de Sampaio T C, Melo R B, Moura T F, Michel S, Barreiros S
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, R. da Quinta Grande, 6, Apt. 127, 2780 Oeiras, Portugal.
Biotechnol Bioeng. 1996 May 5;50(3):257-64. doi: 10.1002/(SICI)1097-0290(19960505)50:3<257::AID-BIT4>3.0.CO;2-F.
We studied a model transesterification reaction catalyzed by subtilisin Carlsberg suspended in toluene, n-hexane, diisopropyl ether, and mixtures of these solvents. To account for solvent effects due to differences in water partitioning between the enzyme and the bulk solvents, we measured water sorption isotherms for the enzyme in each solvent. We measured catalytic activity as a function of enzyme hydration and obtained bell-shaped curves with maxima at the same enzyme hydration in all the solvents. However, the activity maxima were different in all the media, being the lowest in toluene. Differences in the partitioning of substrates and product between the bulk solvent phase and the enzyme active site were accounted for but could not explain the lower catalytic activity observed in toluene. The fact that toluene is very similar to one of the substrates suggested the possibility of competitive inhibition by this solvent. We derived a model allowing for differences in solvation of the substrates, by using thermodynamic activities instead of concentrations, as well as for competitive inhibition by toluene. The model fit the experimental data well, confirming that toluene had a direct adverse effect on the catalytic activity of the enzyme.
我们研究了由悬浮在甲苯、正己烷、二异丙醚以及这些溶剂混合物中的枯草杆菌蛋白酶卡尔伯格催化的模型酯交换反应。为了解释由于酶与大量溶剂之间水分配差异导致的溶剂效应，我们测量了酶在每种溶剂中的水吸附等温线。我们测量了催化活性作为酶水合作用的函数，并在所有溶剂中获得了在相同酶水合作用下具有最大值的钟形曲线。然而，所有介质中的活性最大值不同，在甲苯中最低。考虑了底物和产物在大量溶剂相和酶活性位点之间的分配差异，但无法解释在甲苯中观察到的较低催化活性。甲苯与其中一种底物非常相似这一事实表明了这种溶剂存在竞争性抑制的可能性。我们推导了一个模型，该模型考虑了底物溶剂化的差异，通过使用热力学活度而非浓度，以及甲苯的竞争性抑制。该模型很好地拟合了实验数据，证实甲苯对酶的催化活性有直接不利影响。
