Transition state and rate-limiting step of the reaction catalyzed by the human dual-specificity phosphatase, VHR

Zhong-Yin Zhang, L. Wu, L. Chen

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

The dual-specificity phosphatases are unusual catalysts in that they can utilize protein substrates containing phosphotyrosine as well as phosphoserine/threonine. The dual-specificity phosphatases and the protein- tyrosine phosphatases (PTPases) share the active site motif (H/V)C(X)5R(S/T), but display little amino acid sequence identity outside of the active site. Although the dual-specificity phosphatases and the PTPases appear to bring about phosphate monoester hydrolysis through a similar mechanism, it is not clear what causes the difference in the active-site specificity between the two groups of enzymes. In this paper, we show that the human dual-specificity phosphatase, VHR [for VH1-Related; Ishibashi et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12170-12174], is rather promiscuous toward small phosphate monoesters (including both aryl and alkyl phosphates of primary alcohols) with effectively identical k(cat)/K(m) and k(cat) values while the pK(a) values of the leaving groups (phenols or alcohols) varied from 7 to 16. Linear free-energy relationship analysis of k(cat) and k(cat)/K(m) of the enzyme-catalyzed hydrolysis reaction suggests that a uniform mechanism is utilized for both the aryl and alkyl substrates. The very small dependency of k(cat)/K(m) on the leaving group pK(a) can be accounted for by the protonation of the leaving group. Pre-steady-state burst kinetic analysis of the VHR-catalyzed hydrolysis of p-nitrophenyl phosphate provides direct kinetic evidence for the involvement of a phosphoenzyme intermediate in the dual specificity phosphatase-catalyzed reaction. The rate-limiting step for the VHR-catalyzed hydrolysis of p-nitrophenyl phosphate corresponds to the decomposition of the phosphoenzyme intermediate. Results from kinetic solvent isotope effects on the formation (k(H2O)/k(D2O) = 0.52) and the breakdown (k(H2O)/k(D2O) = 1.15) of the phosphoenzyme intermediate are consistent with a highly dissociative metaphosphate-like transition state for both steps, where bond formation to the incoming nucleophile is minimal and bond breaking between phosphorus and the leaving group is substantial. To promote and stabilize the dissociative transition state, the proton from the putative general acid Asp92 is largely transferred to the bridge oxygen atom in the transition state.

Original languageEnglish (US)
Pages (from-to)16088-16096
Number of pages9
JournalBiochemistry
Volume34
Issue number49
DOIs
StatePublished - 1995
Externally publishedYes

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Dual-Specificity Phosphatases
Hydrolysis
Catalytic Domain
Protein Tyrosine Phosphatases
Phosphates
Kinetics
Alcohols
Phosphoserine
Nucleophiles
Phosphotyrosine
Phenols
Protonation
Substrates
Enzymes
Threonine
Isotopes
Phosphorus
Free energy
Protons
Amino Acid Sequence

ASJC Scopus subject areas

  • Biochemistry

Cite this

Transition state and rate-limiting step of the reaction catalyzed by the human dual-specificity phosphatase, VHR. / Zhang, Zhong-Yin; Wu, L.; Chen, L.

In: Biochemistry, Vol. 34, No. 49, 1995, p. 16088-16096.

Research output: Contribution to journalArticle

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abstract = "The dual-specificity phosphatases are unusual catalysts in that they can utilize protein substrates containing phosphotyrosine as well as phosphoserine/threonine. The dual-specificity phosphatases and the protein- tyrosine phosphatases (PTPases) share the active site motif (H/V)C(X)5R(S/T), but display little amino acid sequence identity outside of the active site. Although the dual-specificity phosphatases and the PTPases appear to bring about phosphate monoester hydrolysis through a similar mechanism, it is not clear what causes the difference in the active-site specificity between the two groups of enzymes. In this paper, we show that the human dual-specificity phosphatase, VHR [for VH1-Related; Ishibashi et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12170-12174], is rather promiscuous toward small phosphate monoesters (including both aryl and alkyl phosphates of primary alcohols) with effectively identical k(cat)/K(m) and k(cat) values while the pK(a) values of the leaving groups (phenols or alcohols) varied from 7 to 16. Linear free-energy relationship analysis of k(cat) and k(cat)/K(m) of the enzyme-catalyzed hydrolysis reaction suggests that a uniform mechanism is utilized for both the aryl and alkyl substrates. The very small dependency of k(cat)/K(m) on the leaving group pK(a) can be accounted for by the protonation of the leaving group. Pre-steady-state burst kinetic analysis of the VHR-catalyzed hydrolysis of p-nitrophenyl phosphate provides direct kinetic evidence for the involvement of a phosphoenzyme intermediate in the dual specificity phosphatase-catalyzed reaction. The rate-limiting step for the VHR-catalyzed hydrolysis of p-nitrophenyl phosphate corresponds to the decomposition of the phosphoenzyme intermediate. Results from kinetic solvent isotope effects on the formation (k(H2O)/k(D2O) = 0.52) and the breakdown (k(H2O)/k(D2O) = 1.15) of the phosphoenzyme intermediate are consistent with a highly dissociative metaphosphate-like transition state for both steps, where bond formation to the incoming nucleophile is minimal and bond breaking between phosphorus and the leaving group is substantial. To promote and stabilize the dissociative transition state, the proton from the putative general acid Asp92 is largely transferred to the bridge oxygen atom in the transition state.",
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T1 - Transition state and rate-limiting step of the reaction catalyzed by the human dual-specificity phosphatase, VHR

AU - Zhang, Zhong-Yin

AU - Wu, L.

