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. 2000 Jan 28;275(4):2265-8.
doi: 10.1074/jbc.275.4.2265.

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

S W Vetter  1 , Y F Keng, D S Lawrence, Z Y Zhang
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Assessment of protein-tyrosine phosphatase 1B substrate specificity using "inverse alanine scanning"

S W Vetter et al. J Biol Chem. .
Free article

Abstract

An "inverse alanine scanning" peptide library approach has been developed to assess the substrate specificity of protein-tyrosine phosphatases (PTPases). In this method each Ala moiety in the parent peptide, Ac-AAAApYAAAA-NH(2), is separately and sequentially replaced by the 19 non-Ala amino acids to generate a library of 153 well defined peptides. The relatively small number of peptides allows the acquisition of explicit kinetic data for all library members, thereby furnishing information about the contribution of individual amino acids with respect to substrate properties. The approach was applied to protein-tyrosine phosphatase 1B (PTP1B) as a first example, and the highly potent peptide substrate Ac-ELEFpYMDYE-NH(2) (k(cat)/K(m) 2.2 +/- 0.05 x 10(7) M(-1) s(-1)) has been identified. More importantly, several heretofore unknown features of the substrate specificity of PTP1B were revealed. This includes the ability of PTP1B to accommodate acidic, aromatic, and hydrophobic residues at the -1 position, a strong nonpreference for Lys and Arg residues in any position, and the first evidence that residues well beyond the +1 position contribute to substrate efficacy.

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