The single sulfur to oxygen substitution in the active site nucleophile of the Yersinia protein-tyrosine phosphatase leads to substantial structural and functional perturbations

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Abstract

Protein-tyrosine phosphatases (PTPases) feature an essential nucleophilic thiol group which attacks the phosphorus atom in a substrate. A single S to O atom substitution in the nucleophile (via Cys to Ser mutation) renders PTPases catalytically inactive. We suggest that the lack of activity in the Cys to Ser mutant may be caused by structural and/or conformational perturbations in the active site. Yersinia PTPase contains a single tryptophan residue, Trp354, which is invariant among all PTPases and is located in the vicinity of the active site nucleophile Cys403. Thus, Trp354 serves as an intrinsic probe of the PTPase active site conformation. We show that although C403S displays a nearly identical circular dichroism spectrum to that of the wild type enzyme, its ultraviolet spectrum in the region attributed to Trio is significantly different from that of the wild-type enzyme. In addition, the intrinsic fluorescence intensity of C403S is enhanced 3-fold and exhibits different ionic strength dependency from that of the wild-type enzyme. Trp354 also has different accessibilities to quenchers in the wild-type and the C403S mutant PTPases. Furthermore, unfolding experiments demonstrate that the structure of C403S is significantly less stable than the wild-type PTPase and displays a different sensitivity to urea and guanidine hydrochloride. Finally, binding of tungstate enhances the fluorescence of the wild-type Yersinia PTPase with a K(d) of 55 μM, whereas binding of tungstate quenches the fluorescence of the C403S mutant with a K(d) of 690 μM. Collectively, these results indicate that the single sulfur to oxygen change in the active site nucleophile leads to substantial structural/conformational and functional alterations in the Yersinia PTPase.

Original languageEnglish (US)
Pages (from-to)1362-1369
Number of pages8
JournalBiochemistry
Volume36
Issue number6
DOIs
StatePublished - Feb 11 1997
Externally publishedYes

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Yersinia
Nucleophiles
Protein Tyrosine Phosphatases
Sulfur
Catalytic Domain
Substitution reactions
Oxygen
Fluorescence
Enzymes
Atoms
Guanidine
Mutant Proteins
Circular Dichroism
Ionic strength
Sulfhydryl Compounds
Tryptophan
Osmolar Concentration
Phosphorus
Conformations
Urea

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "The single sulfur to oxygen substitution in the active site nucleophile of the Yersinia protein-tyrosine phosphatase leads to substantial structural and functional perturbations",
abstract = "Protein-tyrosine phosphatases (PTPases) feature an essential nucleophilic thiol group which attacks the phosphorus atom in a substrate. A single S to O atom substitution in the nucleophile (via Cys to Ser mutation) renders PTPases catalytically inactive. We suggest that the lack of activity in the Cys to Ser mutant may be caused by structural and/or conformational perturbations in the active site. Yersinia PTPase contains a single tryptophan residue, Trp354, which is invariant among all PTPases and is located in the vicinity of the active site nucleophile Cys403. Thus, Trp354 serves as an intrinsic probe of the PTPase active site conformation. We show that although C403S displays a nearly identical circular dichroism spectrum to that of the wild type enzyme, its ultraviolet spectrum in the region attributed to Trio is significantly different from that of the wild-type enzyme. In addition, the intrinsic fluorescence intensity of C403S is enhanced 3-fold and exhibits different ionic strength dependency from that of the wild-type enzyme. Trp354 also has different accessibilities to quenchers in the wild-type and the C403S mutant PTPases. Furthermore, unfolding experiments demonstrate that the structure of C403S is significantly less stable than the wild-type PTPase and displays a different sensitivity to urea and guanidine hydrochloride. Finally, binding of tungstate enhances the fluorescence of the wild-type Yersinia PTPase with a K(d) of 55 μM, whereas binding of tungstate quenches the fluorescence of the C403S mutant with a K(d) of 690 μM. Collectively, these results indicate that the single sulfur to oxygen change in the active site nucleophile leads to substantial structural/conformational and functional alterations in the Yersinia PTPase.",
author = "Zhong-Yin Zhang and Li Wu",
year = "1997",
month = "2",
day = "11",
doi = "10.1021/bi9624043",
language = "English (US)",
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pages = "1362--1369",
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TY - JOUR

