Belzar Klara J, Zhou Aiwu, Carrell Robin W, Gettins Peter G W, Huntington James A
Department of Haematology, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Rd., Cambridge CB2 2XY, United Kingdom.
J Biol Chem. 2002 Mar 8;277(10):8551-8. doi: 10.1074/jbc.M110807200. Epub 2001 Dec 10.
Antithrombin requires allosteric activation by heparin for efficient inhibition of its target protease, factor Xa. A pentasaccharide sequence found in heparin activates antithrombin by inducing conformational changes that affect the reactive center of the inhibitor resulting in optimal recognition by factor Xa. The mechanism of transmission of the activating conformational change from the heparin-binding region to the reactive center loop remains unresolved. To investigate the role of helix D elongation in the allosteric activation of antithrombin, we substituted a proline residue for Lys(133). Heparin binding affinity was reduced by 25-fold for the proline variant compared with the control, and a significant decrease in the associated intrinsic fluorescence enhancement was also observed. Rapid kinetic studies revealed that the main reason for the reduced affinity for heparin was an increase in the rate of the reverse conformational change step. The pentasaccharide-accelerated rate of factor Xa inhibition for the proline variant was 10-fold lower than control, demonstrating that the proline variant cannot be fully activated toward factor Xa. We conclude that helix D elongation is critical for the full conversion of antithrombin to its high affinity, activated state, and we propose a mechanism to explain how helix D elongation is coupled to allosteric activation.
抗凝血酶需要肝素的变构激活才能有效抑制其靶蛋白酶Xa因子。肝素中发现的一种五糖序列通过诱导构象变化来激活抗凝血酶，这种构象变化会影响抑制剂的反应中心，从而使Xa因子能够实现最佳识别。从肝素结合区域到反应中心环的激活构象变化的传递机制仍未得到解决。为了研究螺旋D延长在抗凝血酶变构激活中的作用，我们用脯氨酸残基取代了赖氨酸（133）。与对照相比，脯氨酸变体的肝素结合亲和力降低了25倍，并且还观察到相关的固有荧光增强显著降低。快速动力学研究表明，对肝素亲和力降低的主要原因是反向构象变化步骤的速率增加。脯氨酸变体的五糖加速Xa因子抑制速率比对照低10倍，表明脯氨酸变体不能完全被激活以对抗Xa因子。我们得出结论，螺旋D延长对于抗凝血酶完全转化为其高亲和力的激活状态至关重要，并且我们提出了一种机制来解释螺旋D延长如何与变构激活相耦合。
