Rezaie Alireza R
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.
Biochemistry. 2006 Apr 25;45(16):5324-9. doi: 10.1021/bi060056r.
It has been demonstrated that a unique pentasaccharide fragment of heparin (H5) activates AT by exposing an exosite on the serpin that is a recognition site for interaction with the basic autolysis loop (residues 143-154) of fXa. In support of this, the substitution of Arg-150 of fXa with Ala (R150A) impaired the reactivity of the mutant with AT by 1 order of magnitude specifically in the presence H5. To understand the mechanism by which heparin activation of AT improves the reactivity of the serpin with fXa, the H5-catalyzed reaction of AT with fXa, fXa R150A, and fXa S195A was studied using rapid kinetic, surface plasmon resonance, and competitive binding methods. The pseudo-first-order rate constants for the H5-catalyzed AT inhibition of both fXa and fXa R150A exhibited a linear dependence on the serpin concentration, thereby yielding second-order rate constants of 1.0 x 10(6) and 1.5 x 10(5) M(-)(1) s(-)(1), respectively. On the other hand, an approximately 70-saccharide, high-affinity heparin-catalyzed AT inhibition of both fXa derivatives showed a saturable dependence on the inhibitor concentration, yielding an identical rate constant of approximately 20 s(-)(1), but different ternary fXa-heparin-AT dissociation constants (K(E,ATH)) of approximately 130 and approximately 1780 nM for wild-type and R150A fXa, respectively. Competitive kinetic and surface plasmon resonance binding studies using the catalytically inactive S195A mutant of fXa yielded dissociation constants of 255 and 610 nM, respectively, for the mutant protease interaction with the AT-H5 complex. These results suggest that H5 enhances the reactivity of AT with fXa primarily by lowering the K(E,ATH) for the formation of a Michaelis-type serpin-protease encounter complex.
已证实，肝素的一种独特五糖片段（H5）通过在丝氨酸蛋白酶抑制剂（serpin）上暴露一个外部位点来激活抗凝血酶（AT），该外部位点是与凝血因子Xa（fXa）的碱性自溶环（第143 - 154位氨基酸残基）相互作用的识别位点。支持这一观点的是，将fXa的精氨酸-150（R150）替换为丙氨酸（R150A），会使突变体与AT的反应性在H5存在时特异性降低1个数量级。为了解肝素激活AT从而提高丝氨酸蛋白酶抑制剂与fXa反应性的机制，使用快速动力学、表面等离子体共振和竞争性结合方法研究了H5催化的AT与fXa、fXa R150A和fXa S195A的反应。H5催化的AT对fXa和fXa R150A的抑制作用的伪一级速率常数均表现出对丝氨酸蛋白酶抑制剂浓度的线性依赖性，因此分别得到二级速率常数为1.0×10⁶和1.5×10⁵ M⁻¹ s⁻¹。另一方面，一种约70糖的高亲和力肝素催化的AT对两种fXa衍生物的抑制作用表现出对抑制剂浓度的饱和依赖性，得到相同的速率常数约为20 s⁻¹，但野生型和R150A fXa的三元fXa - 肝素 - AT解离常数（K(E,ATH)）分别约为130和1780 nM。使用fXa的催化无活性突变体S195A进行的竞争性动力学和表面等离子体共振结合研究表明，突变型蛋白酶与AT - H5复合物相互作用的解离常数分别为255和610 nM。这些结果表明，H5主要通过降低形成米氏型丝氨酸蛋白酶抑制剂 - 蛋白酶相遇复合物的K(E,ATH)来增强AT与fXa的反应性。
