Kashima A, Inoue Y, Sugio S, Maeda I, Nose T, Shimohigashi Y
Research Division, The Green Cross Corp., Hirakata, Japan.
Eur J Biochem. 1998 Jul 1;255(1):12-23. doi: 10.1046/j.1432-1327.1998.2550012.x.
The dipeptide D-leucyl-L-phenylalanyl p-fluorobenzylamide (D-Leu-Phe-NH-BzlF) inhibits chymotrypsin strongly in a competitive manner with the Ki value of 0.61 microM [Shimohigashi, Y., Maeda, I., Nose, T., Ikesue, K., Sakamoto, H., Ogawa, T., Ide, Y., Kawahara, M., Nezu, T., Terada, Y., Kawano, K. & Ohno, M. (1996) J. Chem. Soc. Perkin Trans. 1, 2479-2485]. The structure/activity studies have suggested a unique inhibitory conformation, in which the C-terminal benzyl group fits the chymotrypsin S1 site and the hydrophobic core constructed by the side chains of D-Leu-Phe fits the S2 or S1' site. To verify this assumption, the molecular structure of the complex between the dipeptide and gamma-chymotrypsin has been determined crystallographically. Gamma-chymotrypsin itself was first crystallized and refined at 1.6-A resolution. The refined structure was virtually identical to the conformation reported and the electron density at the active site was interpreted as a pentapeptide Thr-Pro-Gly-Val-Tyr derived from autolysis of the enzyme (residues 224-228). The chymotrypsin-dipeptide complex was obtained by soaking the crystals of gamma-chymotrypsin in a solution saturated with the dipeptide inhibitor. The crystal structure of the complex has been refined at 1.8-A resolution to a crystallographic R-factor of 18.1%. The structure of gamma-chymotrypsin in the complex agreed fairly well with that of gamma-chymotrypsin per se with a rmsd of 0.13 A for all the C alpha carbons. Two inhibitor molecules were assigned in an asymmetric unit, i.e. one in the active site and the other at the interface of two symmetry-related enzyme molecules. In both sites dipeptides adopted very similar folded conformations, in which side chains of D-Leu-Phe are spatially proximal. In the active site where the binding of dipeptide was judged to be a direct cause of inhibition, C-terminal p-fluorobenzylamide group of the dipeptide, NH-BzlF, was found in the S1 hydrophobic pocket. At the bottom of this pocket, the p-fluorine atom hydrogen bonded with a water molecule, probably to enhance the inhibitory activity. The stereospecific interaction of R and S isomers of the dipeptide with C-terminal NH-C*H(CH3)-C6H5 was well explained by the space available for methyl replacement in the complex. The hydrophobic core constructed by side chains of D-Leu-Phe was found at the broad S2 site. Interestingly, a novel interaction was found between the inhibitor Phe residue and chymotrypsin His57, the phenyl of Phe and the imidazole of His being in a pi-pi stacking interaction at a distance 3.75 A.
二肽D-亮氨酰-L-苯丙氨酰对氟苄酰胺（D-Leu-Phe-NH-BzlF）以竞争性方式强烈抑制胰凝乳蛋白酶，其Ki值为0.61微摩尔[下茂桥洋、前田一、野泽敏、池末久、坂本浩、小川哲、井手洋、川原正、根津哲、寺田洋、川野和夫及大野正（1996年）《化学学会志，珀金 Transactions 1》，2479 - 2485页]。结构/活性研究表明存在一种独特的抑制构象，其中C端苄基适合胰凝乳蛋白酶的S1位点，由D-Leu-Phe侧链构建的疏水核心适合S2或S1'位点。为验证这一假设，已通过晶体学方法确定了该二肽与γ-胰凝乳蛋白酶复合物的分子结构。首先使γ-胰凝乳蛋白酶自身结晶并在1.6埃分辨率下进行精修。精修后的结构与所报道的构象基本相同，活性位点的电子密度被解释为源自酶自溶的五肽Thr-Pro-Gly-Val-Tyr（残基224 - 228）。通过将γ-胰凝乳蛋白酶晶体浸泡在二肽抑制剂饱和溶液中获得胰凝乳蛋白酶 - 二肽复合物。该复合物的晶体结构已在1.8埃分辨率下精修至晶体学R因子为18.1%。复合物中γ-胰凝乳蛋白酶的结构与γ-胰凝乳蛋白酶本身的结构相当吻合，所有Cα碳原子的均方根偏差为0.13埃。在一个不对称单元中确定了两个抑制剂分子，即一个在活性位点，另一个在两个对称相关酶分子的界面处。在这两个位点，二肽都采用非常相似的折叠构象，其中D-Leu-Phe的侧链在空间上相邻。在被判定二肽结合是抑制直接原因的活性位点，发现二肽的C端对氟苄酰胺基团NH-BzlF位于S1疏水口袋中。在该口袋底部，对氟原子与一个水分子形成氢键，可能是为了增强抑制活性。二肽的R和S异构体与C端NH-C*H(CH3)-C6H5的立体特异性相互作用通过复合物中甲基取代可用空间得到了很好的解释。由D-Leu-Phe侧链构建的疏水核心位于宽阔的S2位点。有趣的是，发现抑制剂苯丙氨酸残基与胰凝乳蛋白酶His57之间存在一种新的相互作用，苯丙氨酸的苯基与His的咪唑形成了距离为3.75埃的π-π堆积相互作用。
