Kovach Ildiko M, Kelley Paul, Eddy Carol, Jordan Frank, Baykal Ahmet
Department of Chemistry, The Catholic University of America, Washington, D.C. 20064, USA.
Biochemistry. 2009 Aug 4;48(30):7296-304. doi: 10.1021/bi900098s.
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 alpha-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 alpha-thrombin by PPACK are (1.1 +/- 0.2) x 10(7) M(-1) s(-1) and (2.4 +/- 1.3) x 10(-8) M, respectively, at pH 7.00 in 0.05 M phosphate buffer and 0.15 M NaCl at 25.0 +/- 0.1 degrees C, in good agreement with previous reports. The activation parameters at pH 7.00 in 0.05 M phosphate buffer and 0.15 M NaCl are as follows: DeltaH = 10.6 +/- 0.7 kcal/mol, and DeltaS = 9 +/- 2 cal mol(-1) degrees C(-1). 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 degrees 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, respectively. They inhibit thrombin more than 6 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 degrees 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 degrees 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 and 8.5. The peak at low field is an indication of the presence of a short-strong hydrogen bond (SSHB) at the active site in the adduct. The deuterium isotope effect on this hydrogen bridge is 2.2 +/- 0.2 (phi = 0.45). The presence of an SSHB is also established with a signal at 17.34 ppm for a dealkylated phosphate adduct of thrombin.
凝血酶是血液级联系统中的关键丝氨酸蛋白酶。苯丙氨酸 - 脯氨酸 - 精氨酸 - 氯甲基酮（PPACK）、磷酸盐和膦酸酯抑制剂与凝血酶的活性位点丝氨酸形成共价键。PPACK是一种基于机制的抑制剂，磷酸盐/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 - 凝血酶抑制作用对这些抑制剂浓度、pH值和温度的依赖性。在25.0±0.1℃下，0.05 M磷酸盐缓冲液和0.15 M氯化钠中，pH 7.00时，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，与先前报道吻合良好。在0.05 M磷酸盐缓冲液和0.15 M氯化钠中，pH 7.00时的活化参数如下：ΔH = 10.6±0.7 kcal/mol，ΔS = 9±2 cal mol⁻¹℃⁻¹。抑制二级速率常数的pH依赖性呈钟形。在25.0±0.1℃时，pKa1和pKa2值分别为7.3±0.2和8.8±0.3。一种磷酸盐和一种膦酸酯抑制剂给出了更高的值，pKa1分别为7.8和8.0，pKa2分别为9.3和8.6。它们抑制凝血酶的效率比PPACK低6个数量级以上。在25.0±0.1℃下，pH 7.0和8.3时二级速率常数的氘溶剂同位素效应在所有三种情况下的实验误差范围内均为1，表明凝血酶与抑制剂结合的速率决定步骤中不存在质子转移，但在pH 6.7和30℃下抑制加合物的600 MHz ¹H NMR谱中，相对于TSP，在18.10 ppm处出现一个峰，而在pH 5.3至8.5之间的酶溶液的¹H NMR谱中不存在该峰。低场处的峰表明加合物活性位点存在短强氢键（SSHB）。该氢桥的氘同位素效应为2.2±0.2（φ = 0.45）。凝血酶脱烷基化磷酸盐加合物在17.34 ppm处的信号也证实了SSHB的存在。
