA library, the Cdc42 GTPase was isolated. Furthermore, SigDR468A was co-purified with Cdc42 from yeast lysates. It is concluded that the Salmonella SigD protein deprived of its phosphatase activity is able to disrupt yeast morphogenesis by interfering with Cdc42 function, opening the possibility that the SigD N-terminal region might directly modulate small GTPases from the host during infection.

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Characterization and kinetic analysis of the intracellular domain of human protein tyrosine phosphatase β (HPTP β) using synthetic phosphopeptides

Cited by 198 publications

References 51 publications

The rate of hydrolysis of phosphomonoester dianions and the exceptional catalytic proficiencies of protein and inositol phosphatases

The rate of hydrolysis of phosphomonoester dianions and the exceptional catalytic proficiencies of protein and inositol phosphatases

Molecular Docking and High-Throughput Screening for Novel Inhibitors of Protein Tyrosine Phosphatase-1B

Inhibition of Cdc42-dependent signalling in Saccharomyces cerevisiae by phosphatase-dead SigD/SopB from Salmonella typhimurium

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