Hosea N A, Radić Z, Tsigelny I, Berman H A, Quinn D M, Taylor P
Department of Pharmacology, University of California at San Diego, La Jolla 92093-0636, USA.
Biochemistry. 1996 Aug 20;35(33):10995-1004. doi: 10.1021/bi9611220.
Through site-specific mutagenesis, we examined the determinants on acetylcholinesterase which govern the specificity and reactivity of three classes of substrates: enantiomeric alkyl phosphonates, trifluoromethyl acetophenones, and carboxyl esters. By employing cationic and uncharged pairs of enantiomeric alkyl methylphosphonyl thioates of known absolute stereochemistry, we find that an aspartate residue near the gorge entrance (D74) is responsible for the enhanced reactivity of the cationic organophosphonates. Removal of the charge with the mutation D74N causes a near equal reduction in the reaction rate constants for the Rp and Sp enantiomers and exerts a greater influence on the cationic organophosphonates than on the charged trimethylammonio trifluoromethyl acetophenone and acetylthiocholine. This pattern of reactivity suggests that the orientation of the leaving group for both enantiomers is directed toward the gorge exit and in apposition to Asp 74. Replacement of tryptophan 86 with alanine in the choline subsite also diminishes the reaction rates for cationic organophosphonates, although to a lesser extent than with the D74N mutation, while not affecting the reactions with the uncharged compounds. Hence, reaction with cationic OPs depends to a lesser degree on Trp 86 than on Asp 74. Docking of Sp and Rp cycloheptyl methylphosphonyl thiocholines and thioethylates in AChE as models of the reversible complex and transition state using molecular dynamics affords structural insight into the spatial arrangement of the substituents surrounding phosphorus prior to and during reaction. The leaving group of the Rp and Sp enantiomers, regardless of charge, is directed to the gorge exit and toward Asp 74, an orientation unique to tetrahedral ligands.
通过位点特异性诱变，我们研究了乙酰胆碱酯酶上决定三类底物特异性和反应活性的因素，这三类底物分别是对映体烷基膦酸酯、三氟甲基苯乙酮和羧酸酯。通过使用已知绝对立体化学结构的阳离子和不带电荷的对映体烷基甲基膦酰硫酯对，我们发现峡谷入口附近的一个天冬氨酸残基（D74）负责阳离子有机膦酸酯反应活性的增强。将D74突变为D74N以去除电荷，会使Rp和Sp对映体的反应速率常数几乎同等程度降低，并且对阳离子有机膦酸酯的影响比对带电荷的三甲基铵三氟甲基苯乙酮和乙酰硫代胆碱更大。这种反应活性模式表明，两种对映体离去基团的取向都指向峡谷出口并与天冬氨酸74相对。在胆碱亚位点用丙氨酸取代色氨酸86也会降低阳离子有机膦酸酯的反应速率，尽管程度比D74N突变小，同时不影响与不带电荷化合物的反应。因此，与阳离子有机磷化合物的反应对色氨酸86的依赖程度低于对天冬氨酸74的依赖程度。使用分子动力学将Sp和Rp环庚基甲基膦酰硫代胆碱和硫代乙酯对接至乙酰胆碱酯酶中，作为可逆复合物和过渡态的模型，从而对反应前和反应过程中磷周围取代基的空间排列有了结构上的认识。Rp和Sp对映体的离去基团，无论带不带电荷，都指向峡谷出口并朝向天冬氨酸74，这是四面体配体特有的取向。
