Koeppe Julia R, Komives Elizabeth A
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0378, USA.
Biochemistry. 2006 Jun 27;45(25):7724-32. doi: 10.1021/bi060405h.
Thrombin is a dual action serine protease in the blood clotting cascade. Similar to other clotting factors, thrombin is mainly present in the blood in a zymogen form, prothrombin. Although the two cleavage events required to activate thrombin are well-known, little is known about why the thrombin precursors are inactive proteases. Although prothrombin is much larger than thrombin, prethrombin-2, which contains all of the same amino acids as thrombin, but has not yet been cleaved between Arg320 and Ile321, remains inactive. Crystal structures of both prethrombin-2 and thrombin are available and show almost no differences in the active site conformations. Slight differences were, however, seen in the loops surrounding the active site, which are larger in thrombin than in most other trypsin-like proteases, and have been shown to be important for substrate specificity. To explore whether the dynamics of the active site loops were different in the various zymogen forms of thrombin, we employed amide H/(2)H exchange experiments to compare the exchange rates of regions of thrombin with the same regions of prothrombin, prethrombin-2, and meizothrombin. Many of the surface loops showed less exchange in the zymogen forms, including the large loop corresponding to anion binding exosite 1. Conversely, the autolysis loop and sodium-binding site exchanged more readily in the zymogen forms. Prothrombin and prethrombin-2 gave nearly identical results while meizothrombin in some regions more closely resembled active thrombin. Thus, cleavage of the Arg320-Ile321 peptide bond is the key to formation of the active enzyme, which involves increased dynamics of the substrate-binding loops and decreased dynamics of the catalytic site.
凝血酶是血液凝固级联反应中的一种双功能丝氨酸蛋白酶。与其他凝血因子类似，凝血酶在血液中主要以酶原形式即凝血酶原存在。尽管激活凝血酶所需的两个裂解事件已为人熟知，但关于凝血酶前体为何是无活性蛋白酶却知之甚少。虽然凝血酶原比凝血酶大得多，但凝血酶原2包含与凝血酶相同的所有氨基酸，只是在精氨酸320和异亮氨酸321之间尚未裂解，它仍然无活性。凝血酶原2和凝血酶的晶体结构均已可得，且活性位点构象几乎没有差异。然而，在活性位点周围的环中观察到了细微差异，这些环在凝血酶中比大多数其他胰蛋白酶样蛋白酶中的更大，并且已证明对底物特异性很重要。为了探究凝血酶各种酶原形式中活性位点环的动力学是否不同，我们采用酰胺H/（2）H交换实验来比较凝血酶区域与凝血酶原、凝血酶原2和中凝血酶相同区域的交换率。许多表面环在酶原形式中显示出较少的交换，包括对应于阴离子结合外位点1的大环。相反，自溶环和钠结合位点在酶原形式中更容易交换。凝血酶原和凝血酶原2给出了几乎相同的结果，而中凝血酶在某些区域更类似于活性凝血酶。因此，精氨酸320 - 异亮氨酸321肽键的裂解是活性酶形成的关键，这涉及底物结合环动力学的增加和催化位点动力学的降低。