AU - Chen, L.

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N2 - The dual-specificity phosphatases are unusual catalysts in that they can utilize protein substrates containing phosphotyrosine as well as phosphoserine/threonine. The dual-specificity phosphatases and the protein- tyrosine phosphatases (PTPases) share the active site motif (H/V)C(X)5R(S/T), but display little amino acid sequence identity outside of the active site. Although the dual-specificity phosphatases and the PTPases appear to bring about phosphate monoester hydrolysis through a similar mechanism, it is not clear what causes the difference in the active-site specificity between the two groups of enzymes. In this paper, we show that the human dual-specificity phosphatase, VHR [for VH1-Related; Ishibashi et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12170-12174], is rather promiscuous toward small phosphate monoesters (including both aryl and alkyl phosphates of primary alcohols) with effectively identical k(cat)/K(m) and k(cat) values while the pK(a) values of the leaving groups (phenols or alcohols) varied from 7 to 16. Linear free-energy relationship analysis of k(cat) and k(cat)/K(m) of the enzyme-catalyzed hydrolysis reaction suggests that a uniform mechanism is utilized for both the aryl and alkyl substrates. The very small dependency of k(cat)/K(m) on the leaving group pK(a) can be accounted for by the protonation of the leaving group. Pre-steady-state burst kinetic analysis of the VHR-catalyzed hydrolysis of p-nitrophenyl phosphate provides direct kinetic evidence for the involvement of a phosphoenzyme intermediate in the dual specificity phosphatase-catalyzed reaction. The rate-limiting step for the VHR-catalyzed hydrolysis of p-nitrophenyl phosphate corresponds to the decomposition of the phosphoenzyme intermediate. Results from kinetic solvent isotope effects on the formation (k(H2O)/k(D2O) = 0.52) and the breakdown (k(H2O)/k(D2O) = 1.15) of the phosphoenzyme intermediate are consistent with a highly dissociative metaphosphate-like transition state for both steps, where bond formation to the incoming nucleophile is minimal and bond breaking between phosphorus and the leaving group is substantial. To promote and stabilize the dissociative transition state, the proton from the putative general acid Asp92 is largely transferred to the bridge oxygen atom in the transition state.

AB - The dual-specificity phosphatases are unusual catalysts in that they can utilize protein substrates containing phosphotyrosine as well as phosphoserine/threonine. The dual-specificity phosphatases and the protein- tyrosine phosphatases (PTPases) share the active site motif (H/V)C(X)5R(S/T), but display little amino acid sequence identity outside of the active site. Although the dual-specificity phosphatases and the PTPases appear to bring about phosphate monoester hydrolysis through a similar mechanism, it is not clear what causes the difference in the active-site specificity between the two groups of enzymes. In this paper, we show that the human dual-specificity phosphatase, VHR [for VH1-Related; Ishibashi et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12170-12174], is rather promiscuous toward small phosphate monoesters (including both aryl and alkyl phosphates of primary alcohols) with effectively identical k(cat)/K(m) and k(cat) values while the pK(a) values of the leaving groups (phenols or alcohols) varied from 7 to 16. Linear free-energy relationship analysis of k(cat) and k(cat)/K(m) of the enzyme-catalyzed hydrolysis reaction suggests that a uniform mechanism is utilized for both the aryl and alkyl substrates. The very small dependency of k(cat)/K(m) on the leaving group pK(a) can be accounted for by the protonation of the leaving group. Pre-steady-state burst kinetic analysis of the VHR-catalyzed hydrolysis of p-nitrophenyl phosphate provides direct kinetic evidence for the involvement of a phosphoenzyme intermediate in the dual specificity phosphatase-catalyzed reaction. The rate-limiting step for the VHR-catalyzed hydrolysis of p-nitrophenyl phosphate corresponds to the decomposition of the phosphoenzyme intermediate. Results from kinetic solvent isotope effects on the formation (k(H2O)/k(D2O) = 0.52) and the breakdown (k(H2O)/k(D2O) = 1.15) of the phosphoenzyme intermediate are consistent with a highly dissociative metaphosphate-like transition state for both steps, where bond formation to the incoming nucleophile is minimal and bond breaking between phosphorus and the leaving group is substantial. To promote and stabilize the dissociative transition state, the proton from the putative general acid Asp92 is largely transferred to the bridge oxygen atom in the transition state.

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