Berman H A, Decker M M
Biochim Biophys Acta. 1986 Jul 25;872(1-2):125-33. doi: 10.1016/0167-4838(86)90155-x.
This study examines the importance of electrostatic interactions on ligand association at the active center of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). The active-center serine was covalently modified with the dimensionally equivalent isosteric beta-(trimethylammonium)ethyl and 3,3-dimethylbutyl methylphosphonofluoridates. Reactivation of the 3,3-dimethylbutyl methylphosphono-conjugate by the bisquaternary mono-oxime HI-6, after accounting for the capacity for spontaneous reactivation, proceeded at a rate that was 20-fold greater than that for the cationic conjugate. Decidium, a fluorescent bisquaternary ligand that binds with its trimethylammonium moiety within the active center, exhibited affinity for the 3,3-dimethylbutyl conjugate that was within 2-fold that for the native enzyme, but 100-fold greater than for the cationic conjugate. Whereas association of n-alkyl mono- and bisquaternary ligands with the uncharged conjugate was virtually unaltered with respect to the native enzyme, the affinities of edrophonium, phenyltrimethylammonium and N-methylacridinium were reduced 100-fold for the uncharged conjugate relative to native enzyme. These results indicate that the orientations of the 3,3-dimethylbutyl and beta-(trimethylammonium)ethyl moieties with respect to the surface of the enzyme are not equivalent, that modification of the active center does not preclude cation association of active-center-selective ligands, and that aromatic cations associate at an anionic locus which is unique from that at which decidium and the n-alkyl mono- and bisquaternary cations associate. As such, the results point to the presence of a heterogeneity of cation binding sites within a circumscribed distance from the modified serine, and do not sustain the view proposed by Hasan et al. (J. Biol. Chem. 255 (1980) 3898-3904; 256, (1981) 7781-7785) that electrostatic interactions at the active center are subordinate to steric constraints imposed by a dimensionally restricted trimethyl site.
本研究考察了静电相互作用对乙酰胆碱酯酶（乙酰胆碱乙酰水解酶，EC 3.1.1.7）活性中心配体结合的重要性。活性中心丝氨酸用尺寸相当的等排体β-(三甲基铵)乙基和3,3-二甲基丁基甲基膦酰氟进行共价修饰。在考虑了自发复活能力后，双季铵单肟HI-6对3,3-二甲基丁基共轭物的复活速率比阳离子共轭物快20倍。Decidium是一种荧光双季铵配体，其在活性中心内与其三甲基铵部分结合，对3,3-二甲基丁基共轭物的亲和力比天然酶高2倍以内，但比阳离子共轭物高100倍。虽然正烷基单季铵和双季铵配体与不带电荷的共轭物的结合相对于天然酶几乎没有变化，但相对于天然酶，依酚氯铵、苯基三甲基铵和N-甲基吖啶鎓对不带电荷的共轭物的亲和力降低了100倍。这些结果表明，3,3-二甲基丁基和β-(三甲基铵)乙基部分相对于酶表面的取向不等同，活性中心的修饰并不排除活性中心选择性配体的阳离子结合，并且芳香阳离子在一个与Decidium以及正烷基单季铵和双季铵阳离子结合位点不同的阴离子位点结合。因此，结果表明在距修饰丝氨酸限定距离内存在阳离子结合位点的异质性，并不支持Hasan等人提出的观点（《生物化学杂志》255（1980）3898 - 3904；256，（1981）7781 - 7785），即活性中心的静电相互作用从属于由尺寸受限的三甲基位点施加的空间限制。