T1 - The single sulfur to oxygen substitution in the active site nucleophile of the Yersinia protein-tyrosine phosphatase leads to substantial structural and functional perturbations

AU - Zhang, Zhong-Yin

AU - Wu, Li

PY - 1997/2/11

Y1 - 1997/2/11

N2 - Protein-tyrosine phosphatases (PTPases) feature an essential nucleophilic thiol group which attacks the phosphorus atom in a substrate. A single S to O atom substitution in the nucleophile (via Cys to Ser mutation) renders PTPases catalytically inactive. We suggest that the lack of activity in the Cys to Ser mutant may be caused by structural and/or conformational perturbations in the active site. Yersinia PTPase contains a single tryptophan residue, Trp354, which is invariant among all PTPases and is located in the vicinity of the active site nucleophile Cys403. Thus, Trp354 serves as an intrinsic probe of the PTPase active site conformation. We show that although C403S displays a nearly identical circular dichroism spectrum to that of the wild type enzyme, its ultraviolet spectrum in the region attributed to Trio is significantly different from that of the wild-type enzyme. In addition, the intrinsic fluorescence intensity of C403S is enhanced 3-fold and exhibits different ionic strength dependency from that of the wild-type enzyme. Trp354 also has different accessibilities to quenchers in the wild-type and the C403S mutant PTPases. Furthermore, unfolding experiments demonstrate that the structure of C403S is significantly less stable than the wild-type PTPase and displays a different sensitivity to urea and guanidine hydrochloride. Finally, binding of tungstate enhances the fluorescence of the wild-type Yersinia PTPase with a K(d) of 55 μM, whereas binding of tungstate quenches the fluorescence of the C403S mutant with a K(d) of 690 μM. Collectively, these results indicate that the single sulfur to oxygen change in the active site nucleophile leads to substantial structural/conformational and functional alterations in the Yersinia PTPase.

AB - Protein-tyrosine phosphatases (PTPases) feature an essential nucleophilic thiol group which attacks the phosphorus atom in a substrate. A single S to O atom substitution in the nucleophile (via Cys to Ser mutation) renders PTPases catalytically inactive. We suggest that the lack of activity in the Cys to Ser mutant may be caused by structural and/or conformational perturbations in the active site. Yersinia PTPase contains a single tryptophan residue, Trp354, which is invariant among all PTPases and is located in the vicinity of the active site nucleophile Cys403. Thus, Trp354 serves as an intrinsic probe of the PTPase active site conformation. We show that although C403S displays a nearly identical circular dichroism spectrum to that of the wild type enzyme, its ultraviolet spectrum in the region attributed to Trio is significantly different from that of the wild-type enzyme. In addition, the intrinsic fluorescence intensity of C403S is enhanced 3-fold and exhibits different ionic strength dependency from that of the wild-type enzyme. Trp354 also has different accessibilities to quenchers in the wild-type and the C403S mutant PTPases. Furthermore, unfolding experiments demonstrate that the structure of C403S is significantly less stable than the wild-type PTPase and displays a different sensitivity to urea and guanidine hydrochloride. Finally, binding of tungstate enhances the fluorescence of the wild-type Yersinia PTPase with a K(d) of 55 μM, whereas binding of tungstate quenches the fluorescence of the C403S mutant with a K(d) of 690 μM. Collectively, these results indicate that the single sulfur to oxygen change in the active site nucleophile leads to substantial structural/conformational and functional alterations in the Yersinia PTPase.

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