VISUALIZATION OF THE CYSTEINYL-PHOSPHATE INTERMEDIATE OF A PROTEIN-TYROSINE-PHOSPHATASE BY X-RAY CRYSTALLOGRAPHY

Protein-tyrosine phosphatases (PTPs) are signal transduction enzymes t hat catalyze the dephosphorylation of phosphotyrosine residues via the formation of a transient cysteinyl-phosphate intermediate, The mechan ism 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-Angstrom resolution reveals that a conformatio nally flexible loop (the WPD loop) is closed over the entrance to the catalytic site, sequestering the phosphocysteine intermediate and cata lytic site water molecules and preventing nonspecific phosphoryltransf er reactions to extraneous phosphoryl accepters. 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 nu cleophilic water molecule would be coordinated by the side chain of Gl n-262, In combination with our previous structural data, we can now vi sualize each of the reaction steps of the PTP catalytic pathway. The h ydrolysis of the cysteinyl-phosphate intermediate of PTPs is reminisce nt of GTP hydrolysis by the GTPases, in that both families of enzymes utilize an invariant Gin residue to coordinate the attacking nucleophi lic water molecule.