Phospho-azatyrosine, a less effective protein-tyrosine phosphatase substrate than phosphotyrosine

Azatyrosine (AzaTyr, 4) is a natural product isolated from Streptomyces chi hanesis, whose structure is characterized by a nitrogen atom in the aryl ri ng of a tyrosyl residue. This seemingly minor modification to the tyrosyl r esidue results in profound physiological effects, as AzaTyr has been shown to promote permanent reversion of ras-dependent transformed cells to the no rmal phenotype in culture and to inhibit chemical induction of carcinogenes is in transgenic mice bearing oncogenic human ras. The mechanisms underlyin g these effects are not known, however ras-pathways involve an intricate ba lance between both protein-tyrosine kinases (PTKs) and protein-tyrosine pho sphatases (PTPs). The present study was undertaken to examine the general u tility of AzaTyr as a structural motif for PTP inhibitor design by examinin g the phospho-azatyrosine (pAzaTyr)-containing peptide Ac-Asp-Ala-Asp-Glu-p AzaTyr-Leu-amide (8) in a PTP1 enzyme system. Kinetic analysis indicated th at 8 binds with a K-m value of 210 muM and a catalytic turnover rate, k(cat ) of 52 s(-1). This represents a greater than 50-fold reduction in binding affinity relative to the parent phosphotyrosine-containing peptide, indicat ing that the aryl nitrogen adversely affects binding affinity. The much low er PTP affinity of the pAzaTyr-containing peptide reduces the potential uti lity of the AzaTyr pharmacophore for PTP inhibitor design. These results ar e discussed from the point of view that incorporation of AzaTyr residues in to proteins could result in perturbation of protein-tyrosine phosphorylatio n/dephosphorylation cascades that control signal transduction processes, in cluding,as-dependent pathways. Published by Elsevier Science Ltd.