丁基胆碱酯酶催化的n -甲基lindoyl乙酸酯水解:反应后体积变化及滞后行为分析

Patrick Masson , Marie-Thérèse Froment , Sébastien Fort , Fabien Ribes , Nicole Bec , Claude Balny , Lawrence M Schopfer
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引用次数: 30

摘要

野生型人丁基胆碱酯酶(BuChE)和外周位点突变体(D70G, Y332A, D70G/Y332A)水解中性底物n -甲基lindoxyl乙酸酯(NMIA)遵循Michaelis-Menten动力学。野生型的Km为0.14 mM, D70G、Y332A和D70G/Y332A的Km为0.07-0.16 mM,表明外周位点不参与NMIA结合。所有酶的kcat值都是相同的数量级:12,000-18,000 min−1。底物结合时的体积变化(−ΔVKm)和与NMIA水解相关的活化体积(ΔVkcat‡)是根据催化常数的压力依赖性计算的。−ΔVKm的值表明NMIA与一个芳香残基结合,假定是W82,活性位点结合位点。结合伴随着从峡谷中释放水分子。残基70控制底物结合时释放的水分子的数量。ΔVkcat‡值对野生型呈阳性,对D70G呈弱阳性,清楚地表明催化步骤伴随着水重新进入峡谷。结果支持了残基D70参与活性位点峡谷构象稳定和水化控制的前提。在稳定状态之前,在几分钟的时间尺度上看到了一个缓慢的瞬变。诱导时间随着NMIA浓度的增加而迅速增加,在底物饱和时达到极限。野生型BuChE水解苯甲酰胆碱(BzCh)和活性位点突变体E197Q和E197Q/G117H水解丁基硫代胆碱(BuSCh)的诱导时间要短得多(约1分钟)。这种缓慢的瞬态被解释为没有动力学协同性的滞后。BuChE的滞后行为是由E和E '两个酶态之间缓慢的构象平衡引起的。NMIA只与E ' '的初始形式结合。适温盐和静水压力使平衡向E′方向移动。E→E′的转变伴随着负活化体积(ΔV0‡= - 45±10 ml/mol), E′的形态比E′的形态更致密,E′的水合水比E′的形态更有结构。
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Butyrylcholinesterase-catalyzed hydrolysis of N-methylindoxyl acetate: analysis of volume changes upon reaction and hysteretic behavior

Hydrolysis of the neutral substrate N-methylindoxyl acetate (NMIA) by wild-type human butyrylcholinesterase (BuChE) and peripheral site mutants (D70G, Y332A, D70G/Y332A) was found to follow the Michaelis–Menten kinetics. Km was 0.14 mM for wild-type, and 0.07–0.16 mM for D70G, Y332A and D70G/Y332A, indicating that the peripheral site is not involved in NMIA binding. The values of kcat were of the same order for all enzymes: 12,000–18,000 min−1.

Volume changes upon substrate binding (−ΔVKm) and the activation volumes (ΔVkcat) associated with hydrolysis of NMIA were calculated from the pressure dependence of the catalytic constants. Values of −ΔVKm indicate that NMIA binds to an aromatic residue, presumed to be W82, the active site binding locus. Binding is accompanied by a release of water molecules from the gorge. Residue 70 controls the number of water molecules that are released upon substrate binding. The values of ΔVkcat, which are positive for wild-type and faintly positive for D70G, clearly indicate that the catalytic steps are accompanied by re-entry of water into the gorge. Results support the premise that residue D70 is involved in the conformational stabilization of the active site gorge and in control of its hydration.

A slow transient, preceding the steady state, was seen on a time scale of several minutes. The induction time rapidly increased with NMIA concentration to reach a limit at substrate saturation. Much shorter induction times (<1 min) were seen for hydrolysis of benzoylcholine (BzCh) by wild-type BuChE and for hydrolysis of butyrylthiocholine (BuSCh) by the active site mutants E197Q and E197Q/G117H. This slow transient was interpreted in terms of hysteresis without kinetic cooperativity. The hysteretic behavior of BuChE results from a slow conformational equilibrium between two enzyme states E and E′. NMIA binds only to the primed form E′. Kosmotropic salts and hydrostatic pressure were found to shift the equilibrium toward E′. The E→E′ transition is accompanied by a negative activation volume (ΔV0=−45±10 ml/mol), and the E′ form is more compact than E. Hydration water in the gorge of E′ appears to be more structured than in the unprimed form.

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