Amino acid residues that affect interaction of tissue-type plasminogen activator with plasminogen activator inhibitor 1.

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Abstract
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 allthree 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 .fwdarw. Glu and Arg-299 .fwdarw. Glu) are partially resistant to inhibition by PAI-1 and associate with the serpin at intermediate rates. Other point mutations (Lys-296 .fwdarw. Glu, His-297 .fwdarw. Glu, and Pro-301 .fwdarw. 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.

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