The active site specificity of the Yersinia protein-tyrosine phosphatase

Yersinia protein-tyrosine phosphatase substrates have been synthesized employing an expedient methodology that incorporates phosphorylated non amino acid residues into an active site-directed peptide. While the peptidic portion of these compounds serves an enzyme targeting role, the nonpeptidic component provides a critical assessment of the range of functionality that can be accommodated within the active site region. We have found that the Yersinia phosphatase hydrolyzes both L- and D-stereoisomers of phosphotyrosine in active site-directed peptides, with the former serving as a 10-fold more efficient substrate than the latter. In addition, this enzyme catalyzes the hydrolysis of a variety of aromatic and aliphatic phosphates. Indeed, a peptide bearing the achiral phosphotyrosine analog, phosphotyramine, is not only the most efficient substrate described in this study, it is also one of the most efficient substrates ever reported for the Yersinia phosphatase. Straight chain peptide-bound aliphatic phosphates of the general structure, (Glu)(4)-NH-(CH2)(n)-OPO32- (n = 2-8), are also hydrolyzed, where the most efficient substrate contains seven methylene groups. Finally, a comparison of the substrate efficacy of the peptide-bound species with that of the corresponding non-peptidic analogs, reveals that the peptide component enhances k(cat)/K-m by up to nearly 3 orders of magnitude.