Details

Autor(en) / Beteiligte

• Madison, Edwin L.
• Goldsmith, Elizabeth J.
• Gerard, Robert D.
• Gething, Mary-Jane H.
• Sambrook, Joseph F.
• Bassel-Duby, Rhonda S.

Titel

Amino Acid Residues that Affect Interaction of Tissue-Type Plasminogen Activator with Plasminogen Activator Inhibitor 1

Ist Teil von

• Proceedings of the National Academy of Sciences - PNAS, 1990-05, Vol.87 (9), p.3530-3533

Ort / Verlag

Washington, DC: National Academy of Sciences of the United States of America

Erscheinungsjahr

1990

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• Fibrinolysis is regulated in part by the interaction between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1, a serine protease inhibitor of the serpin family). It is known from our earlier work that deletion of a loop of amino acids (residues 296-302) from the serine protease domain of t-PA suppresses the interaction between the two proteins without altering the reactivity of t-PA towards its substrate, plasminogen. To define more precisely the role of individual residues within this loop, we have used site-directed mutagenesis to replace Lys-296, Arg-298, and Arg-299 with negatively charged glutamic residues. Replacement of all three positively charged amino acids generates a variant of t-PA that associates inefficiently with PAI-1 and is highly resistant to inhibition by the serpin. Two t-PAs with point mutations (Arg-298 → Glu and Arg-299 → Glu) are partially resistant to inhibition by PAI-1 and associate with the serpin at intermediate rates. Other point mutations (Lys-296 → Glu, His-297 → Glu, and Pro-301 → Gly) do not detectably affect the interaction of t-PA with PAI-1. None of these substitutions has a significant effect on the rate of catalysis by t-PA or on the affinity of the enzyme for its substrate, plasminogen. On the basis of these results, we propose a model in which positively charged residues located in a surface loop near the active site of t-PA form ionic bonds with complementary negatively charged residues C-terminal to the reactive center of PAI-1.