Proton bridging in the interactions of thrombin with small inhibitors.
Proton bridging in the interactions of thrombin with small inhibitors.
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DOI：
10.1021/bi900098s
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发表时间：
2009-08-04
期刊：
影响因子：
2.9
通讯作者：
Baykal, Ahmet
中科院分区：
文献类型：
--
作者：
Kovach, Ildiko M.;Kelley, Paul;Eddy, Carol;Jordan, Frank;Baykal, Ahmet
凝血酶是血液级联系统中关键的丝氨酸蛋白酶。Phe-Pro-Arg-氯甲基酮（PPACK）、磷酸酯和膦酸酯抑制剂与凝血酶的活性位点Ser形成共价键。PPACK是一种基于机制的抑制剂，与磷酸酯/膦酸酯形成加合物，模拟凝血酶催化反应中形成的中间体。因此，研究了人α-凝血酶抑制对这些抑制剂浓度、pH和温度的依赖性。PPACK抑制人α-凝血酶的二级速率常数ki/Ki和抑制常数Ki分别为（1.1 ± 0.2）× 107 M−1 s−1和（2.4 ± 1.3）× 10−8 M，在pH 7.00的0.05 M磷酸盐缓冲液、0.15 M NaCl和25.0 ± 0.1 μ C中，与以前的报道一致。在pH7.00、0.05M磷酸盐缓冲液和0.15MNaCl条件下，活化参数分别为：ΔH_∞ = 10.6 ± 0.7kcal/mol和ΔS_∞ = 9 ± 2cal/mol deg.抑制的二级速率常数的pH依赖性是钟形的。在25.0 ± 0.1 °C下，pKa 1和pKa 2的值分别为7.3 ± 0.2和8.8 ± 0.3。磷酸盐和膦酸酯抑制剂的pKa 1值较高，分别为7.8和8.0，pKa 2值为9.3和8.6。它们抑制凝血酶的效率比PPACK低六个数量级。在pH 7.0和8.3、25.0 ± 0.1°C下的二级速率常数的氘溶剂同位素效应在所有三种情况下在实验误差内是一致的，表明在凝血酶与抑制剂缔合的速率决定步骤中不存在质子转移。但是在pH 6.7和30 °C下的抑制加合物的600 MHz 1H NMR光谱中，相对于TSP在18.10 ppm处出现PPACK峰，其在pH 5.3-8.5之间的酶溶液的1H NMR光谱中不存在。低场下的峰指示在加合物中的活性位点处存在SSHB。氢桥的氘同位素效应为2.2 ± 0.2（氘= 0.45）。对于凝血酶的脱烷基化磷酸盐加合物，还通过在17.34 ppm处的信号确定了SSHB的存在。
Thrombin is the pivotal serine protease enzyme in the blood cascade system. Phe-Pro-Arg-chloromethylketone (PPACK), phosphate and phosphonate ester inhibitors form a covalent bond with the active-site Ser of thrombin. PPACK, a mechanism-based inhibitor, and the phosphate/phosphonate esters form adducts that mimic intermediates formed in reactions catalyzed by thrombin. Therefore, the dependence of the inhibition of human α-thrombin on the concentration of these inhibitors, pH, and temperature was investigated. The second-order rate constant, ki/Ki, and the inhibition constant, Ki, for inhibition of human α-thrombin by PPACK are (1.1 ± 0.2) × 107 M−1 s−1 and (2.4 ± 1.3) × 10−8 M at pH 7.00 in 0.05 M phosphate buffer, 0.15 M NaCl, and 25.0 ± 0.1˚C, and in good agreement with previous reports. The activation parameters at pH 7.00, 0.05M phosphate buffer 0.15 M NaCl, are ΔH‡ = 10.6 ± 0.7 kcal/mol and ΔS‡ = 9 ± 2 cal/mol deg. The pH dependence of the second-order rate constants of inhibition is bell shaped. Values of pKa1 and pKa2 are 7.3 ± 0.2 and 8.8 ± 0.3, respectively, at 25.0 ± 0.1 °C. A phosphate and a phosphonate ester inhibitor gave higher values, 7.8 and 8.0, for pKa1 and 9.3 and 8.6 for pKa2. They inhibit thrombin over six orders of magnitude less efficiently than PPACK does. The deuterium solvent isotope effect for the second-order rate constant at pH 7.0 and 8.3 at 25.0 ± 0.1°C is unity within experimental error in all three cases, indicating the absence of proton transfer in the rate-determining step for the association of thrombin with the inhibitors. But in a 600 MHz 1H NMR spectrum of the inhibition adduct at pH 6.7 and 30 °C, a peak at 18.10 ppm with respect to TSP appears with PPACK, which is absent in the 1H NMR spectrum of a solution of the enzyme between pH 5.3–8.5. The peak at low field is an indication of the presence an SSHB at the active site in the adduct. The deuterium isotope effect on this hydrogen bridge is 2.2 ± 0.2 (ϕ = 0.45). The presence of an SSHB is also established with a signal at 17.34 ppm for a dealkylated phosphate adduct of thrombin.
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