Examination of the transition state of the low-molecular mass small tyrosine phosphatase 1. Comparisons with other protein phosphatases

Alvan C. Hengge, Yu Zhao, Li Wu, Zhong-Yin Zhang

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Abstract

The reactions of p-nitrophenyl phosphate (pNPP) with the low-molecular mass tyrosine phosphatase Stp1 and with the mutants D128N, D128A, D128E, and S18A have been studied by measurement of heavy-atom isotope effects in the substrate. The isotope effects were measured at the nonbridging oxygen atoms [18(V/K)(nonbridge)], at the bridging oxygen atom (the site of bond cleavage) [18(V/K)(bridge)], and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The results with native Stp1 were 1.0160 ± 0.0005 for 18(V/K)(bridge), 1.0007 ± 0.0001 for 15(V/K), and 1.0018 ± 0.0003 for 18(V/K)(nonbridge). The values for 18(V/K)(nonbridge) and 15(V/K) differ from those previously measured with other protein-tyrosine phosphatases and from those of the aqueous hydrolysis reaction of pNPP. The values indicate that in the transition state of the native Stp1 reaction the leaving group bears a partial negative charge, and there is nucleophilic interaction between the Cys nucleophile, and the phosphoryl group, causing some decrease in the nonbridge P-O bond order. The transition state remains highly dissociative with respect to the degree of bond cleavage to the leaving group. Mutation of the general acid from aspartic acid to glutamic acid slows catalysis but causes no change in the isotope effects and thus does not alter the degree of proton transfer to the leaving group in the transition state. Mutations of this residue to asparagine or alanine give values for 18(V/K)(bridge) of about 1.029, for 15(V/K) of about 1.003, and for 18(V/K)(nonbridge) of 1.0010 (D128A) to 1.0024 (D128N). These data indicate a dissociative transition state with the leaving group departing as the nitrophenolate anion and indicate more nucleophilic participation than in the aqueous hydrolysis of the pNPP dianion, just as in the native enzyme. The isotope effects with the S18A mutant, in which a hydrogen bonding stabilization of the anionic Cys nucleophile has been removed, were within experimental error of those with the native enzyme, indicating that this alteration has no effect on the transition state for phosphoryl transfer from pNPP.

Original languageEnglish (US)
Pages (from-to)7928-7935
Number of pages8
JournalBiochemistry
Volume36
Issue number25
DOIs
StatePublished - Jun 24 1997
Externally publishedYes

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Phosphoprotein Phosphatases
Molecular mass
Phosphoric Monoester Hydrolases
Isotopes
Tyrosine
Atoms
Nucleophiles
Hydrolysis
Oxygen
Nitrophenols
Mutation
Protein Tyrosine Phosphatases
Proton transfer
Asparagine
Enzymes
Hydrogen Bonding
Catalysis
Aspartic Acid
Alanine
Anions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Examination of the transition state of the low-molecular mass small tyrosine phosphatase 1. Comparisons with other protein phosphatases. / Hengge, Alvan C.; Zhao, Yu; Wu, Li; Zhang, Zhong-Yin.

In: Biochemistry, Vol. 36, No. 25, 24.06.1997, p. 7928-7935.

Research output: Contribution to journalArticle

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title = "Examination of the transition state of the low-molecular mass small tyrosine phosphatase 1. Comparisons with other protein phosphatases",
abstract = "The reactions of p-nitrophenyl phosphate (pNPP) with the low-molecular mass tyrosine phosphatase Stp1 and with the mutants D128N, D128A, D128E, and S18A have been studied by measurement of heavy-atom isotope effects in the substrate. The isotope effects were measured at the nonbridging oxygen atoms [18(V/K)(nonbridge)], at the bridging oxygen atom (the site of bond cleavage) [18(V/K)(bridge)], and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The results with native Stp1 were 1.0160 ± 0.0005 for 18(V/K)(bridge), 1.0007 ± 0.0001 for 15(V/K), and 1.0018 ± 0.0003 for 18(V/K)(nonbridge). The values for 18(V/K)(nonbridge) and 15(V/K) differ from those previously measured with other protein-tyrosine phosphatases and from those of the aqueous hydrolysis reaction of pNPP. The values indicate that in the transition state of the native Stp1 reaction the leaving group bears a partial negative charge, and there is nucleophilic interaction between the Cys nucleophile, and the phosphoryl group, causing some decrease in the nonbridge P-O bond order. The transition state remains highly dissociative with respect to the degree of bond cleavage to the leaving group. Mutation of the general acid from aspartic acid to glutamic acid slows catalysis but causes no change in the isotope effects and thus does not alter the degree of proton transfer to the leaving group in the transition state. Mutations of this residue to asparagine or alanine give values for 18(V/K)(bridge) of about 1.029, for 15(V/K) of about 1.003, and for 18(V/K)(nonbridge) of 1.0010 (D128A) to 1.0024 (D128N). These data indicate a dissociative transition state with the leaving group departing as the nitrophenolate anion and indicate more nucleophilic participation than in the aqueous hydrolysis of the pNPP dianion, just as in the native enzyme. The isotope effects with the S18A mutant, in which a hydrogen bonding stabilization of the anionic Cys nucleophile has been removed, were within experimental error of those with the native enzyme, indicating that this alteration has no effect on the transition state for phosphoryl transfer from pNPP.",
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T1 - Examination of the transition state of the low-molecular mass small tyrosine phosphatase 1. Comparisons with other protein phosphatases

AU - Hengge, Alvan C.

