Thermodynamic Characteristics of Plasminogen Activation by Indirect Activators

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L. I. Sokolovskaya^* and G. L. Volkov

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, ul. Leontovicha 9, 01030 Kiev, Ukraine; fax: (38-044) 235-5172; E-mail: sokludmila@yandex.ru

^* To whom correspondence should be addressed.

Received July 14, 2004; Revision received June 15, 2005

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Several indirect plasminogen (Pg) activators are known including streptokinase and the monoclonal antibody IV-Ic, whose mechanism of activation is well studied. To characterize thermodynamically the activation of Pg by streptokinase (SK) and the monoclonal antibody (mAB) IV-Ic, the activation energies were calculated for various reaction stages. Activation energy of 7.4 kcal/mol was determined for the interaction of the chromogenic substrate S-2251 with plasmin (Pm) and activated equimolar complexes Pm-SK and Pg*SK at the steady-state reaction stage, and 18.7 kcal/mol with the complexes Pg*IV-Ic. A 2.5-fold increase in the energy of activation for the Pg*IV-Ic complex suggests a more intricate mechanism of its interaction with the substrate. At the stage of increasing active center concentrations and the formation of activated complexes Pg*SK and Pg*mAB IV-Ic, the activation energy was found to be 10.5 and 38 kcal/mol, respectively. At this reaction stage the conformational rearrangement of Pg molecule with the formation of active center is the limiting stage determining the reaction rate. Unexpectedly high energy of activation at the second stage of interaction between mAB IV-Ic and Pg suggests several simultaneous reactions and complexity of conformation rearrangement in the Pg molecule in activated complexes, thus requiring large energy expense. Formation of the active center is probably accompanied by its transition within a narrow temperature range into another conformation state with the change in activation parameters of the reaction. Quantitative evaluation of the studied reactions from the perspective of thermodynamics of the enzymatic reactions gives more comprehensive characteristics of the activation mechanism.

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KEY WORDS: energy of activation, plasminogen, plasmin, streptokinase, monoclonal antibody

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