Binding of chloromethyl ketone substrate analogues to crystalline papain.
Binding of chloromethyl ketone substrate analogues to crystalline papain.
复制标题
氯甲基酮底物类似物与结晶木瓜蛋白酶的结合。
作者：
J. Drenth;K. H. Kalk;H. Swen
木瓜蛋白酶（EC 3.4.22.2）是一种蛋白水解酶，其三维结构已通过X射线衍射以2.8 A分辨率测定（Drenth，J.，Jansonius，J.N.，Koekoek河Swen，H. M.，和Wothers，B.G.（1968），Nature（伦敦）218，929-932）。活性位点是分子表面上的凹槽，其中半胱氨酸-25的基本巯基位于组氨酸-159的咪唑环旁边。本研究的主要目的是通过差分傅立叶技术确定沟槽中底物的结合模式，以解释酶的底物特异性（P2应具有疏水侧链（Berger和Schechter，1970）），并有助于阐明催化机制。为此，三个氯甲基酮底物类似物与酶通过共价连接到半胱氨酸-25的硫原子反应。产物与母体结构同晶结晶，母体结构不是天然的活性酶，而是氧化木瓜蛋白酶（可能是木瓜蛋白酶-SO2-）和木瓜蛋白酶与连接到半胱氨酸-25的额外半胱氨酸的混合物。虽然这使得差异电子密度图的解释不那么容易，但它为我们提供了底物的酰基部分结合在活性位点凹槽中的方式的清晰图像。P1残基的羰基氧靠近两个潜在的氢键供体基团，半胱氨酸-25的主链NH和谷氨酰胺-19的NH 2。缬氨酸残基133和157负责木瓜蛋白酶在其底物裂解中的偏好。通过去除共价连接的抑制剂分子的硫原子的半胱氨酸-25的亚甲基，我们得到了可接受的模型的酰基酶的结构和四面体中间体。P1残基的羰基氧在四面体中间体中携带形式负电荷，通过与半胱氨酸-25的骨架NH和谷氨酰胺-19的NH 2基团形成两个氢键来稳定。这种情况类似于蛋白水解丝氨酸酶（亨德森，R.，赖特角S.，赫斯，G。P.，Blow，D. M.（1971），冷泉港研讨会。定量36，63-70; Robertus，J.D.，克劳特，奥尔登河一、和Birktoft，J. J.（1972 b），Biochemistry 11，4293-4303）。在组氨酸-159的咪唑环附近发现易断裂肽键的氮原子，表明该环在质子化离去基团的N原子中发挥作用（Lowe，1970）。这种质子转移将通过环围绕C β-C γ键从与硫原子的面内位置到与N原子的面内位置的30度旋转来促进。这种旋转的可能性来自于完全氧化的木瓜蛋白酶与亲本蛋白质的差异电子密度图。
Papain (EC 3.4.22.2) is a proteolytic enzyme, the three-dimensional structure of which has been determined by x-ray diffraction at 2.8 A resolution (Drenth, J., Jansonius, J.N., Koekoek, R., Swen, H. M., and Wothers, B.G. (1968), Nature (London) 218, 929-932). The active site is a groove on the molecular surface in which the essential sulfhydryl group of cysteine-25 is situated next to the imidazole ring of histidine-159. The main object of this study was to determine by the difference-Fourier technique the binding mode for the substrate in the groove in order to explain the substrate specificity of the enzyme (P2 should have a hydrophobic side chain (Berger and Schechter, 1970) and to contribute to an elucidation of the catalytic mechanism. To this end, three chloromethyl ketone substrate analogues were reacted with the enzyme by covalent attachment to the sulfur atom of cysteine-25. The products crystallized isomorphously with the parent structure that is not the native, active enzyme but a mixture of oxidized papain (probably papain-SO2-) and papain with an extra cysteine attached to cysteine-25. Although this made the interpretation of the difference electron density maps less easy, it provided us with a clear picture of the way in which the acyl part of the substrate binds in the active site groove. The carbonyl oxygen of the P1 residue is near two potential hydrogen-bond donating groups, the backbone NH of cysteine-25 and the NH2 of glutamine-19. Valine residues 133 and 157 are responsible for the preference of papain in its substrate splitting. By removing the methylene group that covalently attaches the inhibitor molecules to the sulfur atom of cysteine-25 we obtained acceptable models for the acyl-enzyme structure and for the tetrahedral intermediate. The carbonyl oxygen of the P1 residue, carrying a formal negative charge in the tetrahedral intermediate, is stabilized by formation of two hydrogen bonds with the backbone NH of cysteine-25 and the NH2 group of glutamine-19. This situation resembles that suggested for the proteolytic serine enzymes (Henderson, R., Wright, C. S., Hess, G. P., and Blow, D. M. (1971), Cold Spring Harbor Symp. Quant. Biol. 36, 63-70; Robertus, J. D., Kraut, J., Alden, R. A., and Birktoft, J. J. (1972b), Biochemistry 11, 4293-4303). The nitrogen atom of the scissile peptide bond was found close to the imidazole ring of histidine-159, suggesting a role for this ring in protonating the N atom of the leaving group (Lowe, 1970). This proton transfer would be facilitated by a 30 degrees rotation of the ring around the C beta-Cgamma bond from an in-plane position with the sulfur atom to an in-plane position with the N atom. The possibility of this rotation is derived from a difference electron-density map for fully oxidizied papain vs. the parent protein.