Asp70 in the Peripheral Anionic Site of Human Butyrylcholinesterase

Abstract

The goal of this work was to determine what amino acids at the mouth of the active-site gorge are important for the function of human butyrylcholinesterase. Mutants D70G, Q119Y, G283D, A277W, A277H and A277W/G283D were expressed in human embryonal kidney cells and the secreted enzymes were assayed by steady-state kinetics. The result was that only one amino acid, D70 was found to be important for function. When D70 was mutated to G , the same mutation as in the naturally occurring atypical butyrylcholinesterase, the affinity for positively charged substrates and positively charged inhibitors decreased 5 -30-fold. The D70G mutant had another striking abnormality in that it was virtually devoid of the phenomenon of substrate activation by excess butyrylthiocholine. Thus, though k,.,, was the same for wild-type and D70G mutant, being 24000 min ' at low butyrylthiocholine concentrations (0.01 -0.1 mM), it failed to increase for the D70G mutant at 40 mM butyrylthiocholine, whereas it increased threefold for wild type. The D70C mutant was more sensitive to changes in salt concentration, its catalytic rate decreasing more than that of the wild type. The D70G mutant appeared to have a greater surface negative charge than wild type suggesting that the D70G mutant had a conformation different from that of the wild type. That D70 affects the function of butyrylcholinesterase, together with its location at the mouth of the active-site gorge, supports the hypothesis that D70 is a component of the peripheral anionic site of butyrylcholinesterase. Mutants containing aromatic amino acids at the mouth of the gorge had increased binding affinity for propidium and fasciculin, but unaltered function, suggesting that aromatic amino acids are not important to the function of the peripheral anionic site of butyrylcholinesterase.Keywords: butyrylcholinesterase; peripheral anionic site; aspartate 70; atypical ; propidium.Atypical human butyrylcholinesterase (BuChE) was first recognized as a genetic variant by Kalow (reviewed in [I]). Kalow noted that atypical BuChE had a lower affinity for all positively charged substrates and positively charged inhibitors and that this lower affinity accounted for the reduced rate of hydrolysis of the muscle relaxant drug, succinyldicholine. Succinyldicholine is given intravenously and 90 % of a standard dose is destroyed by wild-type BuChE in serum, so that only a small percentage of the injected dose reaches the nerve-muscle junction. People who have atypical BuChE do not hydrolyze any of the drug during its residence in the blood. Consequently a massive overdose reaches the muscle endplate and people experience hours of muscle paralysis and difficulty with breathing rather than the intended 2 or 3 min. Kalow and Genest [2) devised a phenotyping method to identify atypical BuChE by measuring the percentage inhibition by dibucaine. This spectrophotometric assay has proven to be completely reliable as a predictor of the atypical variant as judged by clinical response to succinyldicholine [I, 3, 41 and...

Importance of aspartate-70 in organophosphate inhibition, oxime re-activation and aging of human butyrylcholinesterase

“…Moreover, our recent results indicate that, after initial interaction with Asp-70, positively charged substrates are correctly oriented to slide on to Trp-82, the actual ' anionic site ' [54]. Asp-70 is also involved in activation by excess substrate [10]. The present results show that Asp-70 is involved in the complex dependence of re-activation on pralidoxime iodide (2-PAM) concentration.…”

“…This residue is located at the rim of the active-site gorge, where it has been found to be a part of the peripheral anionic site (PAS) of BuChE [10]. Moreover, our recent results indicate that, after initial interaction with Asp-70, positively charged substrates are correctly oriented to slide on to Trp-82, the actual ' anionic site ' [54].…”

“…Site-directed mutagenesis of the BuChE gene was performed as Figure 1 Chemical structures of organophosphates used in this study described [10]. The mutated residues, Asp-70 and Ala-277, are located at the mouth of the active-site gorge [5].…”

“…Mutations on residues Asp-70 and Ala-277 were expected to alter affinity for positively charged ligands of BuChE. Recombinant wild-type and mutant human BuChE were expressed in stably transfected human embryonal 293 kidney cells [10]. Secreted recombinant enzymes were collected into Neuman & Tytell's serum-less medium (Gibco) and stored at 4 mC under sterile conditions.…”

X-Ray Crystallographic Studies on Acetylcholinesterase and Related Enzymes

“…Recent studies have suggested that a particular residue, D72 (D74 in mAChE and hAChE, D70 in hBChE), might contribute to the specificity of ChEs for cationic ligands by a trapping mechanism (Hosea et al, 1996;Masson et al, 1996). This residue is strategically placed near the top of the active-site gorge (Fig.…”