title: Visualization of the cysteinyl-phosphate intermediate of a protein-tyrosine phosphatase by x-ray crystallography.
abstract: Protein-tyrosine phosphatases (PTPs) are signal transduction enzymes that catalyze the dephosphorylation of phosphotyrosine residues via the formation of a transient cysteinyl-phosphate intermediate. The mechanism of hydrolysis of this intermediate has been examined by generating a Gln-262 --> Ala mutant of PTP1B, which allows the accumulation and trapping of the intermediate within a PTP1B crystal. The structure of the intermediate at 2.5-A resolution reveals that a conformationally flexible loop (the WPD loop) is closed over the entrance to the catalytic site, sequestering the phosphocysteine intermediate and catalytic site water molecules and preventing nonspecific phosphoryltransfer reactions to extraneous phosphoryl acceptors. One of the catalytic site water molecules, the likely nucleophile, forms a hydrogen bond to the putative catalytic base, Asp-181. In the wild-type enzyme, the nucleophilic water molecule would be coordinated by the side chain of Gln-262. In combination with our previous structural data, we can now visualize each of the reaction steps of the PTP catalytic pathway. The hydrolysis of the cysteinyl-phosphate intermediate of PTPs is reminiscent of GTP hydrolysis by the GTPases, in that both families of enzymes utilize an invariant Gln residue to coordinate the attacking nucleophilic water molecule.
authors.affiliation: Laboratory of Molecular Biophysics, University of Oxford, Rex Richards Building, South Parks Road, Oxford OX1 3QU, United Kingdom.
authors.forename: A D
authors.initials: AD
authors.lastname: Pannifer
authors.forename: A J
authors.initials: AJ
authors.lastname: Flint
authors.forename: N K
authors.initials: NK
authors.lastname: Tonks
authors.forename: D
authors.initials: D
authors.lastname: Barford
mesh_terms: D018360:Crystallography, X-Ray; D003545:Cysteine / Q000031:analogs & derivatives / Q000737:chemistry*; D006860:Hydrogen Bonding; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry*
chemical_list: D017027:Protein Tyrosine Phosphatases; D003545:Cysteine