title: Understanding the non-catalytic behavior of human butyrylcholinesterase silent variants: Comparison of wild-type enzyme, catalytically active Ala328Cys mutant, and silent Ala328Asp variant.
abstract: Conformational dynamics of wild-type human butyrylcholinesterase (BChE), two mutants of residue Ala328, the catalytically active Ala328Cys, and the catalytically inactive (silent) Ala328Asp, and their interactions with butyrylcholine were studied. The aim was to understand the molecular mechanisms by which point mutations may lead to silent BChE variant or alter catalytic activity. Importance of BChE natural variants is due to medical consequences, i.e. prolonged apnea, following administration of the myorelaxant esters, succinylcholine and mivacurium. Comparison of molecular dynamics (MD) simulations for the three model systems showed that: 1) the active mutant Ala328Cys mutant has some changes in configuration of catalytic residues, which do not prevent binding of butyrylcholine to the active site; 2) in the naturally-occurring silent variant Ala328Asp, the Asp328 carboxylate may either form a salt bridge with Lys339 or a H-bond with His438. In the first case, the Ω-loop swings off the gorge, disrupting the π-cation binding site and the catalytic triad. In the second case, binding of cationic substrates in the catalytic center is also impaired. MD simulations carried out in 0.15 M NaCl, close to physiological ionic strength conditions, favored the second situation. It was seen that Asp328 forms a H-bond with the catalytic triad His438, which in turn disrupts the catalytic machinery. Therefore, we concluded that the Ala328Asp variant is not catalytically active because of that dramatic event. Computational results, consistent with in vitro biochemical data and clinical observations, validate our MD approach.
authors.affiliation: Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Moscow, Russia.
authors.forename: Sofya
authors.initials: S
authors.lastname: Lushchekina
authors.affiliation: Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Moscow, Russia; Department of Chemistry, Lomonosov Moscow State University, Moscow, Russia.
authors.forename: Alexander
authors.initials: A
authors.lastname: Nemukhin
authors.affiliation: Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Moscow, Russia; Department of Chemistry, Lomonosov Moscow State University, Moscow, Russia.
authors.forename: Sergei
authors.initials: S
authors.lastname: Varfolomeev
authors.affiliation: Kazan Federal University, Laboratory of Neuropharmacology, Kazan, Russia. Electronic address: pym.masson@free.fr.
authors.forename: Patrick
authors.initials: P
authors.lastname: Masson
mesh_terms: D055162:Biocatalysis; D002091:Butyrylcholinesterase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002794:Choline / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D017433:Protein Structure, Secondary
chemical_list: D050505:Mutant Proteins; C017100:butyrylcholine; D002091:Butyrylcholinesterase; D002794:Choline
keywords: Binding free energy; Human butyrylcholinesterase; Mivacurium; Molecular dynamics; Myorelaxant abnormal response; Natural variant; Prolonged apnea; Silent variant; Succinylcholine; Umbrella Sampling