Mandrich Luigi, Menchise Valeria, Alterio Vincenzo, De Simone Giuseppina, Pedone Carlo, Rossi Mosè, Manco Giuseppe
Istituto di Biochimica delle Proteine, CNR, Via P. Castellino 111, 80131 Naples, Italy.
Proteins. 2008 Jun;71(4):1721-31. doi: 10.1002/prot.21877.
Recent mutagenic and molecular modelling studies suggested a role for glycine 84 in the putative oxyanion loop of the carboxylesterase EST2 from Alicyclobacillus acidocaldarius. A 114 times decrease of the esterase catalytic activity of the G84S mutant was observed, without changes in the thermal stability. The recently solved three-dimensional (3D) structure of EST2 in complex with a HEPES molecule permitted to demonstrate that G84 (together with G83 and A156) is involved in the stabilization of the oxyanion through a hydrogen bond from its main chain NH group. The structural data in this case did not allowed us to rationalize the effect of the mutation, since this hydrogen bond was predicted to be unaltered in the mutant. Since the mutation could shed light on the role of the oxyanion loop in the HSL family, experiments to elucidate at the mechanistic level the reasons of the observed drop in k (cat) were devised. In this work, the kinetic and structural features of the G84S mutant were investigated in more detail. The optimal temperature and pH for the activity of the mutated enzyme were found significantly changed (T = 65 degrees C and pH = 5.75). The catalytic constants K (M) and V(max) were found considerably altered in the mutant, with ninefold increased K (M) and 14-fold decreased V(max), at pH 5.75. At pH 7.1, the decrease in k (cat) was much more dramatic. The measurement of kinetic constants for some steps of the reaction mechanism and the resolution of the mutant 3D structure provided evidences that the observed effects were partly due to the steric hindrance of the S84-OH group towards the ester substrate and partly to its interference with the nucleophilic attack of a water molecule on the second tetrahedral intermediate.
最近的诱变和分子建模研究表明，嗜酸 Alicyclobacillus 酸热酯酶 EST2 的假定氧阴离子环中的甘氨酸 84 具有一定作用。观察到 G84S 突变体的酯酶催化活性降低了 114 倍，而热稳定性没有变化。最近解析的 EST2 与 HEPES 分子复合物的三维 (3D) 结构表明，G84（与 G83 和 A156 一起）通过其主链 NH 基团的氢键参与氧阴离子的稳定。在这种情况下，结构数据无法让我们合理解释突变的影响，因为预测该氢键在突变体中未改变。由于该突变可能有助于阐明氧阴离子环在 HSL 家族中的作用，因此设计了实验以在机制层面阐明观察到的 k (cat) 下降的原因。在这项工作中，对 G84S 突变体的动力学和结构特征进行了更详细的研究。发现突变酶活性的最佳温度和 pH 有显著变化（T = 65 摄氏度，pH = 5.75）。在 pH 5.75 时，发现突变体中的催化常数 K (M) 和 V(max) 有相当大的改变，K (M) 增加了 9 倍，V(max) 降低了 14 倍。在 pH 7.1 时，k (cat) 的下降更为显著。对反应机制某些步骤的动力学常数测量和突变体 3D 结构的解析提供了证据，表明观察到的影响部分是由于 S84-OH 基团对酯底物的空间位阻，部分是由于其干扰了水分子对第二个四面体中间体的亲核攻击。
