Szegletes T, Mallender W D, Thomas P J, Rosenberry T L
Department of Pharmacology, Mayo Foundation for Medical Education and Research, Department of Research, Mayo Clinic Jacksonville, Florida 32224, USA.
Biochemistry. 1999 Jan 5;38(1):122-33. doi: 10.1021/bi9813577.
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 were 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 KS 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.
乙酰胆碱酯酶（AChE）中配体相互作用的两个位点最早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确认：一个位于活性位点峡谷底部的酰化位点和一个位于其开口处的外周位点。我们最近提出了一种空间位阻模型，该模型展示了诸如碘化丙啶等小分子外周位点配体如何抑制底物水解[Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206 - 4216]。在该模型中，结合的外周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。在此，我们首先提供证据表明，结合的外周位点配体不仅会降低底物的速率常数，还会降低其水解产物的解离速率常数。将先前AChE催化底物水解的反应方案扩展到包括产物解离步骤，并在程序SCoP中用指定的速率常数模拟了在非平衡条件下碘化丙啶存在时乙酰硫代胆碱的水解速率。接下来，我们表明阳离子底物如乙酰硫代胆碱和7 - 乙酰氧基 - N - 甲基喹啉鎓（M7A）既结合到外周位点也结合到酰化位点。神经毒素束丝菌素被用于专门报告在外周位点的相互作用。对这些底物抑制束丝菌素缔合速率的分析表明，乙酰硫代胆碱在外周位点结合的KS值约为1 mM，M7A结合的KS值为0.2 mM。AChE反应方案进一步扩展，将底物结合到外周位点作为催化途径的起始步骤。用该方案对空间位阻模型的模拟结果与观察到的乙酰硫代胆碱和M7A的底物抑制以及乙酰硫代胆碱和M7A混合物中的相互竞争性抑制合理一致。底物抑制是通过底物结合到外周位点时产物解离受阻来解释的。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。
