The X-ray Crystal Structures of Yersinia Tyrosine Phosphatase with Bound Tungstate and Nitrate

Abstract

X-ray crystal structures of the Yersinia tyrosine phosphatase (PTPase) in complex with tungstate and nitrate have been solved to 2.4-Å resolution. Tetrahedral tungstate, WO 4 2؊ , is a competitive inhibitor of the enzyme and is isosteric with the substrate and product of the catalyzed reaction. Planar nitrate, NO 3 ؊ , is isosteric with the PO 3 moiety of a phosphotransfer transition state. The crystal structures of the Yersinia PTPase with and without ligands, together with biochemical data, permit modeling of key steps along the reaction pathway. These energy-minimized models are consistent with a general acid-catalyzed, in-line displacement of the phosphate moiety to Cys 403 on the enzyme, followed by attack by a nucleophilic water molecule to release orthophosphate. This nucleophilic water molecule is identified in the crystal structure of the nitrate complex. The active site structure of the PTPase is compared to alkaline phosphatase, which employs a similar phosphomonoester hydrolysis mechanism. Both enzymes must stabilize charges at the nucleophile, the PO 3 moiety of the transition state, and the leaving group. Both an associative (bond formation preceding bond cleavage) and a dissociative (bond cleavage preceding bond formation) mechanism were modeled, but a dissociative-like mechanism is favored for steric and chemical reasons. Since nearly all of the 47 invariant or highly conserved residues of the PTPase domain are clustered at the active site, we suggest that the mechanism postulated for the Yersinia enzyme is applicable to all the PTPases.

Visualization of the Cysteinyl-phosphate Intermediate of a Protein-tyrosine Phosphatase by X-ray Crystallography

“…Critical residues appear to be Asp-181 and Gln-262 and one of the buried water molecules, W2. The position of this water molecule is reminiscent of a water molecule (WAT3) observed in a Yersinia PTPnitrate (NO 3 ) complex (28). Nitrate adopts a planar trigonal geometry, mimicking the transition state during a phosphoryl transfer reaction.…”

Characterization of the Net1 Cell Cycle-dependent Regulator of the Cdc14 Phosphatase from Budding Yeast

“…The Net1 binding site has been mapped to a position within the conserved domain of Cdc14 (residues 1-374) which encompasses the entire active site region (residues 279 -291). The structures (4,(42)(43)(44)(45) of several different PTPs reveal that tungstate, a general PTP inhibitor that mimics the transition state, is bound within the active site in a manner similar to the phosphate of substrates. Sodium tungstate prevents Net1 binding in a concentration-dependent manner (Fig.…”

Sodium tungstate modulates ATM function upon DNA damage

“…It can also significantly reduce weight gain and adiposity by increasing energy dissipation and fatty acid oxidation rates in obese rats [13]. The molecular mechanisms of tungstate action remain poorly understood, but available data suggests that they involve the inhibition of phosphatases [14][15][16][17]. This notion has been linked to the capacity of this molecule to regulate translational control pathways [18].…”

“…Tungstate inhibits several types of phosphatases [14][15][16][17], therefore it is reasonable to hypothesize that the inhibition of a protein phosphatase explains the effect of this compound on the modulation of ATM signalling. Protein phosphatase 2A (PP2A) and protein phosphatase 5 (PP5) are two of the main phosphatases involved in ATM dephosphorylation [31,32].…”