Restoration of serine protease-inhibitor interaction by protein engineering

Edwin L. Madison, Elizabeth J. Goldsmith, Mary Jane H Gething, Joseph F. Sambrook, Robert D. Gerard

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)21423-21426
Number of pages4
JournalJournal of Biological Chemistry
Volume265
Issue number35
StatePublished - Dec 15 1990

Fingerprint

Protein Engineering
Serine Proteinase Inhibitors
Plasminogen
Plasminogen Activator Inhibitor 1
Restoration
Plasminogen Inactivators
Plasminogen Activators
Arginine
Glutamic Acid
Proteins
Fibrinolysin
Endothelial cells
Tissue Plasminogen Activator
Substrates
Enzymes
Mutation
Genes
Chemical activation
Suicide
Plasmas

ASJC Scopus subject areas

  • Biochemistry

Cite this

Madison, E. L., Goldsmith, E. J., Gething, M. J. H., Sambrook, J. F., & Gerard, R. D. (1990). Restoration of serine protease-inhibitor interaction by protein engineering. Journal of Biological Chemistry, 265(35), 21423-21426.

Restoration of serine protease-inhibitor interaction by protein engineering. / Madison, Edwin L.; Goldsmith, Elizabeth J.; Gething, Mary Jane H; Sambrook, Joseph F.; Gerard, Robert D.

In: Journal of Biological Chemistry, Vol. 265, No. 35, 15.12.1990, p. 21423-21426.

Research output: Contribution to journalArticle

Madison, EL, Goldsmith, EJ, Gething, MJH, Sambrook, JF & Gerard, RD 1990, 'Restoration of serine protease-inhibitor interaction by protein engineering', Journal of Biological Chemistry, vol. 265, no. 35, pp. 21423-21426.
Madison EL, Goldsmith EJ, Gething MJH, Sambrook JF, Gerard RD. Restoration of serine protease-inhibitor interaction by protein engineering. Journal of Biological Chemistry. 1990 Dec 15;265(35):21423-21426.
Madison, Edwin L. ; Goldsmith, Elizabeth J. ; Gething, Mary Jane H ; Sambrook, Joseph F. ; Gerard, Robert D. / Restoration of serine protease-inhibitor interaction by protein engineering. In: Journal of Biological Chemistry. 1990 ; Vol. 265, No. 35. pp. 21423-21426.
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