Neuenschwander Pierre F, Williamson Stephen R, Nalian Armen, Baker-Deadmond Kimberly J
Department of Biochemistry, Biomedical Research Program, The University of Texas Health Science Center, Tyler, Texas 75708, USA.
J Biol Chem. 2006 Aug 11;281(32):23066-74. doi: 10.1074/jbc.M603743200. Epub 2006 Jun 9.
Reactivity of factor IXa with basic pancreatic trypsin inhibitor is enhanced by low molecular weight heparin (enoxaparin). Previous studies by us have suggested that this effect involves allosteric modulation of factor IXa. We examined the reactivity of factor IXa with several isolated Kunitz-type inhibitor domains: basic pancreatic trypsin inhibitor, the Kunitz inhibitor domain of protease Nexin-2, and the first two inhibitor domains of tissue factor pathway inhibitor. We find that enhancement of factor IXa reactivity by enoxaparin is greatest for basic pancreatic trypsin inhibitor (>10-fold), followed by the second tissue factor pathway inhibitor domain (1.7-fold) and the Kunitz inhibitor domain of protease Nexin-2 (1.4-fold). Modeling studies of factor IXa with basic pancreatic trypsin inhibitor suggest that binding of this inhibitor is sterically hindered by the 99-loop of factor IXa, specifically residue Lys(98). Slow-binding kinetic studies support the formation of a weak initial enzyme-inhibitor complex between factor IXa and basic pancreatic trypsin inhibitor that is facilitated by enoxaparin binding. Mutation of Lys(98) to Ala in factor IXa results in enhanced reactivity with all inhibitors examined, whereas almost completely abrogating the enhancing effects of enoxaparin. The results implicate Lys(98) and the 99-loop of factor IXa in defining enzyme inhibitor specificity. More importantly, these results demonstrate the ability of factor IXa to be allosterically modulated by occupation of the heparin-binding exosite.
低分子量肝素（依诺肝素）可增强因子IXa与碱性胰蛋白酶抑制剂的反应活性。我们之前的研究表明，这种效应涉及因子IXa的变构调节。我们检测了因子IXa与几种分离的Kunitz型抑制结构域的反应活性：碱性胰蛋白酶抑制剂、蛋白酶Nexin-2的Kunitz抑制结构域以及组织因子途径抑制剂的前两个抑制结构域。我们发现，依诺肝素对因子IXa反应活性的增强作用在碱性胰蛋白酶抑制剂中最为显著（>10倍），其次是组织因子途径抑制剂的第二个结构域（1.7倍）和蛋白酶Nexin-2的Kunitz抑制结构域（1.4倍）。因子IXa与碱性胰蛋白酶抑制剂的建模研究表明，该抑制剂的结合在空间上受到因子IXa的99环（特别是赖氨酸残基Lys(98)）的阻碍。慢结合动力学研究支持因子IXa与碱性胰蛋白酶抑制剂之间形成弱的初始酶-抑制剂复合物，依诺肝素的结合可促进这一过程。将因子IXa中的Lys(98)突变为丙氨酸会增强其与所有检测到的抑制剂的反应活性，而几乎完全消除依诺肝素的增强作用。这些结果表明Lys(98)和因子IXa的99环在定义酶抑制剂特异性方面发挥作用。更重要的是，这些结果证明了因子IXa可通过占据肝素结合别构位点而受到变构调节。
