Exosite interactions determine the affinity of factor X for the extrinsic Xase complex
Exosite interactions determine the affinity of factor X for the extrinsic Xase complex
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DOI：
10.1074/jbc.m005266200
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发表时间：
2000-09-15
影响因子：
4.8
通讯作者：
Krishnaswamy, S
中科院分区：
文献类型：
--
作者：
Baugh, RJ;Dickinson, CD;Krishnaswamy, S
凝血的启动是由酶复合物（Xase）激活因子X引起的，该酶复合物由胰蛋白酶样丝氨酸蛋白酶（因子VIIa）组成，与磷脂膜上的组织因子（TF）结合。我们已经研究了使用TF重组到磷脂酰胆碱、磷脂酰丝氨酸或纯磷脂酰胆碱囊泡中的Xase的蛋白底物特异性的基础。我们表明，占领Xase内的活性位点VIIa的可逆抑制剂或替代肽基基板是足以排除在活性位点的底物相互作用，但不改变因子X的Xase的亲和力，这是显而易见的经典竞争性抑制肽基基板裂解，但作为经典的非竞争性抑制因子X激活活性位点定向配体。这意味着X酶对因子X的有效识别是由多步反应引起的，该反应需要在酶复合物上与活性位点（外位点）不同的位点处进行初始相互作用，然后进行活性位点相互作用和键裂解。胞外位点相互作用决定蛋白质底物亲和力，而第二个结合步骤影响反应的最大催化速率。我们还表明，竞争性抑制可以通过使用X因子衍生物干扰外位点结合来实现，所述X因子衍生物预期与Xase内的VIIa活性位点具有有限或废除的相互作用。因此，在酶复合物内从VIIa的活性位点移除的外位点处的底物相互作用决定了VIIa-TF复合物对因子X的生产性识别中的亲和力和结合特异性。这可能代表了一种普遍的策略，通过这种策略，通过同源凝固酶实现了独特的蛋白质底物特异性。
The initiation of coagulation results from the activation of factor X by an enzyme complex (Xase) composed of the trypsin-like serine proteinase, factor VIIa, bound to tissue factor (TF) on phospholipid membranes. We have investigated the basis for the protein substrate specificity of Xase using TF reconstituted into vesicles of phosphatidylcholine, phosphatidylserine, or pure phosphatidylcholine. We show that occupation of the active site of VIIa within Xase by a reversible inhibitor or an alternate peptidyl substrate is sufficient to exclude substrate interactions at the active site but does not alter the affinity of Xase for factor X, This is evident as classical competitive inhibition of peptidyl substrate cleavage but as classical noncompetitive inhibition of factor X activation by active site-directed ligands. This implies that the productive recognition of factor X by Xase arises from a multistep reaction requiring an initial interaction at sites on the enzyme complex distinct from the active site (exosites), followed by active site interactions and bond cleavage. Exosite interactions determine protein substrate affinity, whereas the second binding step influences the maximum catalytic rate for the reaction. We also show that competitive inhibition can be achieved by interfering with exosite binding using factor X derivatives that are expected to have limited or abrogated interactions with the active site of VIIa within Xase. Thus, substrate interactions at exosites, sites removed from the active site of VIIa within the enzyme complex, determine affinity and binding specificity in the productive recognition of factor X by the VIIa-TF complex. This may represent a prevalent strategy through which distinctive protein substrate specificities are achieved by the homologous enzymes of coagulation.