Malikayil J A, Burkhart J P, Schreuder H A, Broersma R J, Tardif C, Kutcher L W, Mehdi S, Schatzman G L, Neises B, Peet N P
Hoechst Marion Roussel, Inc., Cincinnati, Ohio 45215, USA.
Biochemistry. 1997 Feb 4;36(5):1034-40. doi: 10.1021/bi9622231.
An inhibitor of alpha-thrombin was designed on the basis of the X-ray crystal structures of thrombin and trypsin. The design strategy employed the geometric and electrostatic differences between the specificity pockets of the two enzymes. These differences arise due to the replacement of Ser 190 in trypsin by Ala 190 in thrombin. The new inhibitor contained a tryptophan side chain instead of the arginine side chain that is present in the prototypical thrombin inhibitors. This inhibitor had a Ki value of 0.25 microM, displayed more than 400-fold specificity for thrombin over trypsin, and doubled the rat plasma APTT at a concentration of 44.9 microM. The X-ray crystal structure of the inhibitor/alpha-thrombin complex was determined. This represents the first reported three-dimensional structure of a thrombin/ inhibitor complex where the specificity pocket of the enzyme is occupied by a chemical moiety other than a guanidino or an amidino group. As was predicted by the molecular model, the tryptophan side chain docks into the specificity pocket of the enzyme. This finding is in contrast with the indole binding region of thrombin reported earlier [Berliner, L. J., & Shen, Y. Y. L. (1977) Biochemistry 16, 4622-4626]. The lower binding affinity of the new inhibitor for trypsin, compared to that for thrombin, appears to be due to (i) the extra energy required to deform the smaller specificity pocket of trypsin to accommodate the bulky indole group and (ii) the favorable electrostatic interactions of the indole group with the more hydrophobic specificity pocket of thrombin. The neutral indole group may be of pharmacological significance because the severe hypotension and respiratory distress observed following the administration of some thrombin inhibitors have been linked to the positively charged guanidino or amidino functionalities.
基于凝血酶和胰蛋白酶的X射线晶体结构设计了一种α-凝血酶抑制剂。设计策略利用了这两种酶特异性口袋之间的几何和静电差异。这些差异是由于胰蛋白酶中190位的丝氨酸被凝血酶中190位的丙氨酸取代而产生的。新抑制剂含有一个色氨酸侧链，而不是原型凝血酶抑制剂中存在的精氨酸侧链。该抑制剂的Ki值为0.25微摩尔，对凝血酶的特异性比对胰蛋白酶高400多倍，在浓度为44.9微摩尔时使大鼠血浆活化部分凝血活酶时间（APTT）加倍。测定了抑制剂/α-凝血酶复合物的X射线晶体结构。这是首次报道的凝血酶/抑制剂复合物的三维结构，其中酶的特异性口袋被胍基或脒基以外的化学部分占据。正如分子模型所预测的，色氨酸侧链嵌入酶的特异性口袋。这一发现与早期报道的凝血酶吲哚结合区域[Berliner, L. J., & Shen, Y. Y. L. (1977) Biochemistry 16, 4622 - 4626]形成对比。与凝血酶相比，新抑制剂对胰蛋白酶的结合亲和力较低，这似乎是由于：（i）使胰蛋白酶较小的特异性口袋变形以容纳庞大的吲哚基团所需的额外能量，以及（ii）吲哚基团与凝血酶更疏水的特异性口袋之间有利的静电相互作用。中性吲哚基团可能具有药理学意义，因为在施用一些凝血酶抑制剂后观察到的严重低血压和呼吸窘迫与带正电荷的胍基或脒基官能团有关。
