ALLOSTERIC MODULATION OF ACETYLCHOLINESTERASE ACTIVITY BY PERIPHERAL LIGANDS INVOLVES A CONFORMATIONAL TRANSITION OF THE ANIONIC SUBSITE
ALLOSTERIC MODULATION OF ACETYLCHOLINESTERASE ACTIVITY BY PERIPHERAL LIGANDS INVOLVES A CONFORMATIONAL TRANSITION OF THE ANIONIC SUBSITE
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DOI：
10.1021/bi00047a008
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发表时间：
1995-11-28
期刊：
影响因子：
2.9
通讯作者：
SHAFFERMAN, A
中科院分区：
文献类型：
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作者：
BARAK, D;ORDENTLICH, A;SHAFFERMAN, A
Asp74、Trp286 和 Tyr72 残基（人乙酰胆碱酯酶 (HuAChE) 的外周阴离子位点 (PAS) 的组成部分）的替换同样影响 PAS 特异性配体丙啶的结合常数和抑制常数，表明抑制活性的变化是与 PAS 结合改变的直接结果。相比之下，活性中心HuAChE突变体W86A和Y133A分别表现出对丙啶抑制的350倍和25倍增加的抵抗力，但结合亲和力没有变化，表明PAS介导的抑制的变构机制涉及这些Trp86和Tyr133残基的构象变化，而不是抑制剂本身对底物进入的物理阻碍。这些发现支持了最近的提议，即变构机制通过阴离子亚位点 Trp86 的活性和非活性构象之间的转变来发挥作用，并且用丙氨酸替换 Tyr133 可以稳定封闭活性中心的非活性 Trp86 构象 [Ordentlich 等人，2017]。 (1995) J.Biol。化学。 270、2082]。为了进一步支持这一机制和 Tyr133 的作用，我们发现 (a) 不可逆抑制剂氟磷酸二异丙酯或对氧磷与 Y133A HuAChE 的非共价复合物的解离常数 (Kd) 相对于野生型酶或其 Y133F 或 W86A 突变体增加了 20-500 倍； (b)通过从Y133A酶（即W86A/Y133A突变体）中去除Trp86来恢复底物如3,3-二甲基丁基硫代乙酸酯的接近。我们认为 Trp86 的构象转变与 HuAChE 的半胱氨酸环 (Cys69-Cys96) 的运动耦合，并且是天然酶的动力学所固有的。
Replacement of residues Asp74, Trp286, and Tyr72, which are constituents of the peripheral anionic site (PAS) of human acetylcholinesterase (HuAChE), affected similarly both the binding and the inhibition constants of the PAS-specific ligand propidium, demonstrating that changes in the inhibitory activity are a direct consequence of altered binding to the PAS. In contrast, the active center HuAChE mutants W86A and Y133A show respective 350- and 25-fold increased resistance to inhibition by propidium but no change in binding affinities, demonstrating that the allosteric mechanism of PAS-mediated inhibition involves a conformational change of-these Trp86 and Tyr133 residues rather than physical obstruction of substrate access by the inhibitor itself. These findings support the recent proposal that the allosteric mechanism operates via transition between active and nonactive conformations of the anionic subsite Trp86 and that replacement of Tyr133 by alanine may stabilize a nonactive Trp86 conformation that occludes the active center [Ordentlich et al. (1995) J. Biol. Chem. 270, 2082]. In further support of this mechanism and the role of Tyr133, we find that (a) the dissociation constants (Kd) for the noncovalent complexes of the irreversible inhibitors diisopropyl phosphorofluoridate or paraoxon with Y133A HuAChE are increased 20-500-fold, relative to either wild-type enzyme or its Y133F or W86A mutants; and (b) access of substrates such as 3,3-dimethylbutyl thioacetate is restored by removal of Trp86 from the Y133A enzyme (i.e., the W86A/Y133A mutant). We suggest that the conformational transition of Trp86 is coupled to the motions of the cysteine loop (Cys69-Cys96) of HuAChE and is inherent to the dynamics of the native enzyme.