Biochemical Neuropharmacology Laboratory, Kazan Federal University, 18 Ul. Kremlevskaya, 420008 Kazan, Russia.
Arbuzov Institute of Organic and Physical Chemistry, Kazan Scientific Center, Russian Academy of Sciences, 8 Ul. Arbuzov, 420088 Kazan, Russia.
Int J Mol Sci. 2023 Jun 21;24(13):10472. doi: 10.3390/ijms241310472.
Cholinesterases (ChEs) display a non-michaelian behavior with positively charged substrates. In the steady-state rate equation, the b factor describes this behavior: if 1 there is substrate activation, if 1 there is substrate inhibition. The mechanistic significance of the factor was investigated to determine whether this behavior depends on acylation, deacylation or on both steps. Kinetics of human acetyl- (AChE) and butyryl-cholinesterase (BChE) were performed under steady-state conditions and using a time-course of complete substrate hydrolysis. For the hydrolysis of short acyl(thio)esters, where acylation and deacylation are partly rate-limiting, steady-state kinetic analysis could not decide which step determines . However, the study of the hydrolysis of an arylacylamide, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), where acetylation is rate-limiting, showed that depends on the acylation step. The magnitude of and opposite values between AChE and BChE for the hydrolysis of acetyl(thio)- versus benzoyl-(thio) esters, then indicated that the productive adjustment of substrates in the active center at high concentration depends on motions of both the Ω and the acyl-binding loops. Benzoylcholine was shown to be a poor substrate of AChE, and steady-state kinetics showed a sudden inhibition at high concentration, likely due to the non-dissociation of hydrolysis products. The poor catalytic hydrolysis of this bulky ester by AChE illustrates the importance of the fine adjustment of substrate acyl moiety in the acyl-binding pocket. Molecular modeling and QM/MM simulations should definitively provide evidence for this statement.
胆碱酯酶（ChE）对带正电荷的底物表现出非米氏行为。在稳态速率方程中，b 因子描述了这种行为：如果 1，则存在底物激活，如果 1，则存在底物抑制。研究了 b 因子的机械意义，以确定这种行为是否取决于酰化、脱酰化或两者都有。在稳态条件下，使用完整底物水解的时程，进行了人乙酰胆碱酯酶（AChE）和丁酰胆碱酯酶（BChE）的动力学研究。对于短酰基（硫代）酯的水解，酰化和脱酰化部分是限速步骤，稳态动力学分析无法确定哪个步骤决定了 b 因子。然而，对芳基酰基酰胺 3-（乙酰氨基）-N，N，N-三甲基苯胺（ATMA）水解的研究表明，b 因子取决于酰化步骤。AChE 和 BChE 对乙酰（硫代）酯与苯甲酰（硫代）酯水解的 b 值和相反值表明，在高浓度下，底物在活性中心的产生活性调整取决于 Ω 和酰基结合环的运动。苯甲酰胆碱是 AChE 的不良底物，稳态动力学显示在高浓度时突然抑制，可能是由于水解产物不分离。AChE 对这种大体积酯的催化水解不良说明了底物酰基部分在酰基结合口袋中精细调整的重要性。分子建模和 QM/MM 模拟应该明确提供证据证明这一说法。
