N.M. Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Kosygina Str. 4, Moscow 119334, Russia; Lomonosov State University, Chemistry Department, Moscow 119991, Russia.
N.M. Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Kosygina Str. 4, Moscow 119334, Russia.
Chem Biol Interact. 2019 Jun 1;306:138-146. doi: 10.1016/j.cbi.2019.04.019. Epub 2019 Apr 19.
A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198·H438·E325) was replaced by S198·H438·N322E in silico. Molecular dynamics for 1.5 μs and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293 cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293 cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198·H438·N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.
设计了一种新型催化三联体的人丁酰胆碱酯酶（BChE）突变体计算机模型，N322E/E325G。该野生型酶的催化三联体（S198·H438·E325）在计算机中被 S198·H438·N322E 取代。1.5 μs 的分子动力学和马尔可夫状态模型分析表明，新的催化三联体应该在突变酶中起作用，表明其具有功能性。对野生型 BChE 和双突变体与依地膦酸酯的反应进行量子力学/分子力学建模表明，突变体对有机磷的反应性很高。在 Expi293 细胞中表达了一个截短的单体（L530 停止）双突变体。对非纯化的转染细胞培养液进行分析。在天然条件下进行聚丙烯酰胺凝胶电泳，然后用 BTC 作为底物进行活性染色，为单体 BChE 突变体具有活性提供了证据。双突变体被依地膦酸酯抑制后，进行聚丙烯酰胺凝胶电泳和活性染色表明，该酶缓慢自我再激活。然而，由于 Expi293 细胞分泌内源性 BChE 四聚体和几种有机磷反应酶，因此在粗细胞培养液中未确定新突变体磷酸化后的催化参数和自我再激活常数。该研究表明，具有新型催化三联体（S198·H438·N322E）的计算机设计的双突变体（N322E/E325G）是设计具有有机磷水解酶活性的新型活性人 BChE 突变体的合适模板。
