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.
Highlights
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 interaction between Asp181 and the apical vanadate oxygen atom is possible as a result of the closed conformation of the WPD loop, similar to that observed in the PTP1B-phosphopeptide complex [7]
Model building shows that if W2 were to form hydrogen bonds with Gln-262, as observed for the apical oxygen atom of the PTP1B-vanadate complex, the water molecule would be ideally positioned for nucleophilic in-line attack on the cysteinyl-phosphate group, being colinear with the P–S␥ bond and 3.5 Å from the phosphorus atom of the cysteinylphosphate
Summary
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 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. The formation of phosphoryl-enzyme intermediates is an essential component of numerous enzymatic mechanisms that involve phosphoryl-transfer reactions, for example the dephosphorylation of Tyr(P) residues catalyzed by protein-tyrosine phosphatases [1]. The Arg-221 side chain reorients to optimize salt bridge interactions with the phosphate bound to the catalytic site This shift is coupled to motion of the WPD loop via a hydrogen bond between NH2 of Arg-221 and the carbonyl oxygen of Pro-180 and hydrophobic interactions between the aliphatic moiety of Arg-221 and the side chain of Trp-179. The structure of a PTP1B1⁄7WO4 complex suggests that after hydrolysis of the phosphoryl-enzyme interme-
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