Intramolecular relationships in cholinesterases revealed by oocyte expression of site-directed and natural variants of human BCHE.

Abstract

Structure‐function relationships of cholinesterases (CHEs) were studied by expressing site‐directed and naturally occurring mutants of human butyrylcholinesterase (BCHE) in microinjected Xenopus oocytes. Site‐directed mutagenesis of the conserved electronegative Glu441,Ile442,Glu443 domain to Gly441,Ile442,Gln443 drastically reduced the rate of butyrylthiocholine (BTCh) hydrolysis and caused pronounced resistance to dibucaine binding. These findings implicate the charged Glu441,Ile442,Glu443 domain as necessary for a functional CHE catalytic triad as well as for binding quinoline derivatives. Asp70 to Gly substitution characteristic of ‘atypical’ BCHE, failed to alter its Km towards BTCh or dibucaine binding but reduced hydrolytic activity to 25% of control. Normal hydrolytic activity was restored to Gly70 BCHE by additional His114 or Tyr561 mutations, both of which co‐appear with Gly70 in natural BCHE variants, which implies a likely selection advantage for these double BCHE mutants over the single Gly70 BCHE variant. Gly70 BCHE variants also displayed lower binding as compared with Asp70 BCHE to cholinergic drugs, certain choline esters and solanidine. These effects were ameliorated in part by additional mutations or in binding solanidine complexed with sugar residues. These observations indicate that structural interactions exist between N′ and C′ terminal domains in CHEs which contribute to substrate and inhibitor binding and suggest a crucial involvement of both electrostatic and hydrophobic domains in the build‐up of the CHE active center.

Human Erythrocyte but Not Brain Acetylcholinesterase Hydrolyses Heroin to Morphine

“…Following 5 min incubation with 650 mol/L of the noted agents and washing to remove unbound drug, we tested the residual capacity of these enzymes to hydrolyse ATCh or BTCh using a spectrophotometric assay adapted for use with microtitre plates. 16 When tested within 5 min of drug removal, codeine did not affect substrate hydrolysis by BuChE, AChE-E5 or AChE-E6. In contrast, heroin enhanced the activity of BuChE by 25% and inhibited the activity of AChE-E5 by 28%.…”

Acetylcholinesterase/paraoxonase genotype and expression predict anxiety scores in Health, Risk Factors, Exercise Training, and Genetics study

“…We also genotyped three known PON1 promoter polymorphisms (indicated by distance in nucleotides from the translation start site at 0): Ϫ108C͞T, Ϫ162A͞G, Ϫ126G͞C, contributing to 22.4%, 2.4%, and none of the variation in PON1 expression, respectively (33). Of the numerous BCHE mutations, we genotyped the D70G substitution yielding the ''atypical'' BChE variant, with enzymatic activity 30% lower than the wild-type enzyme (34). Homozygous carriers of this polymorphism display extreme anxiety after exposure to anti-AChEs (35).…”

Anionic subsites of the catalytic center of acetylcholinesterase from Torpedo and from cobra venom.

“…Recently replacement of Asp-70 by glycine in human butyrylcholinesterase (homologous to Asp-72 in Torpedo AcChoEase) was reported to disrupt anionic site interactions (35,36). There is converging evidence that the region around this residue and Trp-84, both contained in the first disulfide loop of AcChoEase (positions 67-94), is mainly contributing to the cholinium binding site of cholinesterases.…”