Chen Z, Li Y, Mulichak A M, Lewis S D, Shafer J A
Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Arch Biochem Biophys. 1995 Sep 10;322(1):198-203. doi: 10.1006/abbi.1995.1452.
Crystals of human alpha-thrombin complexed with hirugen and the alpha-keto acid thrombin inhibitor APPA (p-amidinophenylpyruvate) that diffract to 1.6 A resolution were obtained by soaking an alpha-thrombin-hirugen crystal in a solution of APPA. The crystal structure was determined using the difference Fourier method and refined to an R factor of 18.7% at 1.6 A resolution. This structure is the highest resolution structure of the thrombin molecule that is currently available. With the exception of the region near Arg77A-Asn78, the structures of the thrombin and hirugen molecules in the ternary complex are similar to those reported for the thrombin-hirugen binary complex. As previously determined for the APPA-trypsin complex, the carbonyl carbon atom of APPA forms a covalent bond with O gamma of Ser195 of thrombin to yield a "transition-state" analog of the tetrahedral intermediate. Comparison of the specificity pocket of the APPA complexes of thrombin and trypsin reveals differences in hydrogen bonding and shows for the first time that the S1 site of thrombin is larger than that of trypsin and as a result thrombin may be able to accommodate a bulkier P1 group than trypsin.
通过将α-凝血酶-水蛭素晶体浸泡在APPA（对脒基苯丙酮酸）溶液中，获得了与水蛭素和α-酮酸凝血酶抑制剂APPA复合的人α-凝血酶晶体，其衍射分辨率达到1.6 Å。使用差值傅里叶方法确定了晶体结构，并在1.6 Å分辨率下精修至R因子为18.7%。该结构是目前可用的凝血酶分子的最高分辨率结构。除了靠近Arg77A-Asn78的区域外，三元复合物中凝血酶和水蛭素分子的结构与报道的凝血酶-水蛭素二元复合物的结构相似。如先前对APPA-胰蛋白酶复合物所确定的，APPA的羰基碳原子与凝血酶的Ser195的Oγ形成共价键，从而产生四面体中间体的“过渡态”类似物。凝血酶和胰蛋白酶的APPA复合物的特异性口袋的比较揭示了氢键的差异，并首次表明凝血酶的S1位点比胰蛋白酶的大，因此凝血酶可能比胰蛋白酶能够容纳更大的P1基团。
