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J Biol Chem, Vol. 275, Issue 19, 14579-14589, May 12, 2000
From the Binding of streptokinase (SK) to plasminogen (Pg)
conformationally activates the zymogen and converts both Pg and plasmin (Pm) into specific Pg activators. The interaction of SK with Pm and its
relationship to the mechanism of Pg activation were evaluated in
equilibrium binding studies with active site-labeled fluorescent Pm
derivatives and in kinetic studies of SK-induced changes in the
catalytic specificity of Pm. SK bound to fluorescein-labeled and native
Pm with dissociation constants of 11 ± 2 pM and
12 ± 4 pM, which represented a 1,000-10,000-fold
higher affinity than determined for Pg. Stoichiometric binding of SK to
native Pm was followed by generation of a two-fragment form of SK
cleaved at Lys59 (SK'), which exhibited an
indistinguishable affinity for labeled Pm, while a truncated,
SK55-414 species had a 120-360-fold reduced affinity.
Binding of SK to native Pm was accompanied by a >50-fold enhancement
in specificity for activation of Pg, which was paralleled by a
surprising 2.6-10-fold loss of specificity of Pm for 8 of 11 tripeptide-pNA substrates. Further studies with Pm labeled
at the active site with 2-anilinonaphthalene-6-sulfonic acid
demonstrated directly that binding of SK to Pm resulted in expression
of a new substrate binding exosite for Pg on the SK·Pm complex. It is
concluded that SK activates Pg in part by preferential binding to the
active zymogen conformation. High affinity binding of SK to Pm enhances
Pg substrate specificity principally through emergence of a substrate
recognition exosite.
Streptokinase Binds to Human Plasmin with High Affinity,
Perturbs the Plasmin Active Site, and Induces Expression of a
Substrate Recognition Exosite for Plasminogen*
§,
Department of Pathology, Vanderbilt
University School of Medicine, Nashville, Tennessee 37232 and the
¶ Medicine Service, Ann Arbor Veterans Affairs Hospital,
Ann Arbor, Michigan 48109
*
This work was supported in part by National Institutes of
Health Grant HL-56181 and National Institutes of Health Research Career
Development Award HL-02832 (to P. E. B.) and a Department of
Veterans Affairs Merit Review Award (to W. P. F.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Pathology, Vanderbilt University School of Medicine, C3321A Medical
Center North, Nashville, TN 37232-2561. Tel.: 615-343-9863; Fax:
615-343-7023; E-mail: paul.bock@mcmail.vanderbilt.edu.
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