Journal of Biological Chemistry
Volume 272, Issue 4, 24 January 1997, Pages 2183-2191
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Protein Chemistry and Structure
A Steady-state Template Model That Describes the Kinetics of Fibrin-stimulated [Glu1]- and [Lys78]Plasminogen Activation by Native tissue-type Plasminogen Activator and Variants That Lack Either the Finger or Kringle-2 Domain*

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The kinetics of activation of both [Glu1]- and [Lys78]Plg(S741C-fluorescein) by native (recombinant) tissue-type plasminogen activator and its deletion variants lacking either the finger or kringle-2 domain were measured by fluorescence within fully polymerized fibrin clots. The kinetics conform to the Michaelis-Menten equation at any fixed fibrin concentration so long as the plasminogen concentration is expressed as either the free or fibrin-bound, but not the total. The apparent kcat and Km values both vary systematically with the concentration of fibrin. Competition kinetics disclosed an active site-dependent interaction between t-PA and [Glu1]Plg(S741C-fluorescein) in the presence, but not the absence, of fibrin. A steady-state template model having the rate equation v/[A]o = kcat(app)·;[Plg]/(Km(app)+ [Plg]) was derived and used to interpret the data. The model indicates that catalytic efficiency is determined by the stability of the ternary activator-fibrin-plasminogen complex rather than the binding of the activator or plasminogen to fibrin. This implies that efforts to improve the enzymatic properties of t-PA might be more fruitfully directed at enhancing the stability of the ternary complex rather than fibrin binding.

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This work was supported by a TALENT (NATO) Stipend N91-238 (to A. J. G. H.) from the Netherlands Organization for Scientific Research (NWO), and by grants from the Heart and Stroke Foundation of Ontario, the Medical Research Council of Canada, and the National Institutes of Health. 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.

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Current address: Academic Medical Center, Dept. of Biochemistry (K1-163), University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.