Shafferman Avigdor, Barak Dov, Stein Dana, Kronman Chanoch, Velan Baruch, Greig Nigel H, Ordentlich Arie
Department Biochemistry and Molecular Biology, Israel Institute for Biological Research, Ness-Ziona, Israel.
Chem Biol Interact. 2008 Sep 25;175(1-3):166-72. doi: 10.1016/j.cbi.2008.03.013. Epub 2008 Apr 8.
Functional architecture of the AChE active center appears to be characterized by both structural "rigidity", necessary to stabilize the catalytic triad as well as by flexibility in accommodating the different, high affinity AChE ligands. These seemingly conflicting structural properties of the active center are demonstrated through combination of structural methods with kinetic studies of the enzyme and its mutant derivatives with plethora of structurally diverse ligands and in particular with series of stereoselective covalent and noncovalent AChE ligands. Thus, steric perturbation of the acyl pocket precipitates in a pronounced stereoselectivity toward methylphosphonates by disrupting the stabilizing environment of the catalytic histidine rather than through steric exclusion demonstrating the functional importance of the "rigid" environment of the catalytic machinery. The acyl pocket, the cation-binding subsite (Trp86) and the peripheral anionic subsite were also found to be directly involved in HuAChE stereoselectivity toward charged chiral phosphonates, operating through differential positioning of the ligand cationic moiety within the active center. Residue Trp86 is also a part of the "hydrophobic patch" which seems flexible enough to accommodate the structurally diverse ligands like tacrine, galanthamine and the two diastereomers of huperzine A. Also, we have recently discovered further aspects of the role of both the unique structure and the flexibility of the "hydrophobic patch" in determining the reactivity and stereoselectivity of HuAChE toward certain carbamates including analogs of physostigmine. In these cases the ligands are accommodated mostly through hydrophobic interactions and their stereoselectivity delineates precisely the steric limits of the pocket. Hence, the HuAChE stereoselectivity provides a sensitive tool in the in depth exploration of the functional architecture of the active center. These studies suggest that the combination of "rigidity" and flexibility within the HuAChE gorge are an essential element of its molecular design.
乙酰胆碱酯酶（AChE）活性中心的功能结构似乎具有两种特性：结构“刚性”，这对于稳定催化三联体是必需的；以及灵活性，以便容纳不同的高亲和力AChE配体。活性中心这些看似相互矛盾的结构特性通过结构方法与对该酶及其突变衍生物进行动力学研究相结合得以证明，这些研究使用了大量结构多样的配体，特别是一系列立体选择性共价和非共价AChE配体。因此，酰基口袋的空间扰动通过破坏催化组氨酸的稳定环境而非通过空间排斥，导致对甲基膦酸酯具有明显的立体选择性，这证明了催化机制“刚性”环境的功能重要性。还发现酰基口袋、阳离子结合亚位点（Trp86）和外周阴离子亚位点直接参与人源乙酰胆碱酯酶（HuAChE）对带电荷手性膦酸酯的立体选择性，其通过配体阳离子部分在活性中心内的不同定位来实现。残基Trp86也是“疏水补丁”的一部分，该“疏水补丁”似乎足够灵活，能够容纳结构多样的配体，如他克林、加兰他敏和石杉碱甲的两种非对映异构体。此外，我们最近还发现了“疏水补丁”独特结构和灵活性在决定HuAChE对某些氨基甲酸酯（包括毒扁豆碱类似物）的反应性和立体选择性方面作用的更多方面。在这些情况下，配体主要通过疏水相互作用被容纳，并且它们的立体选择性精确地描绘了口袋的空间界限。因此，HuAChE的立体选择性为深入探索活性中心的功能结构提供了一个灵敏的工具。这些研究表明，HuAChE峡谷内“刚性”和灵活性的结合是其分子设计的一个基本要素。
