Amino acid residues that affect interaction of tissue-type plasminogen activator with plasminogen activator inhibitor 1.
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.
Madison
E L
EL
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
Goldsmith
E J
EJ
Gerard
R D
RD
Gething
M J
MJ
Sambrook
J F
JF
Bassel-Duby
R S
RS
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D005342:Fibrinolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D015849:Plasminogen Inactivators / Q000378:metabolism*; D011485:Protein Binding; D012689:Sequence Homology, Nucleic Acid; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
D015345:Oligonucleotide Probes; D015849:Plasminogen Inactivators; D010959:Tissue Plasminogen Activator