Distinguishing the specificities of closely related proteases: Role of P3 in substrate and inhibitor discrimination between tissue-type plasminogen activator and urokinase

Song Hua Ke, Gary S. Coombs, Kathy Tachias, Marc Navre, David R. Corey, Edwin L. Madison

Research output: Contribution to journalArticle

54 Scopus citations

Abstract

Elucidating subtle specificity differences between closely related enzymes is a fundamental challenge for both enzymology and drug design. We have addressed this issue for two intimately related serine proteases, tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), by modifying the technique of substrate phage display to create substrate subtraction libraries. Characterization of individual members of the substrate subtraction library accomplished the rapid, direct identification of small, highly selective substrates for t-PA. Comparison of the amino acid sequences of these selective substrates with the consensus sequence for optimal substrates for t-PA, derived using standard substrate phage display protocols, suggested that the P3 and P4 residues are the primary determinants of the ability of a substrate to discriminate between t- PA and u-PA. Mutagenesis of the P3 and P4 residues of plasminogen activator inhibitor type 1, the primary physiological inhibitor of both t-PA and u-PA, confirmed this prediction and indicated a predominant role for the P3 residue. Appropriate replacement of both the P3 and P4 residues enhanced the t-PA specificity of plasminogen activator inhibitor type 1 by a factor of 600, and mutation of the P3 residue alone increased this selectivity by a factor of 170. These results demonstrate that the combination of substrate phage display and substrate subtraction methods can be used to discover specificity differences between very closely related enzymes and that this information can be utilized to create highly selective inhibitors.

Original languageEnglish (US)
Pages (from-to)16603-16609
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number26
DOIs
StatePublished - Jun 27 1997

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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