Acetylthiocholine binds to asp74 at the peripheral site of human acetylcholinesterase as the first step in the catalytic pathway
Acetylthiocholine binds to asp74 at the peripheral site of human acetylcholinesterase as the first step in the catalytic pathway
复制标题
DOI：
10.1021/bi000210o
复制
发表时间：
2000-07-04
期刊：
影响因子：
2.9
通讯作者：
Rosenberry, TL
中科院分区：
文献类型：
--
作者：
Mallender, WD;Szegletes, T;Rosenberry, TL
配体与乙酰胆碱酯酶 (AChE) 结合的研究已经证明了两个相互作用位点。在酰化位点处形成酰基酶中间体，并且催化活性可以通过与外围位点的配体结合来抑制。 AChE-配体复合物的三维结构揭示了一个狭窄而深的活性位点峡谷，并表明对峡谷底部的酰化位点具有特异性的配体必须首先穿过峡谷入口附近的外围位点。在最近试图阐明外周位点在 AChE 催化途径中的作用的研究中，我们发现特异性结合外周位点的配体可以减慢其他配体进入和退出酰化位点的速率，我们将这一特征称为空间封锁 [Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216]。我们还证明，阳离子底物可以在外围位点形成低亲和力复合物，在低底物浓度下加速催化水解，但在高浓度下由于产物释放的空间封锁而导致底物抑制[Szegletes, T., Mallender, W. D., Thomas, P. J., and Rosenberry, T. L. (1999) Biochemistry 38, 122-133]。在本报告中，我们证明了人 AChE 外周位点中与底物乙酰硫胆碱相互作用的关键残基是 D74。我们扩展了我们的动力学模型来评估外围位点的底物亲和力，由平衡解离常数 Ks 表示，根据底物水解速率对底物浓度的依赖性。对于人 AChE，通过拟合该底物抑制曲线获得的 Ks 为 1.9 +/- 0.7 mM，与直接从神经毒素束蛋白与外周位点结合的乙酰硫胆碱抑制测量得到的 Ks 为 1.3 +/- 1.0 mM 一致。对于 Torpedo AChE，通过底物抑制获得的 Ks 为 0.5 +/- 0.2 mM，与用束蛋白测量的 Ks 为 0.4 +/- 0.2 mM 一致。 D72G 突变（对应于人 AChE 中的 D74G）的引入将 Torpedo 酶中的 Ks 增加至 4-10 mM，将人酶中的 Ks 增加至约 33 mM。虽然人 D74G 突变体中的周转数 k(cat) 没有变化，但 D74G 中乙酰硫胆碱对外围位点的亲和力大约降低了 20 倍，导致突变体中的 k(cat)/K-app（二级水解速率常数）相应降低。此外，我们表明 D74 在将由束蛋白与外周位点结合诱导的抑制构象效应传递到酰化位点方面非常重要。这种抑制作用是通过束蛋白结合导致的中性有机磷酸酯 7-[(甲基乙氧基膦酰基)氧基]-4-甲基香豆素 (EMPC) 的一级磷酸化速率常数 k(OP) 的相对降低来测量的，从野生型人 AChE 中的 0.002 降低到 D74G 突变体中的 0.24。
Studies of ligand binding to acetylcholinesterase (AChE) have demonstrated two sites of interaction. An acyl-enzyme intermediate is formed at the acylation site, and catalytic activity can be inhibited by ligand binding to a peripheral site. The three-dimensional structures of AChE-ligand complexes reveal a narrow and deep active site gorge and indicate that ligands specific for the acylation site at the base of the gorge must first traverse the peripheral site near the gorge entrance. In recent studies attempting to clarify the role of the peripheral site in the catalytic pathway for AChE, we showed that ligands which bind specifically to the peripheral site can slow the rates at which other ligands enter and exit the acylation site, a feature we called steric blockade [Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216]. We also demonstrated that cationic substrates can form a low-affinity complex at the peripheral site that accelerates catalytic hydrolysis at low substrate concentrations but results in substrate inhibition at high concentrations because of steric blockade of product release [Szegletes, T., Mallender, W. D., Thomas, P. J., and Rosenberry, T. L. (1999) Biochemistry 38, 122-133]. In this report, we demonstrate that a key residue in the human AChE peripheral site with which the substrate acetylthiocholine interacts is D74. We extend our kinetic model to evaluate the substrate affinity for the peripheral site, indicated by the equilibrium dissociation constant Ks, from the dependence of the substrate hydrolysis rate on substrate concentration. For human AChE, a Ks of 1.9 +/- 0.7 mM obtained by fitting this substrate inhibition curve agreed with a Ks of 1.3 +/- 1.0 mM measured directly from acetylthiocholine inhibition of the binding of the neurotoxin fasciculin to the peripheral site. For Torpedo AChE, a Ks of 0.5 +/- 0.2 mM obtained from substrate inhibition agreed with a Ks of 0.4 +/- 0.2 mM measured with fasciculin. Introduction of the D72G mutation (corresponding to D74G in human AChE) increased the Ks to 4-10 mM in the Torpedo enzyme and to about 33 mM in the human enzyme. While the turnover number k(cat) was unchanged in the human D74G mutant, the roughly 20-fold decrease in acetylthiocholine affinity for the peripheral site in D74G resulted in a corresponding decrease in k(cat)/K-app, the second-order hydrolysis rate constant, in the mutant. In addition, we show that D74 is important in conveying to the acylation site an inhibitory conformational effect induced by the binding of fasciculin to the peripheral site. This inhibitory effect, measured by the relative decrease in the first-order phosphorylation rate constant k(OP) for the neutral organophosphate 7- [(methylethoxyphosphonyl)oxy]-4-methylcoumarin (EMPC) that resulted from fasciculin binding, decreased from 0.002 in wild-type human AChE to 0.24 in the D74G mutant.