Mechanistic studies of protein tyrosine phosphatases

Abstract

Protein-tyrosine phosphatases (PTPs) consist of a superfamily of enzymes that include classical tyrosine-specific PTPs, the dual specificity phosphatases and the low molecular weight phosphatases. PTPs catalyze the hydrolysis of phosphate monoesters by a two-step mechanism through a phosphoenzyme intermediate (E-P). The transition state for the PTP-catalyzed E-P dephosphorylation step was probed by studying the effect of changing the basicity on the reactivity of a series of alcohols towards the E-P. The Bronsted betanuc value of the PTP-catalyzed E-P dephosphorylation indicates that the transition state is highly dissociative and similar to that in an uncatalyzed solution reaction. Furthermore, elimination of the conserved hydroxyl group in the PTP signature motif impairs the E-P dephosphorylation step and renders its transition state less dissociative, suggesting an important role of the hydroxyl group in the PTP active site in stabilizing the dissociative transition state.;The classical PTPs' catalysis involves a cysteinyl phosphate intermediate (E-P), the phosphoryl group of which cannot be transferred to nucleophiles other than water. Unique to the classical PTP family are two invariant Gin residues (Gln446 and Gln450 in Yersinia PTP). Mutations at Gln446 (and to a much smaller extent Gln450) to Ala, Asn or Met (but not Glu) residues disrupt a bifurcated hydrogen bond between the side chain of Gln446 and the nucleophilic water and confer phosphotransferase activity to the Yersinia PTP. This suggests that the conserved Gln446 residue is responsible for maintaining PTPs' strict hydrolytic activity. Gln446 does not appear to be essential for the phosphoenzyme formation step; however, it plays an important role during the hydrolysis of the intermediate by correctly positioning the nucleophilic water in the active site for an in-line attack on the phosphorus atom of the E-P. Gln450 interacts through a bound water molecule with the phosphoryl moiety and may play a role for the precise alignment of active site residues, which are important for substrate binding and transition state stabilization for both chemical steps.;MAP kinase phosphatase-3 (MKP3) is a dual specificity phosphatase that specifically inactivates one subfamily of MAP kinases, the extracellular signal-regulated kinases (ERK1/2). To gain insight into the mechanism of PTP-catalyzed dephosphorylation of its physiological substrate, the MKP3-catalyzed dephosphorylation of the phosphorylated ERK2 protein was analyzed. The results reveal an ordered, distributive mechanism, in which MKP3 hydrolyzes pTyr185 first, dissociates from ERK2/pT183 and then a second MKP3 molecule binds ERK2/pT 183 and hydrolyzes pThr183 to generate fully dephosphorylated ERK2. The bisphosphorylated ERK2 is a highly specific substrate for MKP3 with a kcat/Km more than six orders of magnitude higher than those for an ERK2-derived phosphopeptide. The strikingly high substrate specificity displayed by MKP3 may be a consequence of a substrate-induced fit activation mechanism. The results also indicate that the role of the N-terminal domain is to increase the "effective" concentration of ERK2 near the active site of MKP3. Overall, this work provides a solid framework upon which further studies can be based to reveal the molecular basis of substrate specificity of MAP kinases inactivation by MAP kinase phosphatases.