AU - Zhao, Yu

AU - Wu, Li

AU - Zhang, Zhong-Yin

PY - 1997/6/24

Y1 - 1997/6/24

N2 - The reactions of p-nitrophenyl phosphate (pNPP) with the low-molecular mass tyrosine phosphatase Stp1 and with the mutants D128N, D128A, D128E, and S18A have been studied by measurement of heavy-atom isotope effects in the substrate. The isotope effects were measured at the nonbridging oxygen atoms [18(V/K)(nonbridge)], at the bridging oxygen atom (the site of bond cleavage) [18(V/K)(bridge)], and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The results with native Stp1 were 1.0160 ± 0.0005 for 18(V/K)(bridge), 1.0007 ± 0.0001 for 15(V/K), and 1.0018 ± 0.0003 for 18(V/K)(nonbridge). The values for 18(V/K)(nonbridge) and 15(V/K) differ from those previously measured with other protein-tyrosine phosphatases and from those of the aqueous hydrolysis reaction of pNPP. The values indicate that in the transition state of the native Stp1 reaction the leaving group bears a partial negative charge, and there is nucleophilic interaction between the Cys nucleophile, and the phosphoryl group, causing some decrease in the nonbridge P-O bond order. The transition state remains highly dissociative with respect to the degree of bond cleavage to the leaving group. Mutation of the general acid from aspartic acid to glutamic acid slows catalysis but causes no change in the isotope effects and thus does not alter the degree of proton transfer to the leaving group in the transition state. Mutations of this residue to asparagine or alanine give values for 18(V/K)(bridge) of about 1.029, for 15(V/K) of about 1.003, and for 18(V/K)(nonbridge) of 1.0010 (D128A) to 1.0024 (D128N). These data indicate a dissociative transition state with the leaving group departing as the nitrophenolate anion and indicate more nucleophilic participation than in the aqueous hydrolysis of the pNPP dianion, just as in the native enzyme. The isotope effects with the S18A mutant, in which a hydrogen bonding stabilization of the anionic Cys nucleophile has been removed, were within experimental error of those with the native enzyme, indicating that this alteration has no effect on the transition state for phosphoryl transfer from pNPP.

AB - The reactions of p-nitrophenyl phosphate (pNPP) with the low-molecular mass tyrosine phosphatase Stp1 and with the mutants D128N, D128A, D128E, and S18A have been studied by measurement of heavy-atom isotope effects in the substrate. The isotope effects were measured at the nonbridging oxygen atoms [18(V/K)(nonbridge)], at the bridging oxygen atom (the site of bond cleavage) [18(V/K)(bridge)], and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The results with native Stp1 were 1.0160 ± 0.0005 for 18(V/K)(bridge), 1.0007 ± 0.0001 for 15(V/K), and 1.0018 ± 0.0003 for 18(V/K)(nonbridge). The values for 18(V/K)(nonbridge) and 15(V/K) differ from those previously measured with other protein-tyrosine phosphatases and from those of the aqueous hydrolysis reaction of pNPP. The values indicate that in the transition state of the native Stp1 reaction the leaving group bears a partial negative charge, and there is nucleophilic interaction between the Cys nucleophile, and the phosphoryl group, causing some decrease in the nonbridge P-O bond order. The transition state remains highly dissociative with respect to the degree of bond cleavage to the leaving group. Mutation of the general acid from aspartic acid to glutamic acid slows catalysis but causes no change in the isotope effects and thus does not alter the degree of proton transfer to the leaving group in the transition state. Mutations of this residue to asparagine or alanine give values for 18(V/K)(bridge) of about 1.029, for 15(V/K) of about 1.003, and for 18(V/K)(nonbridge) of 1.0010 (D128A) to 1.0024 (D128N). These data indicate a dissociative transition state with the leaving group departing as the nitrophenolate anion and indicate more nucleophilic participation than in the aqueous hydrolysis of the pNPP dianion, just as in the native enzyme. The isotope effects with the S18A mutant, in which a hydrogen bonding stabilization of the anionic Cys nucleophile has been removed, were within experimental error of those with the native enzyme, indicating that this alteration has no effect on the transition state for phosphoryl transfer from pNPP.

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