Proton inventory studies of α-thrombin-catalyzed reactions of substrates with selected P and P′ sites
Proton inventory studies of α-thrombin-catalyzed reactions of substrates with selected P and P′ sites
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DOI：
10.1021/ja0320166
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发表时间：
2004-05-19
影响因子：
15
通讯作者：
Kovach, IM
中科院分区：
文献类型：
--
作者：
Enyedy, EJ;Kovach, IM
氘的人类alpha-thrombin-catalyzed水解动力学溶剂同位素效应(1)基质与选定P-1-P-3网站,Z-Pro-Arg-7-amido-4-methylcoumarin (7-AMC) N-t-Boc-Val-Pro-Arg-7-AMC, Bz-Phe-Val-Arg-4-nitroanilide(机构)和H-D-Phe-L-Pip-Arg-pNA (DOD) k (cat) = (2.8 - -3.3) + / - 0.1 (DOD) (k (cat) / km) =(0.8 - -2.1) + / - 0.1(2)内部fluorescence-quenched基质(a) (AB) Val-Phe-Pro-Arg-Ser-Phe-Arg-Leu-Lys -Asp-OH (DNP),最优序列,(b) (AB) var - ser - pro - arg - ser - ph - gln - lys (DNP)- asp - oh对因子VIII的识别序列为（DOD）k(cat) = 2.2 +/- 0.2和（DOD）(k(cat)/ k -m) =(0.8-0.9) +/- 0.1，在pL （L = H， D）最大值为8.4-9.0和(25.0-26.0)+/- 0.1度。最合理的模型拟合部分同位素效应（质子库存）数据是在所有底物浓度下，假设速率决定的酰化作用，根据最小的卡方值和分馏因子的一致性选择的。Z-Pro-Arg-7-AMC的数据与k（cat）和k(cat)/ k -m的过渡态单质子桥值phi(TS) = 0.39 +/- 0.05和溶剂重组组分phi(S) = 0.8 +/- 0.1和phi(S) = 1.22相一致。三肽酰胺的数据符合碗形曲线；例如n -t- bc - val - pro - arg -7- amc: phi(1)(TS) = phi(2)(TS) = 0.57 +/- 0.01, k(cat) = 1, k(cat)/ k -m = 1.6 +/- 0.1。非肽（2b）的质子库存是线性的。h - d - ph - l - pip - arg - pna和十肽（2a）的k（cat）数据与催化质子桥接的两个相同的分离因子最一致，phi(1)(TS) = phi(2)(TS) = 0.68 +/- 0.02，而后者的一个大的反分量（phi(S) = 3.1 +/- 0.5），表明在离开基团时溶剂发生了实质性的重组。几乎所有底物的k(cat)/ k -m质子库存曲线都呈圆顶状，其逆同位素效应分量（phi(S) = 1.2-2.4）源于凝血酶与底物结合过程中的溶剂重组。这些来自中等效应的巨大贡献完全符合凝血酶的双重止血和溶栓功能所需的构象调整。
Deuterium kinetic solvent isotope effects for the human alpha-thrombin-catalyzed hydrolysis of (1) substrates with selected P-1-P-3 sites, Z-Pro-Arg-7-amido-4-methylcoumarin (7-AMC), N-t-Boc-Val-Pro-Arg-7-AMC, Bz-Phe-Val-Arg-4-nitroanilide (pNA), and H-D-Phe-L-Pip-Arg-pNA, are (DOD)k(cat) = (2.8-3.3) +/- 0.1 and (DOD)(k(cat)/K-m) = (0.8-2.1) +/- 0.1 and (2) internally fluorescence-quenched substrates (a) (AB)Val-Phe-Pro-Arg-Ser-Phe-Arg-Leu-Lys(DNP)-Asp-OH, an optimal sequence, and (b) (AB)Val-Ser-Pro-Arg-Ser-Phe-Gln-Lys(DNP)-Asp-OH, recognition sequence for factor VIII, are (DOD)k(cat) = 2.2 +/- 0.2 and (DOD)(k(cat)/K-m) = (0.8-0.9) +/- 0.1, at the pL (L = H, D) maximum, 8.4-9.0, and (25.0-26.0) +/- 0.1 degreesC. The most plausible models fitting the partial isotope effect (proton inventory) data have been selected on the basis of lowest values of the reduced chi squared and consistency of fractionation factors at all substrate concentrations, assuming rate-determining acylation. The data for Z-Pro-Arg-7-AMC are consistent with a single-proton bridge at the transition state phi(TS) = 0.39 +/- 0.05 and components for solvent reorganization phi(S) = 0.8 +/- 0.1 and phi(S) = 1.22 for k(cat) and k(cat)/K-m, respectively. The data for tripeptide amides fit bowl-shaped curves; an example is N-t-Boc-Val-Pro-Arg-7-AMC: phi(1)(TS) = phi(2)(TS) = 0.57 +/- 0.01 and phi(S) = 1 for k(cat) and 1.6 +/- 0.1 for k(cat)/K-m. Proton inventories for the nonapeptide (2b) are linear. The data for k(cat) for H-D-Phe-L-Pip-Arg-pNA and the decapeptide (2a) are most consistent with two identical fractionation factors for catalytic proton bridging, phi(1)(TS) = phi(2)(TS) = 0.68 +/- 0.02 and a large inverse component (phi(S) = 3.1 +/- 0.5) for the latter, indicative of substantial solvent reorganization upon leaving group departure. Proton inventory curves for k(cat)/K-m for nearly all substrates are dome-shaped with an inverse isotope effect component (phi(S) = 1.2-2.4) originating from solvent reorganization during association of thrombin with substrate. These large contributions from medium effects are in full accord with the conformational adjustments required for the fulfillment of the dual, hemostatic and thrombolytic, functions of thrombin.