Volume 272, Number 19,
Issue of May 9, 1997
pp. 12662-12666
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Proteinase Specificity and Functional Diversity in Point Mutants
of Plasminogen Activator Inhibitor 1
(Received for publication, January 16, 1997, and in revised form, February 25, 1997)
Ann
Gils
and
Paul J.
Declerck
From the Laboratory for Pharmaceutical Biology and
Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke
Universiteit Leuven, B-3000 Leuven, Belgium
Plasminogen activator inhibitor-1
(PAI-1) is a unique member of the serpin superfamily. The alternative
behavior of PAI-1 as an inhibitor, a non-inhibitory substrate, or a
non-reactive latent form has been shown to be dependent on the initial
conformation. In this study, we have evaluated the effect of a
substitution outside the reactive site loop (P18) or in the reactive
site loop (P6 and P10) on proteinase specificity and conformational
transitions in PAI-1. Wild-type PAI-1 (wtPAI-1) revealed the same
conformational distribution pattern toward tissue-type plasminogen
activator (t-PA) as toward urokinase-type plasminogen activator (u-PA)
(i.e. 53 ± 6.9% active, 36 ± 6.8% latent, and
12 ± 1.9% substrate). Inactivation of wtPAI-1 resulted in the
conversion of the labile active form into the latent form while the
stable substrate form remained unchanged. PAI-1-P6 (Val 
Pro at
P6) revealed a target specificity for t-PA (39 ± 7%
versus 3 ± 2% of the theoretical maximal value
toward t-PA and u-PA, respectively), PAI-1-P10 (Ser Pro at P10)
was 4-fold more active toward u-PA than toward t-PA, and PAI-1-P18
(Asn Pro at P18) exhibited inhibitory properties exclusively toward u-PA (41 ± 10%). Surprisingly, inactivation of these
mutants revealed functional and conformational transitions distinct
from those observed for wtPAI-1. Inactivation of
PAI-1-P6(Val Pro) resulted in a total conversion of the active
form into the latent form and in a partial conversion of the substrate
form into the latent form. The active forms of both
PAI-1-P10(Ser Pro) and PAI-1-P18(Asn Pro) are also labile
but, in contrast to the active form of wtPAI-1, convert into substrate
forms. Based on the existence of various conformations of PAI-1, we
propose an alternative reaction scheme describing the putative
interactions between serpins and their target proteinases. The unusual
conformational and functional flexibility of PAI-1 that, according to
the current study, appears not to be restricted to the reactive site
loop further underlines the importance of potential structural
rearrangements (e.g. upon binding to cofactors) in PAI-1 (or serpins in
general) for its functional behavior at particular biological
sites.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
