Polyol-induced activation by excess substrate of the D70G butyrylcholinesterase mutant
Polyol-induced activation by excess substrate of the D70G butyrylcholinesterase mutant
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DOI：
10.1016/s0167-4838(98)00253-2
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发表时间：
1999-01-11
期刊：
影响因子：
--
通讯作者：
Masson, P
中科院分区：
文献类型：
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作者：
Levitsky, V;Xie, WH;Masson, P
野生型人丁酰胆碱酯酶(BUCHE)具有非迈克尔式行为，以丁酰硫胆碱(ETC)为底物进行底物活化。D70G突变体具有与野生型相同的催化常数，但对ETC的亲和力比野生型低10倍，在常规条件下不表现出过量的ETC激活。在本工作中，发现添加多元醇或糖改变了D70G突变体与ETC的动力学行为。在蔗糖浓度为40%时，过量底物对D70G突变体酶有明显的激活作用。因为D70与Y332是氢键的，所以对Y332的突变体进行了研究。突变体Y332F的行为与野生型Buhe相似，而突变体Y332A、Y332A/D70G和D70G的底物活性可忽略不计。野生型、Y332F、Y332A和Y332A/D70G在高浓度糖存在下的行为没有改变。底物活化的解释是在D70的外围位置结合了第二个底物分子。如果D70是参与底物激活的唯一残基，则D70G突变体应该不能底物激活。D70G突变体通过介质工程显示底物激活的能力表明，其他残基参与了初始底物结合和过量底物的激活。渗透分子诱导的Y332和其他暴露于溶剂的残基的构象和/或水化状态的变化可能是D70G突变体的非迈克尔式行为的原因。(C)1999 Elsevier Science B.V.保留所有权利。
Wild-type human butyrylcholinesterase (BuChE) has a non-Michaelian behaviour showing substrate activation with butyrylthiocholine (ETC) as the substrate. The D70G mutant has a catalytic constant identical to that of the wild-type enzyme, but a IO-fold lower affinity for ETC compared to wild-type enzyme, and it does not exhibit activation by excess ETC under conventional conditions. In the present work it was found that addition of polyols or sugars changed the kinetic behaviour of the D70G mutant with ETC. In the presence of 40% sucrose, the D70G mutant enzyme displayed marked activation by excess substrate. Because D70 is hydrogen bonded to Y332, mutants of Y332 were studied. Mutant Y332F had a behaviour similar to that of wild-type BuChE, whereas mutants Y332A, Y332A/D70G and D70G had negligible substrate activation. The behaviour of wild-type, Y332F, Y332A and Y332A/D70G did not change in the presence of high concentrations of sugar. Substrate activation has been explained by binding of a second substrate molecule in the peripheral site at D70. The D70G mutant should be incapable of substrate activation, if D70 were the only residue involved in substrate activation. The ability of the D70G mutant to display substrate activation by medium engineering suggests that other residues are involved in initial substrate binding and activation by excess substrate. Osmolyte-induced change in conformation and/or hydration status of Y332 and other solvent-exposed residues may account for the non-Michaelian behaviour of the D70G mutant. (C) 1999 Elsevier Science B.V. All rights reserved.