Effect of Calcium on the Hydrolysis Activity of Human Butyrylcholinesterase
Effect of Calcium on the Hydrolysis Activity of Human Butyrylcholinesterase
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钙对人丁酰胆碱酯酶水解活性的影响
DOI：
10.1016/j.xphs.2019.12.006
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发表时间：
2020
影响因子：
3.8
通讯作者：
Akihisa Toda
中科院分区：
文献类型：
--
作者：
Teruko Imai;Fatma Goksin Bahaｒ;Kayoko Ohura;Akihisa Toda
本实验的目的是研究钙离子对人丁酰胆碱酯酶（HuBChE）水解阳离子和阴离子底物的影响。在钙离子浓度增加（~20 mM）的情况下，HuBChE 对阿司匹林（一种阴离子底物）的水解显着增加，如 kcat 增加（~18 倍）所示。丁酰硫代胆碱（BTC）是一种阳离子底物，通过底物活化进行双相水解；第二个 BTC 分子导致 inkcat 增加了 3 倍。在较低和较高浓度的 BTC 下，HuBChE 的水解作用因钙离子的添加而略微减慢。其他阳离子底物，具有丁酰基和戊酰基的普萘洛尔衍生物，被 HuBChE 优先水解；在钙离子不存在和存在的情况下，这些化合物的水解速率几乎相同。这些数据表明钙离子对阴离子和阳离子底物的 HuBChE 活性的不同影响。此外，在钙离子存在下阿司匹林水解过程中，我们证明存在 2 个额外的钙结合位点，Km 值为 1.8 和 5.9 mM。这些结合位点表现出比 EF-hand 基序低得多的亲和力，EF-hand 基序之前被鉴定为高亲和力钙结合位点。
The aim of this experiment was to study the effects of calcium ion on the hydrolysis of cationic and anionic substrate by human butyrylcholinesterase (HuBChE). The hydrolysis of aspirin, an anionic substrate, by HuBChE was markedly increased in the presence of increasing concentrations of calcium ion (∼20 mM), as shown by the increasingkcat(∼18-fold). Butyrylthiocholine (BTC), a cationic substrate, was biphasically hydrolyzed with substrate activation; a second BTC molecule caused a 3-fold increase inkcat. At both lower and higher concentrations of BTC, its hydrolysis by HuBChE was slightly slowed down by the addition of calcium ion. Other cationic substrates, propranolol derivatives with butyryl and valeryl groups, wereR-preferentially hydrolyzed by HuBChE; the rate of hydrolysis of these compounds was nearly the same in the absence and presence of calcium ion. These data indicate differential effects of calcium ion on HuBChE activity with anionic and cationic substrates. Furthermore, during the hydrolysis of aspirin in the presence of calcium ions, we demonstrated the existence of 2 additional binding sites for calcium, withKmvalues of 1.8 and 5.9 mM. These binding sites exhibited much lower affinities than the EF-hand motif, previously identified as a high-affinity calcium-binding site.