ABSTRACT
Protein tyrosine phosphatase (PTP) targeted, peptide based chemical probes are valuable tools for studying this important family of enzymes, despite the inherent difficulty of developing peptides targeted towards an individual PTP. Here, we have taken a rational approach to designing a SHP-2 targeted, fluorogenic peptide substrate based on information about the potential biological substrates of SHP-2. The fluorogenic, phosphotyrosine mimetic phosphocoumaryl aminopropionic acid (pCAP) provides a facile readout for monitoring PTP activity. By optimizing the amino acids surrounding the pCAP residue, we obtained a substrate with the sequence Ac-DDPI-pCAP-DVLD-NH2 and optimized kinetic parameters (kcatâ¯=â¯0.059⯱â¯0.008â¯s-1, Kmâ¯=â¯220⯱â¯50⯵M, kcat/Km of 270â¯M-1s-1). In comparison, the phosphorylated coumarin moiety alone is an exceedingly poor substrate for SHP-2, with a kcat value of 0.0038⯱â¯0.0003â¯s-1, a Km value of 1100⯱â¯100⯵M and a kcat/Km of 3â¯M-1s-1. Furthermore, this optimized peptide has selectivity for SHP-2 over HePTP, MEG1 and PTPµ. The data presented here demonstrate that PTP-targeted peptide substrates can be obtained by optimizing the sequence of a pCAP containing peptide.
Subject(s)
Peptides/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Amino Acid Sequence , Coumarins/chemistry , Coumarins/metabolism , Drug Design , Humans , Kinetics , Peptides/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Substrate SpecificityABSTRACT
Two resorufin-based substrates for protein tyrosine phosphatase (PTP) activity have been synthesized. These substrates provide sensitive fluorogenic readouts of PTP activity in vitro and in living cells at both acidic and neutral pH. In addition, the presence of the pathogenic bacteria Staphylococcus aureus was detected visually using a colorimetric readout.
