Title: Tetramerization domain of human butyrylcholinesterase is at the C- terminus Blong RM, Bedows E, Lockridge O Ref: Biochemical Journal, 327:747, 1997 : PubMed
Butyrylcholinesterase (BChE) in human serum consists predominantly of tetramers. Recombinant BChE, however, expressed in Chinese hamster ovary (CHO) cells, consists of approx. 55% dimers, 10-30% tetramers and 15-40% monomers. To determine the origin of the monomer species we added the FLAG epitope (epitope tag, amino acid sequence DYKDDDDK) to the C-terminus of the enzyme, and expressed BChE-FLAG in CHO cells. We found that secreted, active monomers had lost their FLAG epitope, suggesting that the monomers were made by proteolysis of dimers or tetramers at the C-terminus. To estimate the number of amino acids that could be deleted from the C-terminus without losing BChE activity, we expressed deletion mutants. We found that deletion of up to 50 amino acids from the C-terminus yielded active monomers, but that deletion of 51 amino acids destroyed BChE activity and caused the inactive protein to remain within the cell. Deletion of eight or more amino acids from the N-terminus also resulted in inactive protein that remained inside the cell. Monomeric BChE had wild-type Km and kcat values (8 microM and 24000 min-1 for butyrylthiocholine) and showed substrate activation. The Cys-571-->Ala mutant, though incapable of forming the interchain disulphide bond, had nearly the same amount of tetrameric BChE as recombinant wild-type BChE. These results support the conclusion that the tetramerization domain of BChE is at the C-terminus, within the terminal 50 amino acids, and that the interchain disulphide bond is not essential for tetramerization. Molecular modelling suggested that the tetramerization domain was a four-helix bundle, stabilized by interactions of seven conserved aromatic amino acids.
Butyrylcholinesterase [BCHE (acylcholine acyl hydrolase); EC 3.1.1.8] limits the access of drugs, including tacrine, to other proteins. The "atypical" BCHE 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 BCHE 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. BCHE 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" BCHE will be overly sensitive to additional anticholinesterases and especially so when exposed to several anticholinesterases in combination.
Title: Site-directed mutagenesis of active site residues reveals plasticity of human butyrylcholinesterase in substrate and inhibitor interactions Gnatt A, Loewenstein Y, Yaron A, Schwarz M, Soreq H Ref: Journal of Neurochemistry, 62:749, 1994 : PubMed
In search of the molecular mechanisms underlying the broad substrate and inhibitor specificities of butyrylcholinesterase (BCHE), we employed site-directed mutagenesis to modify the catalytic triad residue Ser198, the acyl pocket Leu286 and adjacent Phe329 residues, and Met437 and Tyr440 located near the choline binding site. Mutant proteins were produced in microinjected Xenopus oocytes, and Km values towards butyrylthiocholine and IC50 values for the organophosphates diisopropylfluorophosphonate (DFP), diethoxyphosphinylthiocholine iodide (echothiophate), and tetraisopropylpyrophosphoramide (iso-OMPA) were determined. Substitution of Ser198 by cysteine and Met437 by aspartate nearly abolished activity, and other mutations of Ser198 completely abolished it. Tyr440 and Leu286 mutants remained active, but with higher Km and IC50 values. Rates of inhibition by DFP were roughly parallel to IC50 values for several Leu286 mutants. Both Km and IC50 values increased for Leu286 mutants in the order Asp < Gln < Lys. In contrast, cysteine, leucine, and glutamine mutants of Phe329 displayed unmodified Km values toward butyrylthiocholine, but up to 10-fold decreased IC50 values for DFP, iso-OMPA, and echothiophate. These findings add Tyr440 and Phe329 to the list of residues interacting with substrate and ligands, demonstrate plasticity in the active site region of BCHE, and foreshadow the design of recombinant BCHEs with tailored scavenging properties.
