Sequence requirements in the reactive-center loop of plasminogen-activator inhibitor-1 for recognition of plasminogen activators.
Plasminogen activator inhibitor-1 (PAI-1) is a member of the serpin superfamily of proteins and is the fast acting inhibitor of both urinary plasminogen activator and tissue-type plasminogen activator. We have assessed the functional significance of reactive center residues on the carboxy-terminal side of the cleavage site of recombinant human PAI-1. Using site-directed mutagenesis, the P1'-P5' residues (P1' is the first residue on the carboxy-terminal side of the protease cleavage site) of the wild-type PAI-1 reactive center sequence were replaced with the corresponding sequences of plasminogen activator inhibitor-2, antithrombin, alpha 2-antiplasmin and protease nexin I. Rate constants of inhibition of the serine proteases urinary plasminogen activator, tissue-type plasminogen activator, plasmin and thrombin by the variants were determined. The results suggest a crucial role for both reactive center length and sequence in the inhibition of plasminogen activators by PAI-1. Analysis of substitutions at positions P4' and P5' both confirms and extends our previous work demonstrating a favorable electrostatic interaction between these residues and tissue-type plasminogen activator. None of the variants show dramatic increases in the rate constants of inhibition of other serine proteases, suggesting that these residues alone are not sufficient to confer protease specificity on PAI-1. Apparently, the determinants of the rapid inhibitory specificity of PAI-1 are localized to the P1'-P5' region of the reactive center and these residues act synergistically to produce the exquisite specificity of PAI-1 for plasminogen activators.
Tucker
H M
HM
Department of Biochemistry, University of Texas, Southwestern Medical Center, Dallas, USA.
Gerard
R D
RD
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics* / Q000378:metabolism; D010960:Plasminogen Activators / Q000378:metabolism*
D017395:Plasminogen Activator Inhibitor 1; D010960:Plasminogen Activators