Overlapping drug interaction sites of human butyrylcholinesterase dissected by site-directed mutagenesis

Y Loewenstein-Lichtenstein, D Glick, N Gluzman, M Sternfeld, H Zakut and H Soreq

Department of Biological Chemistry, The Institute of Life Sciences, The Hebrew University of Jerusalem, Israel.

Butyrylcholinesterase [BuChE (acylcholine acyl hydrolase); EC 3.1.1.8] limits the access of drugs, including tacrine, to other proteins. The "atypical" BuChE variant, in which Asp70 at the rim of the active site gorge is substituted by glycine, displayed a more drastically weakened interaction with tacrine than with cocaine, dibucaine, succinylcholine, BW284c51 [1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide], or alpha-solanine. To delineate the protein domains that are responsible for this phenomenon, we mutated residues within the rim of the active site gorge, the region parallel to the peripheral site in the homologous enzyme acetylcholinesterase [AChE (acetylcholine acetyl hydrolase); EC 3.1.1.7], the oxyanion hole, and the choline-binding site. When expressed in microinjected Xenopus laevis oocytes, all mutant DNAs yielded comparable amounts of immunoreactive protein products. Most mutants retained catalytic activity close to that of wild-type BuChE and were capable of binding ligands. However, certain modifications in and around the oxyanion hole caused a dramatic loss in activity. The affinities for tacrine were reduced more dramatically than for all other ligands, including cocaine, in both oxyanion hole and choline-binding site mutants. Modified ligand affinities further demonstrated a peripheral site in residues homologous with those of AChE. BuChE mutations that prevented tacrine interactions also hampered its ability to bind other drugs and inhibitors, which suggests a partial overlap of the binding sites. This predicts that in addition to their genetic predisposition to adverse responses to tacrine, homozygous carriers of "atypical" BuChE will be overly sensitive to additional anticholinesterases and especially so when exposed to several anticholinesterases in combination.

Volume 50, Issue 6, pp. 1423-1431, 12/01/1996
Copyright © 1996 by American Society for Pharmacology and Experimental Therapeutics

This article has been cited by other articles:

				V. Marcel, S. Estrada-Mondaca, F. Magne, J. Stojan, A. Klaebe, and D. Fournier Exploration of the Drosophila Acetylcholinesterase Substrate Activation Site Using a Reversible Inhibitor (Triton X-100) and Mutated Enzymes J. Biol. Chem., April 14, 2000; 275(16): 11603 - 11609. [Abstract] [Full Text] [PDF]

				A. Ordentlich, D. Barak, C. Kronman, N. Ariel, Y. Segall, B. Velan, and A. Shafferman Functional Characteristics of the Oxyanion Hole in Human Acetylcholinesterase J. Biol. Chem., July 31, 1998; 273(31): 19509 - 19517. [Abstract] [Full Text] [PDF]