Energetics of Thrombin-Thrombomodulin Interaction
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- 作者：Alessandro Vindigni ; Christopher E. White ; Elizabeth A. Komives ; and Enrico Di Cera
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：June 3, 1997
- 年：1997
- 卷：36
- 期：22
- 页码：6674 - 6681
- 全文大小：233K
- 年卷期：v.36,no.22(June 3, 1997)
- ISSN：1520-4995
文摘
Temperature and salt dependence studies of thrombin interactionwith thrombomodulin, withand without chondroitin sulfate, and two fragments containing theEGF-like domains 4-5 and 4-5-6reveal the energetic signatures and the mechanism of recognition ofthis physiologically important cofactor.Binding of thrombomodulin is affected drastically by theparticular salt present in solution and is positivelylinked to Na+ binding to thrombin and the conversion ofthe enzyme from the slow to the fast form, butis opposed by Cl- binding to the fibrinogen recognitionsite and especially to the heparin binding site.Binding of thrombomodulin has an unusually large salt dependence(
salt = -4.8) contributed mostly bythe polyelectrolyte-like nature of the chondroitin sulfate moiety thatbinds to the heparin binding site andincreases the affinity of the cofactor by almost 10-fold. On theother hand, the chondroitin sulfate has noeffect on the 
Cp of binding, which isdetermined predominantly by contacts made by the EGF-likedomains 5 and 6 with the fibrinogen recognition site. The modestheat capacity change (-0.2 kcal mol-1K-1) observed when thrombomodulin binds to the fast formsuggests a rigid-body association of thecofactor with the enzyme. In the slow form, however, the heatcapacity change is significantly morepronounced (-0.5 kcal mol-1 K-1) andsignals the presence of a conformational transition of theenzymelinked to binding of the cofactor that mimics the slow
fastconversion. These results demonstrate thatrecognition of thrombomodulin by thrombin is steered electrostaticallyby the highly charged regions ofthe fibrinogen recognition site and the heparin binding site, to whichthe chondroitin sulfate moiety bindsand enhances the affinity of the interaction. The recognitionevent also involves conformational changesof the enzyme in the slow form mediated by binding of the EGF-likedomains 5-6 to the fibrinogenrecognition site. Consistent with this model, binding ofthrombomodulin to the fast form has only asmall effect on the hydrolysis of nine chromogenic substrates carryingsubstitutions at P1, P2, and P3aimed at probing the environment of the specificity sites S1, S2, andS3 of the enzyme. Binding to theslow form, on the other hand, enhances the specificity toward allsubstrates up to 15-fold. For substratescarrying a Gly at P2, binding of thrombomodulin changes the relativespecificity of the slow and fastforms and makes the slow form more specific. Interestingly, theseeffects are not specific ofthrombomodulin and depend solely on binding to the fibrinogenrecognition site of the enzyme. In fact,they are also observed with the hirudin C-terminal fragment 55-65.The characterization of the mechanismof thrombin-thrombomodulin interaction and the effects of thecofactor on the hydrolysis of chromogenicsubstrates probing the interior of the catalytic pocket bear on thethrombomodulin-induced enhancementof protein C cleavage by thrombin. We propose that thisenhancement is due predominantly to an effectof thrombomodulin on the bound protein C in the ternary complex.Therefore, thrombomodulin wouldcarry out its physiological function by making protein C a bettersubstrate for thrombin, rather than makingthrombin a better enzyme for protein C.
salt = -4.8) contributed mostly bythe polyelectrolyte-like nature of the chondroitin sulfate moiety thatbinds to the heparin binding site andincreases the affinity of the cofactor by almost 10-fold. On theother hand, the chondroitin sulfate has noeffect on the Cp of binding, which isdetermined predominantly by contacts made by the EGF-likedomains 5 and 6 with the fibrinogen recognition site. The modestheat capacity change (-0.2 kcal mol-1K-1) observed when thrombomodulin binds to the fast formsuggests a rigid-body association of thecofactor with the enzyme. In the slow form, however, the heatcapacity change is significantly morepronounced (-0.5 kcal mol-1 K-1) andsignals the presence of a conformational transition of theenzymelinked to binding of the cofactor that mimics the slowfastconversion. These results demonstrate thatrecognition of thrombomodulin by thrombin is steered electrostaticallyby the highly charged regions ofthe fibrinogen recognition site and the heparin binding site, to whichthe chondroitin sulfate moiety bindsand enhances the affinity of the interaction. The recognitionevent also involves conformational changesof the enzyme in the slow form mediated by binding of the EGF-likedomains 5-6 to the fibrinogenrecognition site. Consistent with this model, binding ofthrombomodulin to the fast form has only asmall effect on the hydrolysis of nine chromogenic substrates carryingsubstitutions at P1, P2, and P3aimed at probing the environment of the specificity sites S1, S2, andS3 of the enzyme. Binding to theslow form, on the other hand, enhances the specificity toward allsubstrates up to 15-fold. For substratescarrying a Gly at P2, binding of thrombomodulin changes the relativespecificity of the slow and fastforms and makes the slow form more specific. Interestingly, theseeffects are not specific ofthrombomodulin and depend solely on binding to the fibrinogenrecognition site of the enzyme. In fact,they are also observed with the hirudin C-terminal fragment 55-65.The characterization of the mechanismof thrombin-thrombomodulin interaction and the effects of thecofactor on the hydrolysis of chromogenicsubstrates probing the interior of the catalytic pocket bear on thethrombomodulin-induced enhancementof protein C cleavage by thrombin. We propose that thisenhancement is due predominantly to an effectof thrombomodulin on the bound protein C in the ternary complex.Therefore, thrombomodulin wouldcarry out its physiological function by making protein C a bettersubstrate for thrombin, rather than makingthrombin a better enzyme for protein C.