title: Comparative analysis of the proteinase specificity in wild-type and stabilized plasminogen activator inhibitor-1: evidence for contribution of intramolecular flexibility.
abstract: PAI-1, the physiological inhibitor of tissue-type and urokinase-type plasminogen activator, is a unique member of the serpins as it exists in three distinct conformations: an active inhibitory conformation, a non-inhibitory substrate conformation, and a non-reactive latent conformation. Proline substitution of single residues in the P16-P20 region (situated at the proximal hinge of the reactive site loop) of wild-type PAI-1 (wtPAI-1) and a stabilized PAI-1-variant (PAI-1-stab; N150H, K154T, Q301P, Q319L, and M354I, t(1/2)=150), respectively, resulted in two series of PAI-1-variants with different properties. In wtPAI-1 only substitution at P18 resulted in a pronounced u-PA specificity and substrate behaviour towards t-PA. In contrast, in PAI-1-stab substitution at either P18, P19 or P20 resulted in a u-PA specificity and a significantly increased substrate behaviour towards t-PA and u-PA. Importantly, analysis of the kinetics of inhibition did not reveal any differences in the second-order rate constants of inhibition (k approximately 10(7)M(-1)s(-1)). The pronounced differences observed for identical mutations in wtPAI-1 vs PAI-1-stab demonstrate that not merely the sequence of the reactive loop but also intramolecular interactions between the hF/s3A-loop and the main part of the molecule govern the functional and conformational behaviour of PAI-1.
authors.affiliation: Laboratory for Pharmaceutical Biology and Phytopharmacology, K.U.Leuven, Van Evenstraat 4, 3000 Leuven, Belgium.
authors.forename: Bart
authors.initials: B
authors.lastname: De Taeye
authors.forename: Ann
authors.initials: A
authors.lastname: Gils
authors.forename: Nele
authors.initials: N
authors.lastname: Vleugels
authors.forename: Anja
authors.initials: A
authors.lastname: Rabijns
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Declerck
mesh_terms: D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000378:metabolism
chemical_list: D017395:Plasminogen Activator Inhibitor 1; D015842:Serine Proteinase Inhibitors; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator