Masson Patrick, Froment Marie Thérèse, Fort Sébastien, Ribes Fabien, Bec Nicole, Balny Claude, Schopfer Lawrence M
Centre de Recherches du Service de Santé des Armées, Unité d'Enzymologie, BP 87, 24 Av. Maquis du Gresivaudan, 38702 La Tronche Cedex, France.
Biochim Biophys Acta. 2002 Jun 3;1597(2):229-43. doi: 10.1016/s0167-4838(02)00265-0.
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. K(m) 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 k(cat) were of the same order for all enzymes: 12,000-18,000 min(-1). Volume changes upon substrate binding (-DeltaV(K(m))) and the activation volumes (DeltaV++(k(cat)) associated with hydrolysis of NMIA were calculated from the pressure dependence of the catalytic constants. Values of -DeltaV(K(m)) 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 DeltaV++(k(cat)), 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 (DeltaV++(0)= -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.
野生型人丁酰胆碱酯酶（BuChE）和外周位点突变体（D70G、Y332A、D70G/Y332A）对中性底物N-甲基吲哚酚乙酸酯（NMIA）的水解遵循米氏动力学。野生型的K(m)为0.14 mM，D70G、Y332A和D70G/Y332A的K(m)为0.07 - 0.16 mM，这表明外周位点不参与NMIA的结合。所有酶的k(cat)值处于同一数量级：12,000 - 18,000 min⁻¹。根据催化常数对压力的依赖性，计算了底物结合时的体积变化（-ΔV(K(m))）以及与NMIA水解相关的活化体积（ΔV++(k(cat))）。-ΔV(K(m))的值表明NMIA与一个芳香族残基结合，推测该残基为W82，即活性位点结合位点。结合伴随着水分子从酶沟中释放。残基70控制底物结合时释放的水分子数量。野生型的ΔV++(k(cat))值为正，D70G的该值为微弱正值，这清楚地表明催化步骤伴随着水重新进入酶沟。结果支持了残基D70参与活性位点沟的构象稳定及其水合作用控制这一前提。在几分钟的时间尺度上观察到稳态之前有一个缓慢的瞬态过程。诱导时间随NMIA浓度迅速增加，在底物饱和时达到极限。野生型BuChE水解苯甲酰胆碱（BzCh）以及活性位点突变体E197Q和E197Q/G1,17H水解丁酰硫代胆碱（BuSCh）时观察到的诱导时间要短得多（<1分钟）。这种缓慢的瞬态过程被解释为无动力学协同性的滞后现象。BuChE的滞后行为源于两种酶状态E和E'之间缓慢的构象平衡。NMIA仅与引发形式E'结合。发现促溶剂盐和静水压力会使平衡向E'移动。E→E'转变伴随着负的活化体积（ΔV++(0)= -45±10 ml/mol），并且E'形式比E更紧凑。E'酶沟中的水合水似乎比未引发形式的结构更有序。
