The dual-specificity protein phosphatases have recently been shown to act as key regulators of mitogenic signaling pathways as well as of the cell cycle process. The are unusual catalysts in that they can utilize protein substrates containing phosphotyrosine as well as phosphoserine/threonine. The dual-specificity phosphatases and the protein tyrosine phosphatases (PTPase) share the active site motif (H/V)C(X)5R(S/T) but display little amino acid sequence identity outside of the active site. Although the dual-specificity phosphatases and the PTPases appear to bring about phosphate monoester hydrolysis through a similar mechanism, there is very limited information about the structural features that control the substrate specificity for the two groups of enzymes. As a first step in the development of selective dual- specificity phosphatase inhibitors, we have examined the active site substrate specificity of the human dual-specificity phosphatase, VHR [for VH1-Related; Ishibashi et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12170- 12174]. Like the tyrosine-specific PTP1, VHR also preferentially catalyzes the hydrolysis of aromatic phosphates. However, we demonstrate herein that relatively modest changes in the substitution patterns on the phosphorylated aromatic nucleus generates dramatic, and differential, swings in substrate selectivity for VHR and PTP1. For example, VHR appears to be significantly more accommodating than PTP1 toward sterically-demanding substrates. Thus, the active site specificity of these two protein phosphatases is decidedly dissimilar. In addition, we have also identified several low molecular weight compounds that are more efficient substrates than the most potent peptidic substrates ever reported for VHR. Finally, we have shown that the Michaelis constants exhibited by these substrates are accurate assessments of enzyme affinity. Consequently, it should be possible to develop phosphatase- selective inhibitors based upon the distinct substrate specificities of these enzymes.
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