Proton bridging in the interactions of thrombin with hirudin and its mimics.
Proton bridging in the interactions of thrombin with hirudin and its mimics.
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DOI：
10.1021/bi301625a
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发表时间：
2013-04-09
期刊：
影响因子：
2.9
通讯作者：
Zhang D
中科院分区：
文献类型：
--
作者：
Kovach IM;Kakalis L;Jordan F;Zhang D
凝血酶是血液级联系统中关键的丝氨酸蛋白酶，因此是控制其活性的药物设计的目标。凝血酶的最有效的非生理抑制剂是水蛭素，一种天然存在的小蛋白。水蛭素及其合成模拟物利用一系列氢键、盐桥和与凝血酶的疏水相互作用来实现紧密结合，Ki值在纳摩尔至飞摩尔范围内。在600 MHz下进行的一维1H NMR光谱显示，在pH 5.6-8.8缓冲液和5 - 35 °C之间，人α-凝血酶和r-水蛭素之间的复合物中硅烷在15.33 ppm低场处产生共振。在人α-凝血酶与水蛭素IV、水蛭素V、一种Nα（Me）Arg肽、RGD-水蛭素和Nα-2-萘磺酰基-甘氨酰-DL-4-脒基苯基丙氨酰-哌啶乙酸盐（NAPAP）的复合物中也观察到15.17和15.54 ppm之间的共振，而在猪胰蛋白酶和NAPAP的复合物中未观察到这种低场共振。化学位移表明，这些共振代表H键的环境。相应氢键中的H-供体受体距离估计为<2.7 π。将Phe-Pro-Arg-氯甲基酮（PPACK）加入人α-凝血酶与r-水蛭素的复合物中，在18.03 ppm处产生额外的信号，该信号比观察到的信号高0.10 ppm（Kovach，I. M.等人Biochemistry 2009，48，7296-7304）。相反，15.33 ppm处的峰保持不变。凝血酶-水蛭素型复合物的分馏因子在20%误差范围内接近1.0。凝血酶与水蛭素家族抑制剂复合物中最可能的短H键位点是水蛭素C末端的疏水斑块，其中Glu 57 '和Glu 58'嵌入并与Arg 75和Arg 77及其溶剂合物水相互作用（在凝血酶上）。存在于水蛭素抑制剂家族中的Glu 57 '和Glu 58'是纤维蛋白原（凝血酶的主要底物）的关键结合表位，这使得SHB作为纤维蛋白原识别位点处的结合元件引起了很大兴趣。
Thrombin is the pivotal serine protease enzyme in the blood cascade system and thus a target of drug design for control of its activity. The most efficient non-physiologic inhibitor of thrombin is hirudin, a naturally occurring small protein. Hirudin and its synthetic mimics employ a range of hydrogen bonding, salt bridging and hydrophobic interactions with thrombin to achieve tight binding with Ki values in the nano- to femtomolar range. The one-dimensional 1H NMR spectrum carried out at 600 MHz reveals a resonance at 15.33 ppm downfield from silanes in complexes between human α-thrombin and r-hirudin in pH 5.6-8.8 buffers and between 5 and 35 °C. There is also a resonance between 15.17 and 15.54 ppm seen in human α-thrombin complexes with hirunorm IV, hirunorm V, an Nα(Me)Arg-peptide, RGD-hirudin and Nα-2-naphthylsulfonyl-glycyl-DL-4-amidinophenylalanyl-piperidide acetate salt (NAPAP), while there is no such low-field resonance observed in a complex of porcine trypsin and NAPAP. The chemical shifts suggest that these resonances represent H-bonded environments. H-donor acceptor distances in the corresponding H-bonds are estimated to be <2.7 Å. Addition of Phe-Pro-Arg-Chloromethylketone (PPACK) to a complex of human α-thrombin with r-hirudin results in an additional signal at 18.03 ppm, which is 0.10 ppm upfield from one observed (Kovach, I. M. et al. Biochemistry 2009, 48, 7296–7304) for thrombin covalently modified with PPACK. In contrast, the peak at 15.33 ppm remains unchanged. The fractionation factors for the thrombin-hirudin type complexes are near 1.0 within 20% error. The most likely site of the short H-bond in thrombin complexes with the hirudin family of inhibitors is in the hydrophobic patch of the C-terminus of hirudin where Glu57’ and Glu58’ are embedded and interact with Arg75 and Arg77 and their solvate water (on thrombin). Glu57’ and Glu58’ present in the hirudin family of inhibitors is a key binding epitope of fibrinogen, thrombin’s prime substrate, which lends substantial interest to the SHB as a binding element at the fibrinogen recognition site.
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影响因子：
2.9
作者：
DAVIE, EW;FUJIKAWA, K;KISIEL, W
通讯作者：
KISIEL, W
影响因子：
56.9
作者：
Ash, EL;Sudmeier, JL;Bachovchin, WW
通讯作者：
Bachovchin, WW
影响因子：
56.9
作者：
FREY, PA;WHITT, SA;TOBIN, JB
通讯作者：
TOBIN, JB
DOI：
10.1111/j.1432-1033.1990.tb19320.x
发表时间：
1990-10-05
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
BODE, W;TURK, D;STURZEBECHER, J
通讯作者：
STURZEBECHER, J
影响因子：
15
作者：
Enyedy, EJ;Kovach, IM
通讯作者：
Kovach, IM