Berman H A, Leonard K
Department of Biochemical Pharmacology, State University of New York, Buffalo 14260.
Biochemistry. 1990 Nov 27;29(47):10640-9. doi: 10.1021/bi00499a010.
This paper examines covalent reactivity of AchE with respect to cationic and uncharged methylphosphonates and substrates in the absence and presence of cationic ligands selective for the active center and the peripheral anionic site. The organophosphorus inhibitors are enantiomeric alkyl methylphosphonothioates (1-5) containing cycloheptyl and isopropyl phosphono ester groups and S-methyl, S-n-pentyl, and S-[beta-(trimethylammonio)ethyl] leaving groups; these agents differ in their configuration about phosphorus and their steric, hydrophobic, and electrostatic characteristics. The synthetic substrates examined are acetylthiocholine, p-nitrophenyl acetate, and 7-acetoxy-4-methylcoumarin (7AMC). Antagonism of the methylphosphonothioate reaction by cationic ligands is strongly dependent on the nature of both the cation and the methylphosphonate but independent of the configuration about phosphorus. While all cations cause linear mixed inhibition of acetylthiocholine hydrolysis, there are observed a variety of inhibition patterns of 7AMC and p-nitrophenyl acetate hydrolysis that are distinctly nonlinear, as well as patterns in which the reciprocal plots intersect in the upper right quadrant. Strong antagonism of cationic (methylphosphonyl)thiocholines correlates very well with linear inhibition of acetylthiocholine. Ligands that cause only negligible antagonism of the uncharged methylphosphonates display nonlinear inhibition of uncharged substrates. These relationships, since they are most pronounced for peripheral site ligands and are strongly dependent on the charge carried by the reactant, suggest that the peripheral anionic site alters enzyme reactivity through an electrostatic interaction with the net negative active center. Such behavior indicates a potential role for the peripheral anionic site in conserving AchE catalytic efficiency within a narrow range of values.
本文研究了在不存在和存在对活性中心及外周阴离子位点具有选择性的阳离子配体的情况下，乙酰胆碱酯酶（AchE）与阳离子和不带电荷的甲基膦酸酯及底物的共价反应活性。有机磷抑制剂是对映体的烷基甲基硫代膦酸酯（1 - 5），含有环庚基和异丙基膦酸酯基团以及S - 甲基、S - n - 戊基和S - [β - （三甲基铵）乙基]离去基团；这些试剂在磷的构型及其空间、疏水和静电特性方面存在差异。所研究的合成底物是乙酰硫代胆碱、对硝基苯乙酸酯和7 - 乙酰氧基 - 4 - 甲基香豆素（7AMC）。阳离子配体对甲基硫代膦酸酯反应的拮抗作用强烈依赖于阳离子和甲基膦酸酯的性质，但与磷的构型无关。虽然所有阳离子都会导致乙酰硫代胆碱水解的线性混合抑制，但观察到7AMC和对硝基苯乙酸酯水解的多种抑制模式明显是非线性的，以及双倒数图在右上象限相交的模式。阳离子（甲基膦酰基）硫代胆碱的强拮抗作用与乙酰硫代胆碱的线性抑制密切相关。对对硝基苯乙酸酯的拮抗作用可忽略不计的配体对不带电荷的底物表现出非线性抑制。这些关系，由于在外周位点配体中最为明显且强烈依赖于反应物所带电荷，表明外周阴离子位点通过与带净负电荷的活性中心的静电相互作用改变酶的反应活性。这种行为表明外周阴离子位点在将AchE催化效率保持在狭窄值范围内具有潜在作用。
