Krishnan R, Zhang E, Hakansson K, Arni R K, Tulinsky A, Lim-Wilby M S, Levy O E, Semple J E, Brunck T K
Corvas International Inc., San Diego, California 92121, USA.
Biochemistry. 1998 Sep 1;37(35):12094-103. doi: 10.1021/bi980840e.
The crystal structures of three highly potent and selective low-molecular weight rigid peptidyl aldehyde inhibitors complexed with thrombin have been determined and refined to R values 0.152-0. 170 at 1.8-2.1 A resolution. Since the selectivity of two of the inhibitors was >1600 with respect to trypsin, the structures of trypsin-inhibited complexes of these inhibitors were also determined (R = 0.142-0.157 at 1.9-2.1 A resolution). The selectivity appears to reside in the inability of a benzenesulfonamide group to bind at the equivalent of the D-enantiomorphic S3 site of thrombin, which may be related to the lack of a 60-insertion loop in trypsin. All the inhibitors have a novel lactam moiety at the P3 position, while the two with greatest trypsin selectivity have a guanidinopiperidyl group at the P1 position that binds in the S1 specificity site. Differences in the binding constants of these inhibitors are correlated with their interactions with thrombin and trypsin. The kinetics of inhibition vary from slow to fast with thrombin and are fast in all cases with trypsin. The kinetics are examined in terms of the slow formation of a stable transition-state complex in a two-step mechanism. The structures of both thrombin and trypsin complexes show similar well-defined transition states in the S1 site and at the electrophilic carbon atom and Ser195OG. The trypsin structures, however, suggest that the first step in a two-step kinetic mechanism may involve formation of a weak transition-state complex, rather than binding dominated by the P2-P4 positions.
已测定三种与凝血酶复合的高效、选择性低分子量刚性肽基醛抑制剂的晶体结构，并在1.8 - 2.1 Å分辨率下精修至R值为0.152 - 0.170。由于其中两种抑制剂对胰蛋白酶的选择性大于1600，因此也测定了这些抑制剂与胰蛋白酶形成的抑制复合物的结构（在1.9 - 2.1 Å分辨率下R = 0.142 - 0.157）。这种选择性似乎在于苯磺酰胺基团无法结合在凝血酶的D - 对映体S3位点的等效位置，这可能与胰蛋白酶中缺乏60插入环有关。所有抑制剂在P3位置都有一个新型内酰胺部分，而对胰蛋白酶选择性最高的两种抑制剂在P1位置有一个胍基哌啶基团，该基团结合在S1特异性位点。这些抑制剂结合常数的差异与它们与凝血酶和胰蛋白酶的相互作用相关。抑制动力学对凝血酶而言从慢到快变化，而对胰蛋白酶在所有情况下都是快速的。根据两步机制中稳定过渡态复合物的缓慢形成来研究动力学。凝血酶和胰蛋白酶复合物的结构在S1位点以及亲电碳原子和Ser195OG处都显示出相似的明确过渡态。然而，胰蛋白酶的结构表明，两步动力学机制的第一步可能涉及形成弱过渡态复合物，而不是由P2 - P4位置主导的结合。
