Radić Z, Pickering N A, Vellom D C, Camp S, Taylor P
Department of Pharmacology, University of California at San Diego, La Jolla 92093-0636.
Biochemistry. 1993 Nov 16;32(45):12074-84. doi: 10.1021/bi00096a018.
By examining inhibitor interactions with single and multiple site-specific mutants of mouse acetylcholinesterase, we have identified three distinct domains in the cholinesterase structure that are responsible for conferring selectivity for acetyl- and butyrylcholinesterase inhibitors. The first domain is the most obvious; it defines the constraints on the acyl pocket dimensions where the side chains of F295 and F297 primarily outline this region in acetylcholinesterase. Replacement of these phenylalanine side chains with the aliphatic residues found in butyrylcholinesterase allows for the catalysis of larger substrates and accommodates butyrylcholinesterase-selective alkyl phosphates such as isoOMPA. Also, elements of substrate activation characteristic of butyrylcholinesterase are evident in the F297I mutant. Substitution of tyrosines for F295 and F297 further alters the catalytic constants. The second domain is found near the lip of the active center gorge defined by two tyrosines, Y72 and Y124, and by W286; this region appears to be critical for the selectivity of bisquaternary inhibitors, such as BW284C51. The third domain defines the site of choline binding. Herein, in addition to conserved E202 and W86, a critical tyrosine, Y337, found only in the acetylcholinesterases is responsible for sterically occluding the binding site for substituted tricyclic inhibitors such as ethopropazine. Analysis of a series of substituted acridines and phenothiazines defines the groups on the ligand and amino acid side chains in this site governing binding selectivity. Each of the three domains is defined by a cluster of aromatic residues. The two domains stabilizing the quaternary ammonium moieties each contain a negative charge, which contributes to the stabilization energy of the respective complexes.
通过研究抑制剂与小鼠乙酰胆碱酯酶单一位点和多位点特异性突变体的相互作用，我们在胆碱酯酶结构中确定了三个不同的结构域，它们负责赋予对乙酰胆碱酯酶和丁酰胆碱酯酶抑制剂的选择性。第一个结构域最为明显；它定义了酰基口袋尺寸的限制，在乙酰胆碱酯酶中，F295和F297的侧链主要勾勒出该区域。用丁酰胆碱酯酶中发现的脂肪族残基取代这些苯丙氨酸侧链，可催化更大的底物，并容纳丁酰胆碱酯酶选择性的烷基磷酸酯，如异氟磷。此外，F297I突变体中明显具有丁酰胆碱酯酶底物活化的特征。用酪氨酸取代F295和F297会进一步改变催化常数。第二个结构域位于由两个酪氨酸Y72和Y124以及W286所界定的活性中心峡谷的边缘附近；该区域似乎对双季铵抑制剂（如BW284C51）的选择性至关重要。第三个结构域定义了胆碱结合位点。在此，除了保守的E202和W86外，仅在乙酰胆碱酯酶中发现的关键酪氨酸Y337在空间上封闭了诸如乙丙嗪等取代三环抑制剂的结合位点。对一系列取代吖啶和吩噻嗪的分析确定了该位点中配体和氨基酸侧链上决定结合选择性的基团。这三个结构域中的每一个都由一簇芳香族残基所界定。稳定季铵部分的两个结构域各自都含有一个负电荷，这有助于相应复合物的稳定能。
