Department of Physiological Sciences , Oklahoma State University , Stillwater , Oklahoma 74078 , United States.
Department of Entomology and Plant Pathology , Oklahoma State University , Stillwater , Oklahoma 74078 , United States.
Chem Res Toxicol. 2019 Sep 16;32(9):1801-1810. doi: 10.1021/acs.chemrestox.9b00146. Epub 2019 Aug 28.
The single residue mutation of butyrylcholinesterase (BChE) hydrolyzes a number of organophosphosphorus (OP) anticholinesterases. Whereas other BChE active site/proximal mutations have been investigated, none are sufficiently active to be prophylactically useful. In a fundamentally different computer simulations driven strategy, we identified a surface peptide loop (residues 278-285) exhibiting dynamic motions during catalysis and modified it via residue insertions. We evaluated these loop mutants using computer simulations, substrate kinetics, resistance to inhibition, and enzyme reactivation assays using both the choline ester and OP substrates. A slight but significant increase in reactivation was noted with paraoxon with one of the mutants, and changes in and catalytic efficiency were noted in others. Simulations suggested weaker interactions between OP versus choline substrates and the active site of all engineered versions of the enzyme. The results indicate that an improvement of OP anticholinesterase hydrolysis through surface loop engineering may be a more effective strategy in an enzyme with higher intrinsic OP compound hydrolase activity.
丁酰胆碱酯酶（BChE）的单个残基突变可水解多种有机磷（OP）类抗胆碱酯酶。虽然已经研究了其他 BChE 活性位点/近侧突变，但没有一个突变具有足够的活性可用于预防。在一种完全不同的基于计算机模拟的策略中，我们鉴定了一个在催化过程中表现出动态运动的表面肽环（残基 278-285），并通过残基插入对其进行了修饰。我们使用计算机模拟、底物动力学、抑制抗性以及使用胆碱酯和 OP 底物的酶重激活测定来评估这些环突变体。在其中一个突变体中，对氧磷的重激活略有但显著增加，而其他突变体的 和催化效率也发生了变化。模拟表明，与所有工程化酶版本的活性位点相比，OP 与胆碱底物之间的相互作用较弱。结果表明，通过表面环工程提高 OP 类抗胆碱酯酶水解的效率可能是一种更有效的策略，因为该酶具有更高的内在 OP 化合物水解酶活性。
