Assessment of protein-tyrosine phosphatase 1B substrate specificity using "inverse alanine scanning"

An "inverse alanine scanning" peptide library approach has been developed t o assess the substrate specificity of protein-tyrosine phosphatases (PTPase s). In this method each Ala moiety in the parent peptide, Ac-AAAApYAAAA-NH2 , is separately and sequentially replaced by the 19 non-Ala amino acids to generate a library of 158 well defined peptides. The relatively small numbe r of peptides allows the acquisition of explicit kinetic data for all libra ry members, thereby furnishing information about the contribution of indivi dual amino acids with respect to substrate properties. The approach was app lied to protein-tyrosine phosphatase 1B (PTP1B) as a first example, and the highly potent peptide substrate Ac-ELEFpYMDYE-NH2 (k(cat)/K-m 2.2 +/- 0.05 x 10(7) m(-1) s(-1)) has been identified. More importantly, several hereto fore unknown features of the sub strate specificity of PTP1B were revealed. This includes the ability of PTP1B to accommodate acidic, aromatic, and hy drophobic residues at the -1 position, a strong nonpreference for Lys and A rg residues in any position, and the first evidence that residues well beyo nd the +1 position contribute to substrate efficacy.