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首页> 外文期刊>Biochemistry >Selective inhibition of the prothrombinase complex: factor Va alters macromolecular recognition of a tick anticoagulant peptide mutant by factor Xa.
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Selective inhibition of the prothrombinase complex: factor Va alters macromolecular recognition of a tick anticoagulant peptide mutant by factor Xa.

机译：凝血酶原酶复合物的选择性抑制：因子Va通过因子Xa改变壁虱抗凝血肽突变体的大分子识别。

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The prothrombinase complex assembles through reversible interactions between the protease, factor Xa, the cofactor, factor Va, and acidic phospholipid membranes in the presence of calcium ions. Changes in macromolecular recognition by factor Xa which may result from its interaction with factor Va in the prothrombinase complex have been probed using a recombinant derivative of tick anticoagulant peptide where Arg3 has been replaced with Ala (R3A-TAP). In contrast to the wild type inhibitor, R3A-TAP was a weak competitive inhibitor of factor Xa (Ki = 794 nM). The inhibition of the prothrombinase complex by R3A-TAP was characterized by slow, tight-binding kinetics with an increased affinity of approximately 4000-fold (Ki* = 0.195 nM) relative to that of solution-phase factor Xa. Stopped-flow measurements using p-aminobenzamidine (PAB) demonstrated that the reaction between solution-phase factor Xa and R3A-TAP could be adequately described by a single reversible step with rate constants that were consistent with equilibrium binding measurements. The rate-limiting bimolecular combination of R3A-TAP and factor Xa was competitive with PAB binding of the protease. In contrast, the reaction of R3A-TAP with prothrombinase measured using PAB yielded biphasic stopped-flow traces, indicating a multistep pathway for the reaction of the inhibitor with the enzyme complex. The kinetic measurements were consistent with the initial formation of a ternary complex between R3A-TAP, prothrombinase, and PAB followed by two unimolecular steps which lead to PAB dissociation from the enzyme. In this case, prior occupation of the active site by PAB had no effect on the bimolecular reaction between R3A-TAP and prothrombinase. Thus, the interaction of factor Xa with factor Va on the membrane surface alters recognition of R3A-TAP by the protease, leading to changes in the thermodynamics as well as in the observed kinetic mechanism for the reaction. Therefore, a single amino acid substitution in TAP reveals large changes in macromolecular recognition by factor Xa as a consequence of its interaction with the cofactor within the prothrombinase complex.
机译：凝血酶原酶复合物通过在钙离子存在下蛋白酶，Xa因子，辅因子，Va因子和酸性磷脂膜之间的可逆相互作用组装。已经使用壁虱抗凝血肽的重组衍生物（其中Arg3已被Ala替代）检测了Xa因子与凝血酶原酶复合物中因子Va相互作用可能导致的大分子识别变化。与野生型抑制剂相反，R3A-TAP是Xa因子的弱竞争抑制剂（Ki = 794 nM）。 R3A-TAP对凝血酶原酶复合物的抑制作用以缓慢，紧密的结合动力学为特征，相对于溶液相因子Xa的亲和力增加了约4000倍（Ki * = 0.195 nM）。使用对氨基苯甲m（PAB）进行的停流测量表明，溶液相因子Xa和R3A-TAP之间的反应可以通过单个可逆步骤充分描述，其速率常数与平衡结合测量结果一致。 R3A-TAP和因子Xa的限速双分子组合与蛋白酶的PAB结合具有竞争性。相反，使用PAB测得的R3A-TAP与凝血酶原的反应产生了两相停止流痕迹，表明抑制剂与酶复合物反应的多步途径。动力学测量与R3A-TAP，凝血酶原和PAB之间的三元复合物的初始形成相一致，随后是两个单分子步骤，导致PAB从酶中解离。在这种情况下，PAB先前占据活性位点对R3A-TAP与凝血酶原酶之间的双分子反应没有影响。因此，因子Xa与因子Va在膜表面上的相互作用改变了蛋白酶对R3A-TAP的识别，从而导致了热力学以及观察到的反应动力学机制的改变。因此，TAP中的单个氨基酸取代揭示了Xa因子与大分子凝血酶原复合物内辅因子相互作用的结果，大分子识别被Xa因子改变。

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