Butyrylcholinesterase deficiency is characterized by prolonged
apnea after the use of muscle relaxants (
suxamethonium or
mivacurium) in patients who have mutations in the BCHE gene. Here, we report a case of prolonged neuromuscular block after administration of
suxamethonium leading to the discovery of a novel BCHE variant (c.695T>A, p.Val204Asp). Inhibition studies, kinetic analysis and molecular dynamics were undertaken to understand how this mutation disrupts the catalytic triad and determines a "silent" phenotype. Low activity of patient plasma
butyrylcholinesterase with
butyrylthiocholine (BTC) and
benzoylcholine, and values of
dibucaine and
fluoride numbers fit with heterozygous atypical silent genotype. Electrophoretic analysis of plasma BChE of the proband and his mother showed that patient has a reduced amount of tetrameric
enzyme in plasma and that minor fast-moving BChE components: monomer, dimer, and monomer-
albumin conjugate are missing. Kinetic analysis showed that the p.Val204Asp/p.Asp70Gly-p.Ala539Thr BChE displays a pure Michaelian behavior with BTC as the substrate. Both catalytic parameters Km = 265 µM for BTC, two times higher than that of the atypical
enzyme, and a low Vmax are consistent with the absence of activity against
suxamethonium. Molecular dynamic (MD) simulations showed that the overall effect of the mutation p.Val204Asp is disruption of hydrogen bonding between Gln223 and Glu441, leading Ser198 and His438 to move away from each other with subsequent disruption of the catalytic triad functionality regardless of the type of substrate. MD also showed that the
enzyme volume is increased, suggesting a pre-denaturation state. This fits with the reduced concentration of p.Ala204Asp/p.Asp70Gly-p.Ala539Thr tetrameric
enzyme in the plasma and non-detectable fast moving-bands on electrophoresis
gels.