title: Restoration of serine protease-inhibitor interaction by protein engineering.
abstract: Tissue-type plasminogen activator (t-PA) catalyzes the rate-limiting step in the fibrinolytic cascade: conversion of plasminogen to plasmin. Plasma contains several inhibitors of t-PA that limit its activity and prevent systemic activation of plasminogen. The most important of these is endothelial cell plasminogen activator inhibitor (PAI-1), a member of the serine protease inhibitor (serpin) gene family. We have previously demonstrated that mutation of arginine 304 of t-PA to a glutamic acid residue drastically reduces the rate of interaction between the enzyme and its suicide substrate, PAI-1, without affecting the reactivity of the enzyme toward its normal substrate, plasminogen (Madison, E. L., Goldsmith, E. J., Gerard, R.D., Gething, M.J., and Sambrook, J.F. (1989) Nature 339, 721-724). We report here the use of protein modeling to design a compensatory mutation in PAI-1 (glutamic acid 350 to arginine) and create a molecule that rapidly inhibits this "serpin-resistant" variant of t-PA.
authors: Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas 75235-9038.
authors: E L
authors: EL
authors: Madison
authors: E J
authors: EJ
authors: Goldsmith
authors: M J
authors: MJ
authors: Gething
authors: J F
authors: JF
authors: Sambrook
authors: R D
authors: RD
authors: Gerard
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D015849:Plasminogen Inactivators / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering; D013329:Structure-Activity Relationship; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D015849:Plasminogen Inactivators; D010959:Tissue Plasminogen Activator