Altering the nucleophile specificity of a protein-tyrosine phosphatase- catalyzed reaction: Probing the function of the invariant glutamine residues

Yu Zhao, Li Wu, Seong J. Noh, Kun Liang Guan, Zhong Yin Zhang

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

Protein-tyrosine phosphatases (PTPases) catalysis involves a cysteinyl phosphate intermediate, in which the phosphoryl group cannot be transferred to nucleophiles other than water. The dual specificity phosphatases and the low molecular weight phosphatases utilize the same chemical mechanism for catalysis and contain the same (H/V)C(X)5R(S/T) signature motif present in PTPases. Interestingly, the latter two groups of phosphatases do catalyze phosphoryl transfers to alcohols in addition to water. Unique to the PTPase family are two invariant Gln residues which are located at the active site. Mutations at Gln-446 (and to a much smaller extent Gln-450) to Ala, Asn, or Met (but not Glu) residues disrupt a bifurcated hydrogen bond between the side chain of Gln-446 and the nucleophilic water and confer phosphotransferase activity to the Yersinia PTPase. Thus, the conserved Gln- 446 residue is responsible for maintaining PTPases' strict hydrolytic activity and for preventing the PTPases from acting as kinases to phosphorylate undesirable substrates. This explains why phosphoryl transfer from the phosphoenzyme intermediate in PTPases can only occur to water and not to other nucleophilic acceptors. Detailed kinetic analyses also suggest roles for Gln-446 and Gln-450 in PTPase catalysis. Although Gln-446 is not essential for the phosphoenzyme formation step, it plays an important role during the hydrolysis of the intermediate by sequestering and positioning the nucleophilic water in the active site for an in-line attack on the phosphorus atom of the cysteinyl phosphate intermediate. Gln-450 interacts through a bound water molecule with the phosphoryl moiety and may play a role for the precise alignment of active site residues, which are important for substrate binding and transition state stabilization for both of the chemical steps.

Original languageEnglish (US)
Pages (from-to)5484-5492
Number of pages9
JournalJournal of Biological Chemistry
Volume273
Issue number10
DOIs
StatePublished - Mar 6 1998

Fingerprint

Nucleophiles
Protein Tyrosine Phosphatases
Glutamine
Water
Catalysis
Catalytic Domain
Phosphoric Monoester Hydrolases
Phosphotransferases
Dual-Specificity Phosphatases
Phosphates
Yersinia
Substrates
Phosphorus
Hydrogen
Hydrolysis
Hydrogen bonds
Stabilization
Molecular Weight
Molecular weight
Alcohols

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Altering the nucleophile specificity of a protein-tyrosine phosphatase- catalyzed reaction : Probing the function of the invariant glutamine residues. / Zhao, Yu; Wu, Li; Noh, Seong J.; Guan, Kun Liang; Zhang, Zhong Yin.

In: Journal of Biological Chemistry, Vol. 273, No. 10, 06.03.1998, p. 5484-5492.

Research output: Contribution to journalArticle

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abstract = "Protein-tyrosine phosphatases (PTPases) catalysis involves a cysteinyl phosphate intermediate, in which the phosphoryl group cannot be transferred to nucleophiles other than water. The dual specificity phosphatases and the low molecular weight phosphatases utilize the same chemical mechanism for catalysis and contain the same (H/V)C(X)5R(S/T) signature motif present in PTPases. Interestingly, the latter two groups of phosphatases do catalyze phosphoryl transfers to alcohols in addition to water. Unique to the PTPase family are two invariant Gln residues which are located at the active site. Mutations at Gln-446 (and to a much smaller extent Gln-450) to Ala, Asn, or Met (but not Glu) residues disrupt a bifurcated hydrogen bond between the side chain of Gln-446 and the nucleophilic water and confer phosphotransferase activity to the Yersinia PTPase. Thus, the conserved Gln- 446 residue is responsible for maintaining PTPases' strict hydrolytic activity and for preventing the PTPases from acting as kinases to phosphorylate undesirable substrates. This explains why phosphoryl transfer from the phosphoenzyme intermediate in PTPases can only occur to water and not to other nucleophilic acceptors. Detailed kinetic analyses also suggest roles for Gln-446 and Gln-450 in PTPase catalysis. Although Gln-446 is not essential for the phosphoenzyme formation step, it plays an important role during the hydrolysis of the intermediate by sequestering and positioning the nucleophilic water in the active site for an in-line attack on the phosphorus atom of the cysteinyl phosphate intermediate. Gln-450 interacts through a bound water molecule with the phosphoryl moiety and may play a role for the precise alignment of active site residues, which are important for substrate binding and transition state stabilization for both of the chemical steps.",
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