Rezaie A R
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City 73104, USA.
Protein Sci. 1998 Feb;7(2):349-57. doi: 10.1002/pro.5560070215.
A pentasaccharide (PS) fragment of heparin capable of activating antithrombin (AT) markedly accelerates the inhibition of factor Xa by AT, but has insignificant effect on inhibition of thrombin. For inhibition of thrombin, the bridging function of a longer polysaccharide chain is required to accelerate the reaction. To study the basis for the similar reactivity of thrombin with the native or heparin-activated conformers of AT, several residues surrounding the active site pocket of thrombin were targeted for mutagenesis study. Leu99 and Glu192, the variant residues influencing the S2 and S3 subsite specificity of thrombin were replaced with Tyr and Gln. The Tyr60a, Pro60b, Pro60c, and Trp60d residues forming part of the S2 specificity pocket were deleted from the B-insertion loop of the wild-type and Leu99/Glu192 --> Tyr/Gln thrombins. Kinetic studies indicated that the reactivities of all mutants with AT were moderately or severely impaired. Although heparin largely corrected the defect in reactivities, it also markedly elevated the stoichiometries of inhibition with the mutants. Interestingly, PS also accelerated AT inhibition of the mutants 5-68-fold, suggesting that the mutants are able to discriminate between the native and activated conformers of AT. Based on these results and the recent crystal structure determination of AT in complex with PS, a model for thrombin-AT interaction is proposed in which the S2 and S3 subsite residues of thrombin are critical for recognition of the P2 and P3 residues of AT in the native conformation. In the activated conformation, other residues are made accessible for interaction with the protease, and the similar reactivity of thrombin with the native and heparin-activated conformers of AT may be coincidental. The results further suggest that the S2 and S3 subsite residues are crucial in controlling the partitioning of the thrombin-AT intermediate into the alternative inhibitory or substrate pathways of the reaction.
一种能够激活抗凝血酶（AT）的肝素五糖（PS）片段可显著加速AT对Xa因子的抑制作用，但对凝血酶的抑制作用不明显。对于凝血酶的抑制，需要更长多糖链的桥连功能来加速反应。为了研究凝血酶与天然或肝素激活的AT构象具有相似反应性的基础，对凝血酶活性位点口袋周围的几个残基进行了诱变研究。将影响凝血酶S2和S3亚位点特异性的变异残基Leu99和Glu192替换为Tyr和Gln。从野生型和Leu99/Glu192→Tyr/Gln凝血酶的B插入环中删除了构成S2特异性口袋一部分的Tyr60a、Pro60b、Pro60c和Trp60d残基。动力学研究表明，所有突变体与AT的反应性均中度或严重受损。尽管肝素在很大程度上纠正了反应性缺陷，但它也显著提高了突变体的抑制化学计量。有趣的是，PS也将突变体的AT抑制作用加速了5-68倍，这表明突变体能够区分AT的天然构象和激活构象。基于这些结果以及最近AT与PS复合物的晶体结构测定，提出了一种凝血酶-AT相互作用模型，其中凝血酶的S2和S3亚位点残基对于识别天然构象中AT的P2和P3残基至关重要。在激活构象中，其他残基可用于与蛋白酶相互作用，凝血酶与AT的天然构象和肝素激活构象具有相似反应性可能是巧合。结果进一步表明，S2和S3亚位点残基对于控制凝血酶-AT中间体进入反应的替代抑制或底物途径的分配至关重要。
