Substrate binding to the peripheral site of acetylcholinesterase initiates enzymatic catalysis. Substrate inhibition arises as a secondary effect
Substrate binding to the peripheral site of acetylcholinesterase initiates enzymatic catalysis. Substrate inhibition arises as a secondary effect
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DOI：
10.1021/bi9813577
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发表时间：
1999-01-05
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：
Szegletes, T;Mallender, WD;Rosenberry, TL
乙酰胆碱酯酶 (AChE) 中的两个配体相互作用位点首先在配体结合研究中得到证实，后来通过晶体学、位点特异性诱变和分子建模得到证实：活性位点峡谷底部的酰化位点和其口部的外围位点。我们最近引入了一种空间封锁模型，该模型证明了小的外围位点配体（例如丙啶）如何抑制底物水解[Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216]。在此模型中，结合的外围位点配体的唯一作用是降低酰化位点配体的缔合和解离速率常数，而不改变配体与酰化位点结合的平衡常数。在这里，我们首先提供证据表明，结合的外围位点配体不仅降低了底物的速率常数，而且还降低了其水解产物的解离速率常数。以前的 AChE 底物水解反应方案已扩展到包括产物解离步骤，以及在非平衡条件下丙啶存在下的乙酰硫胆碱水解速率，甚至用程序 SCoP 中指定的速率常数进行模拟。接下来我们表明，乙酰硫代胆碱和 7-乙酰氧基-N-甲基喹啉鎓 (M7A) 等阳离子底物与外周位点以及酰化位点结合。神经毒素束蛋白用于专门报告外周部位的相互作用。对这些底物对束蛋白结合率的抑制的分析表明，乙酰硫胆碱外周位点结合的 K-s 值约为 1 mM，M7A 结合的 K-s 值约为 0.2 mM。 AChE 反应方案进一步扩展，包括底物与外围位点的结合作为催化途径的初始步骤。使用该方案模拟的空间封锁模型与观察到的乙酰硫胆碱和 M7A 的底物抑制以及乙酰硫胆碱和 M7A 混合物中的相互竞争性抑制相当一致。底物抑制是通过底物结合到外周位点时产物解离的阻断来解释的。然而，我们的分析表明，AChE 外周位点的主要生理作用是在低底物浓度下加速乙酰胆碱的水解。
Two sites of ligand interaction in acetylcholinesterase (AChE) were first demonstrated in ligand binding studies and later confirmed by crystallography, site-specific mutagenesis, and molecular modeling: an acylation site at the base of the active site gorge and a peripheral site at its mouth. We recently introduced a steric blockade model which demonstrated how small peripheral site ligands such as propidium may inhibit substrate hydrolysis [Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216]. In this model, the only effect of a bound peripheral site ligand is to decrease the association and dissociation rate constants for an acylation site ligand without altering the equilibrium constant for ligand binding to the acylation site. Here, we first provide evidence that not only rate constants for substrates but also dissociation rate constants for their hydrolysis products are decreased by bound peripheral site ligand. Previous reaction schemes for substrate hydrolysis by AChE were extended to include product dissociation steps, and acetylthiocholine hydrolysis rates in the presence of propidium under nonequilibrium conditions Even simulated with assigned rate constants in the program SCoP. We next showed that cationic substrates such as acetylthiocholine and 7-acetoxy-N-methylquinolinium (M7A) bind to the peripheral site as well as to the acylation site. The neurotoxin fasciculin was used to report specifically on interactions at the peripheral site. Analysis of inhibition of fasciculin association rates by these substrates revealed K-s values of about 1 mM for the peripheral site binding of acetylthiocholine and 0.2 mM for the binding of M7A. The AChE reaction scheme was further extended to include substrate binding to the peripheral site as the initial step in the catalytic pathway. Simulations of the steric blockade model with this scheme were in reasonable agreement with observed substrate inhibition for acetylthiocholine and M7A and with mutual competitive inhibition in mixtures of acetylthiocholine and M7A. Substrate inhibition was explained by blockade of product dissociation when substrate is bound to the peripheral site. However, our analyses indicate that the primary physiologic role of the AChE peripheral site is to accelerate the hydrolysis of acetylcholine at low substrate concentrations.