Protein-tyrosine phosphatases (PTPases) feature an essential nucleophilic thiol group which attacks the phosphorus atom in a substrate. A single S to O atom substitution in the nucleophile (via Cys to Ser mutation) renders PTPases catalytically inactive. We suggest that the lack of activity in the Cys to Ser mutant may be caused by structural and/or conformational perturbations in the active site. Yersinia PTPase contains a single tryptophan residue, Trp354, which is invariant among all PTPases and is located in the vicinity of the active site nucleophile Cys403. Thus, Trp354 serves as an intrinsic probe of the PTPase active site conformation. We show that although C403S displays a nearly identical circular dichroism spectrum to that of the wild type enzyme, its ultraviolet spectrum in the region attributed to Trio is significantly different from that of the wild-type enzyme. In addition, the intrinsic fluorescence intensity of C403S is enhanced 3-fold and exhibits different ionic strength dependency from that of the wild-type enzyme. Trp354 also has different accessibilities to quenchers in the wild-type and the C403S mutant PTPases. Furthermore, unfolding experiments demonstrate that the structure of C403S is significantly less stable than the wild-type PTPase and displays a different sensitivity to urea and guanidine hydrochloride. Finally, binding of tungstate enhances the fluorescence of the wild-type Yersinia PTPase with a K(d) of 55 μM, whereas binding of tungstate quenches the fluorescence of the C403S mutant with a K(d) of 690 μM. Collectively, these results indicate that the single sulfur to oxygen change in the active site nucleophile leads to substantial structural/conformational and functional alterations in the Yersinia PTPase.
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