Kazan Federal University, Neuropharmacology Laboratory, 18 ul. Kremlevskaya, 48002, Kazan, Russian Federation.
Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 4 ul. Kosygina, Moscow, 119334, Russian Federation.
Chem Biol Interact. 2019 Sep 1;310:108702. doi: 10.1016/j.cbi.2019.06.015. Epub 2019 Jun 24.
Competing substrate kinetic analysis of human butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) from the time-course of enzyme-catalyzed substrate hydrolysis, using spectrophotometric assays is described. This study is based on the use of a chromogenic reporter "visible" substrate (substrate A), whose complete hydrolysis time course is retarded by a competing "invisible" substrate (substrate B). For BChE, four visible substrates were used, two thiocholine esters, benzoylthiocholine and butyrylthiocholine, and two aryl-acylamides, o-nitro trifluoro acetaminide and 3-(acetamido)-N,N,N-trimethylanilinium. Three different competing invisible substrates were used, phenyl acetate, acetylcholine and butyrylcholine. For AChE, two visible substrates were used, acetylthiocholine and 3-(acetamido)-N,N,N-trimethylanilinium. For AChE, acetylcholine was competing with visible substrates. The ratio (R) of bimolecular rate constants, k/K, for all couples of substrates, invisible/visible (B/A) covered all possible limit situations, R ≪ 1, R ≈ 1 and R ≫ 1. The kinetic approach, based on the method developed by Golicnik and Masson allowed determination of binding and catalytic parameters of cholinesterases for both visible and invisible substrates. This analysis was applied to michaelian and non-michaelian catalytic behaviors (activation and inhibition by excess substrate). Reevaluation of catalytic parameters obtained for acetylcholine and butyrylcholine more than 50 years ago was made. The method is fast, reliable, and particularly suitable for poorly soluble substrates and for substrates B when no direct spectrophotometric assays exist. Moreover, replacing substrate B by a reversible inhibitor, mechanism of cholinesterase inhibition was possible to study. It is therefore, useful for screening libraries of new substrates and inhibitors, and/or screening of new cholinesterase mutants. This method can be applied to any other enzymes.
用分光光度法测定人丁酰胆碱酯酶（BChE）和乙酰胆碱酯酶（AChE）的竞争性底物动力学分析，描述了从酶促底物水解的时间过程。这项研究基于使用显色报告“可见”底物（底物 A），其完全水解时间过程被竞争“不可见”底物（底物 B）延迟。对于 BChE，使用了四种可见底物，两种硫代胆碱酯，苯甲酰硫代胆碱和丁酰硫代胆碱，以及两种芳基酰基酰胺，邻硝基三氟乙酰胺和 3-（乙酰氨基）-N，N，N-三甲基苯胺。使用了三种不同的竞争不可见底物，苯乙酸酯，乙酰胆碱和丁酰胆碱。对于 AChE，使用了两种可见底物，乙酰硫代胆碱和 3-（乙酰氨基）-N，N，N-三甲基苯胺。对于 AChE，乙酰胆碱与可见底物竞争。所有双分子速率常数（k/K）的比率（R），对于所有不可见/可见（B/A）底物对，均涵盖了所有可能的极限情况，R ≪ 1，R ≈ 1 和 R ≫ 1。基于 Golicnik 和 Masson 开发的方法的动力学方法允许确定两种可见和不可见底物的胆碱酯酶的结合和催化参数。这种分析适用于米氏和非米氏催化行为（过量底物的激活和抑制）。对 50 多年前获得的乙酰胆碱和丁酰胆碱的催化参数进行了重新评估。该方法快速、可靠，特别适用于难溶性底物和不存在直接分光光度法测定的底物 B。此外，通过用可逆抑制剂替代底物 B，可以研究胆碱酯酶抑制的机制。因此，它对于筛选新的底物和抑制剂文库以及/或者筛选新的胆碱酯酶突变体很有用。该方法可应用于任何其他酶。
