Aspartate‐70 to glycine substitution confers resistance to naturally occurring and synthetic anionic‐site ligands on inovo produced human butyrylcholinesterase

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<jats:title>Abstract</jats:title><jats:p>The “atypical” allelic variant of human butyrylcholinesterase (BuChE) can be characterized by its failure to bind the local anesthetic dibucaine, the muscle relaxant succinylcholine, and the naturally occurring steroidal alkaloid solanidine, all assumed to bind to the charged anionic site component within the normal BuChE enzyme. A single nuclcotide substitution conferring a change of aspartate‐70 into glycine was recently reported in the CHE gene encoding BuChE from several individuals having the “atypical” BuChE phenotype, whereas in two other DNA samples, this mutation appeared together with a second alteration conferring a change of serine‐425 into proline. To separately assess the contribution of each of these mutations toward anionic site interactions in BuChE, three transcription constructs were engineered with each of these substitutions alone or both of them together. <jats:italic>Xenopus</jats:italic> oocyte microinjection of normal or mutated synthetic BuChEmRNA transcripts was employed in conjunction with biochemical analyzes of theresultant recombinant BuChE variants. The presence of the Gly‐70 mutation alone was found to render the enzyme resistant to 100 μM solanidine and 5 mM succinylcholine; concentrations sufficient to inhibit the “normal”, Asp‐70 containing BuChE by over 50%. Furthermore, when completely inhibited by the organophosphorous poison diisopropylfluorophosphate (DFP), Gly‐70 BuChE failed to be reactivated by 10 mM of the cholinesterase‐specific oxime pyridine 2‐aldoxime methiodide (2‐PAM); a concentration restoring about 50% of activity in the “normal” Asp‐70 recombinant enzyme. The Pro‐425 mutation alone had no apparent influence on BuChE interactions with any of these ligands. However, it conferred synergistic effects on some of the anionic site changes induced by the Gly‐70 mutation. Thus, in addition to being resistant to soianidine and to 2‐PAM reactivation, the Gly‐70/Pro‐25 variant completely failed to bind either dibucaine (1 mM) or succinylcholine (1,000 mM), 20‐fold higher concentrations than those inhibiting the single Gly‐70 mutant. In view of the evolutionary conserved Asp‐70 and Ser‐425 peptide domains in all known cholinesterases, these findings suggest that the Asp‐70 residue serves as part of the anionic site responsible for binding charged substrates throughout the family of cholinesterases and that genetically linked mutations in these two peptide domains may confer selection advantage through their synergistic effects on BuChE resistance to inhibitors.</jats:p>

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