Lushchekina S, Delacour H, Lockridge O, Masson P
Emanuel Institute of Biochemical Physics, Moscow, Russia.
Bégin Military Teaching Hospital, Saint Mandé, France.
Int J Risk Saf Med. 2015;27 Suppl 1:S80-1. doi: 10.3233/JRS-150699.
Prolonged apnoea following injection of ester-containing myoralaxants was first described in 1953. Because a large part of administered succinylcholine is shortly hydrolyzed by plasma butyrylcholinesterase (BChE) under normal conditions, prolonged apnoea was attributed to deficiency in BChE. It was found that BChE deficiency was due to genetic variations. Human BChE gene shows a large polyallelism. About 75 natural mutations of the BCHE gene have been documented so far [1]. Most of them cause alteration in BChE activity through point mutation effect on catalytic activity. Frame shifts and stop codons may also affect expression, or cause truncations in the sequence.
Recently, two novel BChE "silent" variants, Val204Asp [2] and Ala34Val [3], causing prolonged neuromuscular block after administration of mivacurium, were discovered. Mutations were genetically and kinetically characterized. The aim of the current study was to understand how these mutations determine "silent" phenotype.
Molecular dynamics studies were carried out with NAMD 2.9 software at the Lomonosov supercomputer. Charmm 36 force field was used, periodical boundary conditions, 1 atm pressure, 298 K. 100 ns molecular dynamics runs were performed for the wild-type BChE and its mutants Val204Asp and Ala34Val.
Unlike wild-type BChE, which retained its operative catalytic triad through the whole MD simulation, the catalytic triad of mutants was disrupted, making chemical step impossible. Val204Asp mutation leads to reorganization of hydrogen bonding network around the catalytic triad, which in turn increases the distance between catalytic residue main chains. Mutation Ala34Val, located on the protein surface, leads to increased fluctuations in the Ω-loop and subsequent disruption of the gorge structure, including disruption of the catalytic triad and formation of new hydrogen bonds involving catalytic center residues.
Comparative study of the "silent" Ala328Asp mutant and the catalytically active mutant Ala328Cys shows that MD approach can discriminate between the differential effects of point mutations at a same position.
1953年首次报道了注射含酯类肌松药后出现的长时间呼吸暂停。在正常情况下，大部分给予的琥珀酰胆碱会很快被血浆丁酰胆碱酯酶（BChE）水解，因此长时间呼吸暂停被归因于BChE缺乏。后来发现BChE缺乏是由基因变异引起的。人类BChE基因表现出高度的多态性。迄今为止，已记录了约75种BCHE基因的自然突变[1]。其中大多数通过对催化活性的点突变影响BChE活性。移码突变和终止密码子也可能影响表达，或导致序列截断。
最近，发现了两种新型的BChE“沉默”变体，Val204Asp [2]和Ala34Val [3]，它们在给予米库氯铵后会导致长时间的神经肌肉阻滞。对这些突变进行了遗传学和动力学特征分析。本研究的目的是了解这些突变如何决定“沉默”表型。
在罗蒙诺索夫超级计算机上使用NAMD 2.9软件进行分子动力学研究。采用Charmm 36力场，周期性边界条件，1个大气压，298 K。对野生型BChE及其突变体Val204Asp和Ala34Val进行了100 ns的分子动力学模拟。
与野生型BChE在整个分子动力学模拟过程中保留其有效的催化三联体不同，突变体的催化三联体被破坏，使得化学步骤无法进行。Val204Asp突变导致催化三联体周围氢键网络的重组，进而增加了催化残基主链之间的距离。位于蛋白质表面的Ala34Val突变导致Ω环的波动增加，随后峡部结构被破坏，包括催化三联体的破坏和涉及催化中心残基的新氢键的形成。
“沉默”的Ala328Asp突变体与催化活性突变体Ala328Cys的比较研究表明，分子动力学方法可以区分同一位置点突变的不同效应。
