Desai U R, Petitou M, Björk I, Olson S T
Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
J Biol Chem. 1998 Mar 27;273(13):7478-87. doi: 10.1074/jbc.273.13.7478.
To determine the role of individual saccharide residues of a specific heparin pentasaccharide, denoted DEFGH, in the allosteric activation of the serpin, antithrombin, we studied the effect of deleting pentasaccharide residues on this activation. Binding, spectroscopic, and kinetic analyses demonstrated that deletion of reducing-end residues G and H or nonreducing-end residue D produced variable losses in pentasaccharide binding energy of approximately 15-75% but did not affect the oligosaccharide's ability to conformationally activate the serpin or to enhance the rate at which the serpin inhibited factor Xa. Rapid kinetic studies revealed that elimination of the reducing-end disaccharide marginally affected binding to the native low-heparin-affinity conformational state of antithrombin but greatly affected the conversion of the serpin to the activated high-heparin- affinity state, although the activated conformation was still favored. In contrast, removal of the nonreducing- end residue D drastically affected the initial low-heparin-affinity interaction so as to favor an alternative activation pathway wherein the oligosaccharide shifted a preexisiting equilibrium between native and activated serpin conformations in favor of the activated state. These results demonstrate that the nonreducing-end residues of the pentasaccharide function both to recognize the native low-heparin-affinity conformation of antithrombin and to induce and stabilize the activated high-heparin-affinity conformation. Residues at the reducing-end, however, poorly recognize the native conformation and instead function primarily to bind and stabilize the activated antithrombin conformation. Together, these findings establish an important role of the heparin pentasaccharide sequence in preferential binding and stabilization of the activated conformational state of the serpin.
为了确定特定的肝素五糖（标记为DEFGH）中单个糖残基在丝氨酸蛋白酶抑制剂抗凝血酶变构激活中的作用，我们研究了删除五糖残基对这种激活作用的影响。结合、光谱和动力学分析表明，删除还原端残基G和H或非还原端残基D会导致五糖结合能出现约15 - 75%的不同程度损失，但不影响寡糖对丝氨酸蛋白酶抑制剂进行构象激活的能力，也不影响丝氨酸蛋白酶抑制剂抑制因子Xa的速率。快速动力学研究表明，消除还原端二糖对与抗凝血酶天然低肝素亲和力构象状态的结合影响较小，但极大地影响了丝氨酸蛋白酶抑制剂向活化的高肝素亲和力状态的转变，尽管活化构象仍然占优势。相比之下，去除非还原端残基D会极大地影响最初的低肝素亲和力相互作用，从而有利于一种替代激活途径，其中寡糖改变了天然和活化丝氨酸蛋白酶抑制剂构象之间预先存在的平衡，有利于活化状态。这些结果表明，五糖的非还原端残基既能识别抗凝血酶的天然低肝素亲和力构象，又能诱导和稳定活化的高肝素亲和力构象。然而，还原端的残基对天然构象的识别较差，主要作用是结合并稳定活化的抗凝血酶构象。总之，这些发现确立了肝素五糖序列在优先结合和稳定丝氨酸蛋白酶抑制剂活化构象状态方面的重要作用。
