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

The aim of this study was to define the structural elements that determine the differences in substrate recognition capacity of two protein-tyrosine phosphatases (PTPs), PTP1B and PTP alpha, both suggested to be negative regulators of insulin signaling. Since the Ac-DADE(pY)L-NH2 peptide is well recognized by PTP1B, but less efficiently by PTP alpha, it was chosen as a tool for these analyses, C alpha regiovariation analyses and primary sequence alignments indicate that residues 47, 48, 258, and 259 (PTP1B numbering) define a selectivity-determining region. By analyzing a set of DADE(pY)L analogs with a series of PTP mutants in which these four residues were exchanged between PTP1B and PTP alpha, either in combination or alone, we here demonstrate 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) with a glycine almost turns PTP alpha into a PTP1B-like enzyme. By using a novel set of PTP inhibitors and x-ray crystallography, we further provide evidence that Gln(259) in PTP alpha plays a dual role leading to restricted substrate recognition (directly via steric hindrance) and reduced catalytic activity (indirectly via Gln(262)). Both effects may indicate that PTPa: regulates highly selective signal transduction processes.