Saxena A, Redman A M, Jiang X, Lockridge O, Doctor B P
Division of Biochemistry, Walter Reed Army Institute of Research, Washington, DC 20307, USA.
Biochemistry. 1997 Dec 2;36(48):14642-51. doi: 10.1021/bi971425+.
Amino acid sequence alignments of cholinesterases revealed that 6 of 14 aromatic amino acid residues lining the active center gorge of acetylcholinesterase are replaced by aliphatic amino acid residues in butyrylcholinesterase. The Y337 (F330) in mammalian acetylcholinesterase, which is replaced by A328 in human butyrylcholinesterase, is implicated in the binding of ligands such as huperzine A, edrophonium, and acridines and one end of bisquaternary compounds such as BW284C51 and decamethonium. Y337 may sterically hinder the binding of phenothiazines such as ethopropazine, which contains a bulky exocyclic substitution. Inhibition studies of (-)-huperzine A with human butyrylcholinesterase mutants, where A328 (KI = 194.6 microM) was modified to either F (KI = 0.6 microM, as in Torpedo acetylcholinesterase) or Y (KI = 0.032 microM, as in mammalian acetylcholinesterase), confirmed previous observations made with acetylcholinesterase mutants that this residue is important for binding huperzine A. Inhibition studies of ethopropazine with butyrylcholinesterase mutants, where A328 (KI = 0.18 microM) was modified to either F (KI = 0.82 microM) or Y (KI = 0.28 microM), suggested that A328 was not solely responsible for the selectivity of ethopropazine. Volume calculations for the active site gorge showed that the poor inhibitory activity of ethopropazine toward acetylcholinesterase was due to the smaller dimension of the active site gorge which was unable to accommodate the bulky inhibitor molecule. The volume of the butyrylcholinesterase active site gorge is approximately 200 A3 larger than that of the acetylcholinesterase gorge, which allows the accommodation of ethopropazine in two different orientations as demonstrated by rigid-body refinement and molecular dynamics calculations.
胆碱酯酶的氨基酸序列比对显示，在乙酰胆碱酯酶活性中心峡谷内衬的14个芳香族氨基酸残基中有6个被丁酰胆碱酯酶中的脂肪族氨基酸残基取代。哺乳动物乙酰胆碱酯酶中的Y337（F330）在人丁酰胆碱酯酶中被A328取代，它与石杉碱甲、依酚氯铵和吖啶等配体以及双季铵化合物如BW284C51和十烃季铵的一端结合有关。Y337可能在空间上阻碍含有庞大环外取代基的吩噻嗪类如乙丙嗪的结合。用人类丁酰胆碱酯酶突变体进行的（-）-石杉碱甲抑制研究中，其中A328（KI = 194.6 microM）被修饰为F（KI = 0.6 microM，如电鳐乙酰胆碱酯酶）或Y（KI = 0.032 microM，如哺乳动物乙酰胆碱酯酶），证实了之前对乙酰胆碱酯酶突变体的观察，即该残基对石杉碱甲的结合很重要。用丁酰胆碱酯酶突变体进行的乙丙嗪抑制研究中，其中A328（KI = 0.18 microM）被修饰为F（KI = 0.82 microM）或Y（KI = 0.28 microM），表明A328并非乙丙嗪选择性的唯一原因。活性中心峡谷的体积计算表明，乙丙嗪对乙酰胆碱酯酶的抑制活性较差是由于活性中心峡谷尺寸较小，无法容纳庞大的抑制剂分子。丁酰胆碱酯酶活性中心峡谷的体积比乙酰胆碱酯酶峡谷大约200 ų，这使得乙丙嗪能够以两种不同的取向容纳，如刚体精修和分子动力学计算所示。
