Masson Patrick, Schopfer Lawrence M, Froment Marie-Thérèse, Debouzy Jean-Claude, Nachon Florian, Gillon Emilie, Lockridge Oksana, Hrabovska Anna, Goldstein Boris N
Centre de Recherches du Service de Santé des Armées, BP. 87, 38702 La Tronche cédex, France.
Chem Biol Interact. 2005 Dec 15;157-158:143-52. doi: 10.1016/j.cbi.2005.10.019. Epub 2005 Oct 27.
Butyrylcholinesterase (BChE) displays hysteretic behavior with certain neutral and charged substrates in the approach to steady state. Previous studies led us to interpret this phenomenon in terms of slow transitions between two enzyme conformers E and E'. This kinetic peculiarity is observed in human, horse and rat BChE. Oscillations that superimpose on the hysteretic lag are observed when benzoylcholine and N-alkyl derivatives of benzoylcholine are used as substrate. Hysteresis of BChE can be modulated by medium parameters (pH, salts, temperature, and pressure). Though mutant enzymes show different hysteretic behavior, so far attempts to provide a molecular mechanism of BChE hysteresis from mutagenesis studies have been unproductive. However, the substrate dependence of the hysteretic induction times, using wild-type BChE and several mutants, allowed us to build a general, mechanistic model for the hysteresis. In this model, substrate can bind to E, E', or both conformers, and ES and/or E'S can be catalytically active. The exact pathway followed depends on both the nature of the substrate and the structure of the BChE mutant under study. We propose that oscillations develop when substrate exists in different, slowly interconvertible, conformational and/or aggregation forms, of which only the minor form is capable of reacting with BChE. In support of this proposal, NMR studies have provided direct evidence for slow equilibria between monomeric and micellar forms of long-chain, alkyl derivatives of benzoyl-(N-substituted) choline. There is no direct evidence that hysteresis plays a role in BChE function(s). However, the "new view" of protein dynamics proposes that proteins are normally in equilibrium between pre-existing, functional and non-functional conformers; and that binding a ligand to the functional form shifts that equilibrium towards the functional conformation. Therefore, a physiological or toxicological relevance for the hysteresis in BChE cannot be ruled out.
丁酰胆碱酯酶（BChE）在接近稳态时，与某些中性和带电荷的底物表现出滞后行为。先前的研究使我们从两种酶构象E和E'之间的缓慢转变来解释这一现象。这种动力学特性在人、马和大鼠的BChE中均有观察到。当使用苯甲酰胆碱和苯甲酰胆碱的N-烷基衍生物作为底物时，会观察到叠加在滞后延迟上的振荡。BChE的滞后现象可由介质参数（pH、盐、温度和压力）调节。尽管突变酶表现出不同的滞后行为，但迄今为止，通过诱变研究提供BChE滞后分子机制的尝试均未成功。然而，利用野生型BChE和几种突变体，滞后诱导时间的底物依赖性使我们能够建立一个通用的滞后机制模型。在这个模型中，底物可以与E、E'或两者结合，并且ES和/或E'S可以具有催化活性。具体遵循的途径取决于底物的性质和所研究的BChE突变体的结构。我们提出，当底物以不同的、缓慢相互转化的构象和/或聚集形式存在时，就会产生振荡，其中只有次要形式能够与BChE反应。为支持这一观点，核磁共振研究提供了直接证据，证明苯甲酰-（N-取代）胆碱的长链烷基衍生物的单体和胶束形式之间存在缓慢平衡。没有直接证据表明滞后现象在BChE功能中起作用。然而，蛋白质动力学的“新观点”提出，蛋白质通常在预先存在的功能和非功能构象之间处于平衡状态；并且将配体与功能形式结合会使平衡向功能构象移动。因此，不能排除BChE滞后现象在生理或毒理学方面的相关性。
