Residue 259 is a key determinant of substrate specificity of protein-tyrosine phosphatases 1B and alpha

The aim of this study was to define the structural elements that determine the differences in substrate recognition capacity of two protein-tyrosine p hosphatases (PTPs), PTP1B and PTP alpha, both suggested to be negative regu lators of insulin signaling. Since the Ac-DADE(pY)L-NH2 peptide is well rec ognized by PTP1B, but less efficiently by PTP alpha, it was chosen as a too l for these analyses, C alpha regiovariation analyses and primary sequence alignments indicate that residues 47, 48, 258, and 259 (PTP1B numbering) de fine a selectivity-determining region. By analyzing a set of DADE(pY)L anal ogs with a series of PTP mutants in which these four residues were exchange d between PTP1B and PTP alpha, either in combination or alone, we here demo nstrate that the key selectivity-determining residue is 259, In PTP alpha, this residue is a glutamine causing steric hindrance and in PTP1B a glycine allowing broad substrate recognition. Significantly, replacing Gln(259) wi th a glycine almost turns PTP alpha into a PTP1B-like enzyme. By using a no vel set of PTP inhibitors and x-ray crystallography, we further provide evi dence that Gln(259) in PTP alpha plays a dual role leading to restricted su bstrate recognition (directly via steric hindrance) and reduced catalytic a ctivity (indirectly via Gln(262)). Both effects may indicate that PTPa: reg ulates highly selective signal transduction processes.