Abstract
Several of the residues constituting the peripheral anionic site (PAS) in human acetylcholinesterase (HuAChE) were identified by a combination of kinetic studies with 19 single and multiple HuAChE mutants, fluorescence binding studies with the Trp-286 mutant, and by molecular modeling. Mutants were analyzed with three structurally distinct positively charged PAS ligands, propidium, decamethonium, and di(p-allyl-N-dimethylaminophenyl)pentane-3-one (BW284C51), as well as with selective active center inhibitors, hexamethonium and edrophonium. Single mutations of residues Tyr-72, Tyr-124, Glu-285, Trp-286, and Tyr-341 resulted in up to 10-fold increase in inhibition constants for PAS ligands, whereas for multiple mutants up to 400-fold increase was observed. The 6th PAS element residue Asp-74 is unique in its ability to affect conformation of both the active site and the PAS (Shafferman, A., Velan, B., Ordentlich, A., Kronman, C., Grosfeld, H., Leitner, M., Flashner, Y., Cohen, S., Barak, D., and Ariel, N. (1992) EMBO J. 11, 3561-3568) as demonstrated by the several hundred-fold increase in Ki for D74N inhibition by the bisquaternary ligands decamethonium and BW284C51. Based on these studies, singular molecular models for the various HuAChE inhibitor complexes were defined. Yet, for the decamethonium complex two distinct conformations were generated, accommodating the quaternary ammonium group by interactions with either Trp-286 or with Tyr-341. We propose that the PAS consists of a number of binding sites, close to the entrance of the active site gorge, sharing residues Asp-74 and Trp-286 as a common core. Binding of ligands to these residues may be the key to the allosteric modulation of HuAChE catalytic activity. This functional degeneracy is a result of the ability of the Trp-286 indole moiety to interact either via stacking, aromatic-aromatic, or via pi-cation attractions and the involvement of the carboxylate of Asp-74 in charge-charge or H-bond interactions.
Highlights
Several of the residues constituting the peripheral ligands bind at theactive center while others associate with a anionic site (PAS) in human acetylcholinesterase peripheral anionic site (PAS),remote from the active center (for (HuAChE)were identified by a combination of kinetic reviews, see Hucho et al, 1991; Massoulieet al., 1993)
The functional significance of PAS is a controversial issue. It was implicated in the catalytic pathway of acetylcholine (ACh) hydrolysis (Haas et al, 19921, in ionic strength monitoring (Berman and &, 1992),and in substrateinhibition (Radic bisquaternary ligands decamethonium and BW284CS1. et al, 1991).A decrease in affinity for a selective PAS ligand
Formations were generated, accommodatingthe quater- PAS ligands, which include gallamine, d-tubocuranary ammonium group by interactions with either Trp- rine, decamethonium, and propidium, bind in a mutually ex286 or with -341.We propose that thePAS consists of clusive manner, their modes of inhibition of catalysis are not a number of binding sites, close to the entrance of the equivalent and appear to depend upon the structure of the active site gorge, sharing residuesAsp74 and “280 as ligand (Roufogalis and Quist, 1972; Rosenbeny, 1975)
Summary
T6ca-hrpSerhiueeibndnshytoilrbua-ittpteohwsresinatanh(nFdtaigchIenr. tih1ydi)lbiti3hnt,ioio8urcs-m-hdAdoiilaliilomndienikidooien-d5eid(-tipe(cr3(os’Apt-uTitddCriiui)emm(sSe)rti,eghpmyeoltahra)tymealmd(somnhs-iuehurbyemsd)twrrpoaretxoreyep.-yl-zwyemTroeeeouvnsaelldui(gaFatiengdt.h1be)in.Pedfrfioenpcgit,dsisouefmvsetwrraualcsttyiunprcaellsumdoefodrdeaifvsiecaranstiieboxlnecsliuonsfhitvihbeeiPteoAnrSsphenyl)-dimethylammonium chloride (edrophonium), di(p-allyl-N-di- ligand (Taylor and Lappi, 1975). The bisquaternary ligands methylaminophenyl)-pentane-3-one (BW2&34C51), 1,lO-bis(trimethy1- decamethonium and BW284C51 that span the distance beammoniumMecane (decamethonium), and 1,6-bis(trimethylammoni- tween the active center and PAS (Kmpka, 1966; Hodgeet al., um)hexane (hexamethonium) were all purchased from Sigma. Determinatwn ofAChEActivity and Analysis of KineticData-AChE activity with ATC was assayed accordingto Ellman et al (1961).Standard assays were performed in the presence of 0.1 mg/ml bovine serum. 1992)were used to map their interactions with residues along the active center gorge. Hexamethonium which is an active center bisquaternary ligand, too short to reach the PAS (Lullalbumin, 0.3 n m 5,5’-dithiobis(2-nitrobenzoicacid), 5 or 50 m~ sodium mann et al, 1971), was included since its alkyl chain is exphosphate buffer, pH 8.0,and varying substrate (ATC)concentrations. For comparison we have included the cationic active center ligand edrophonium. 1992b, 1992c; Ordentlich et al, 1993)to participate in active center interactions
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