Hrabovská Anna, Debouzy Jean-Claude, Froment Marie-Thérese, Devínsky Ferdinand, Pauliková Ingrid, Masson Patrick
Comenius University, Faculty of Pharmacy, Department of Cell and Molecular Biology of Drugs, Bratislava, Slovakia.
FEBS J. 2006 Mar;273(6):1185-97. doi: 10.1111/j.1742-4658.2006.05144.x.
The purpose of this work was to study the catalytic properties of rat butyrylcholinesterase with benzoylcholine (BzCh) and N-alkyl derivatives of BzCh (BCHn) as substrates. Complex hysteretic behaviour was observed in the approach to steady-state kinetics for each ester. Hysteresis consisted of a long lag phase with damped oscillation. The presence of a long lag phase, with no oscillations, in substrate hydrolysis by rat butyrylcholinesterase was also observed with N-methylindoxyl acetate as substrate. Hysteretic behaviour was explained by the existence of two interconvertible butyrylcholinesterase forms in slow equilibrium, while just one of them is catalytically active. The damped oscillations were explained by the existence of different substrate conformational states and/or aggregates (micelles) in slow equilibrium. Different substrate conformational states were confirmed by 1H-NMR. The K(m) values for substrates decreased as the length of the alkyl chain increased. High affinity of the enzyme for the longest alkyl chain length substrates was explained by multiple hydrophobic interactions of the alkyl chain with amino acid residues lining the active site gorge. Molecular modelling studies supported this interpretation; docking energy decreased as the length of the alkyl chain increased. The long-chain substrates had reduced k(cat) values. Docking studies showed that long-chain substrates were not optimally oriented in the active site for catalysis, thus explaining the slow rate of hydrolysis. The hydrolytic rate of BCH12 and longer alkyl chain esters vs. substrate concentration showed a premature plateau far below V(max). This was due to the loss of substrate availability. The best substrates for rat butyrylcholinesterase were short alkyl homologues, BzCh - BCH4.
这项工作的目的是研究以苯甲酰胆碱（BzCh）和BzCh的N-烷基衍生物（BCHn）为底物时大鼠丁酰胆碱酯酶的催化特性。在每种酯达到稳态动力学的过程中都观察到了复杂的滞后行为。滞后现象包括一个带有阻尼振荡的长延迟阶段。以乙酸N-甲基吲哚酚为底物时，在大鼠丁酰胆碱酯酶催化的底物水解过程中也观察到了没有振荡的长延迟阶段。滞后行为被解释为存在两种处于缓慢平衡状态的可相互转化的丁酰胆碱酯酶形式，而其中只有一种具有催化活性。阻尼振荡被解释为存在处于缓慢平衡状态的不同底物构象状态和/或聚集体（胶束）。通过1H-NMR证实了不同的底物构象状态。底物的K(m)值随着烷基链长度的增加而降低。酶对最长烷基链长度底物的高亲和力被解释为烷基链与活性位点峡谷内衬氨基酸残基之间的多重疏水相互作用。分子建模研究支持了这一解释；对接能量随着烷基链长度的增加而降低。长链底物的k(cat)值降低。对接研究表明，长链底物在活性位点中没有处于催化的最佳取向，因此解释了水解速率较慢的原因。BCH12和更长烷基链酯的水解速率与底物浓度的关系显示，在远低于V(max)时就出现了过早的平台期。这是由于底物可用性的丧失。大鼠丁酰胆碱酯酶的最佳底物是短烷基同系物，即BzCh - BCH4。
