Full and partial deuterium solvent isotope effect studies of alpha-thrombin-catalyzed reactions of natural substrates.
Full and partial deuterium solvent isotope effect studies of alpha-thrombin-catalyzed reactions of natural substrates.
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天然底物的α-凝血酶催化反应的全部和部分氘溶剂同位素效应研究。
DOI：
10.1021/ja043258o
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Kovach,IldikoM
中科院分区：
文献类型：
--
作者：
Zhang,Daoning;Kovach,IldikoM
凝血酶催化纤维蛋白原活化为纤维蛋白肽 A 的质子库存研究与可能在活性位点的 Ser195OγH 和 His57Nε2 以及 His57Nδ1 和 Asp102COOβ- 之间在过渡态形成的双质子桥最一致，在底物酶饱和下分馏因子为 0.66 ± 0.03，在底物饱和时分馏因子为 0.64 ± 0.03 0.2Km、pH 8.0、pD 8.6 和 25.0 ± 0.1 °C 时的纤维蛋白原浓度。强烈的逆溶剂同位素效应（SIE）是由逆滞后时间和凝血图最大斜率产生的，这也与阴离子和阳离子有关。 D2O 中的血凝块要粗得多，如凝血曲线所示，相对于 H2O，其滞后时间短 3−9 倍，斜率更陡。颗粒越细，纤维蛋白网和/或纤维蛋白周围的水结构中互锁的氢键越弱。含有 Na+ 和 Cl- 离子的缓冲液中的反滞后时间质子库存和凝血曲线的最大斜率最适合缓冲液中氘含量的指数依赖性，并在 pH 8.0 和 25.0 ± 0.1 °C 下给出分馏因子 5.6 ± 0.5 和 7.8 ± 0.6。在底物浓度 = 0.2Km 的 0.3 M 氯化钠和 0.3 M 氯化胆碱中，凝血酶催化的蛋白 C (PC) 活化为 APC 与逆动力学 SIE (KSIE) 分别为 0.75 ± 0.09 和 1.02 ± 0.06 相关。相比之下，与首次报道的天然底物相比，凝血酶催化的显色底物水解可产生更大的 KSIE（Enyedy, E. I.; Kovach. I. MJ. Am. Chem. Soc.2004,126, 6017−6024）和更复杂的质子库存。本研究阐明了凝血酶催化的两种生理反应初始阶段的速率决定过渡态特征的差异。
Proton inventory studies of the thrombin-catalyzed fibrinogen activation to fibrinopeptide A are most consistent with a two-proton bridge forming at the transition state probably between Ser195OγH and His57Nε2 and His57Nδ1 and Asp102COOβ- at the active site, with fractionation factors 0.66 ± 0.03 under enzyme saturation with substrate and 0.64 ± 0.03 at fibrinogen concentration at 0.2Km, at pH 8.0, pD 8.6, and 25.0 ± 0.1 °C. Strongly inverse solvent isotope effects (SIEs) result from inverse lag times and maximal slopes of blood clotting plots, which are also anion and cation dependent. The blood clot is much coarser in D2O, as indicated in clotting curves with 3−9 times shorter lag time and steeper slopes with respect to H2O. The finer the particles, the weaker the H-bonds interlocking the fibrin mesh and/or in water structure around fibrin. Proton inventories of inverse lag times and maximal slopes of blood clotting curves in buffers containing Na+and Cl-ions give the best fit to an exponential dependence on deuterium content in the buffer and give fractionation factors 5.6 ± 0.5 and 7.8 ± 0.6 at pH 8.0 and 25.0 ± 0.1 °C. The thrombin-catalyzed activation of protein C (PC) to APC is associated with inverse kinetic SIEs (KSIEs) of 0.75 ± 0.09 and 1.02 ± 0.06 in 0.3 M NaCl and 0.3 M choline chloride, respectively, at substrate concentrations = 0.2Km. In comparison, thrombin-catalyzed hydrolysis of chromogenic substrates gives greater KSIEs (Enyedy, E. I.; Kovach. I. MJ. Am. Chem. Soc.2004,126, 6017−6024) and more complex proton inventories than the ones reported here for the first time for natural substrates. The present study illuminates differences in the character of the rate-determining transition state for the initial phase of the two physiological reactions catalyzed by thrombin.