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J. Biol. Chem., Vol. 275, Issue 49, 38561-38570, December 8, 2000
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From the Joseph Stokes Research Institute, Children's Hospital of
Philadelphia and Department of Pediatrics, University of
Pennsylvania, Philadelphia, Pennsylvania 19104
The prothrombinase complex, composed of the
proteinase, factor Xa, bound to factor Va on membranes, catalyzes
thrombin formation by the specific and ordered proteolysis of
prothrombin at Arg323-Ile324, followed by
cleavage at Arg274-Thr275. We have used a
fluorescent derivative of meizothrombin des fragment 1 (mIIa
Exosite Binding Tethers the Macromolecular Substrate to the
Prothrombinase Complex and Directs Cleavage at Two Spatially Distinct
Sites*
F1) as a
substrate analog to assess the mechanism of substrate recognition in
the second half-reaction of bovine prothrombin activation. Cleavage of
mIIaF1 exhibits pseudo-first order kinetics regardless of the
substrate concentration relative to Km. This
phenomenon arises from competitive product inhibition by thrombin,
which binds to prothrombinase with exactly the same affinity as
mIIaF1. As thrombin is known to bind to an exosite on
prothrombinase, initial interactions at an exosite likely play a role
in the enzyme-substrate interaction. Occupation of the active site of
prothrombinase by a reversible inhibitor does not exclude the binding
of mIIaF1 to the enzyme. Specific recognition of mIIaF1 is
achieved through an initial bimolecular reaction with an enzymic
exosite, followed by an active site docking step in an intramolecular
reaction prior to bond cleavage. By alternate substrate studies, we
have resolved the contributions of the individual binding steps to
substrate affinity and catalysis. This pathway for substrate binding is
identical to that previously determined with a substrate analog for the
first half-reaction of prothrombin activation. We show that differences
in the observed kinetic constants for the two cleavage reactions arise
entirely from differences in the inferred equilibrium constant for the
intramolecular binding step that permits elements surrounding the
scissile bond to dock at the active site of prothrombinase. Therefore,
substrate specificity is achieved by binding interactions with an
enzymic exosite that tethers the protein substrate to prothrombinase
and directs cleavage at two spatially distinct scissile bonds.
*
This work was supported by National Institutes of Health
Grants HL-52883 and HL-62523 (to S. K.)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 all correspondence should be addressed: Joseph Stokes
Research Inst., Children's Hospital of Philadelphia, 310 Abramson, 3516 Civic Center Blvd., Philadelphia, PA 19104. Tel.: 215-590-3346; Fax: 215-590-2320; E-mail: skrishna@mail.med.upenn.edu.
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