Molecular recognition in acetylcholinesterase catalysis: free-energy correlations for substrate turnover and inhibition by trifluoro ketone transition-state analogs.
Ten meta-substituted aryl trifluoromethyl ketones (m-XC6H4COCF3; X = H, CH3, CF3, C2H5, isopropyl, t-butyl, NH2, NMe2, N+Me3, NO2) have been evaluated as inhibitors of acetylcholinesterases from Electrophorus electricus and Torpedo californica. Trifluoro ketones that have small meta substituents (X = H, CH3, CF3, C2H5, NH2, NO2) are rapid reversible inhibitors, whereas the remaining compounds in this study show time-dependent inhibition. Dissociation constants (Ki values) for these compounds span a range of approximately 10(7)-fold, with trifluoroacetophenone (X = H) being the least potent and m-(N,N,N-trimethylammonio)trifluoroacetophenone (X = Me3N+) being the most potent inhibitor. For the latter compound Ki values are 1.5 and 15 fM for inhibitions of the respective acetylcholinesterases (Nair, H. K., Lee, K., & Quinn, D. M. (1993) J. Am. Chem. Soc. 115, 9939-9941). Linear correlations of log(kcat/Km) for substrate turnover versus pKi of inhibitors have slopes of approximately 0.6, which suggest that aryl trifluoro ketones bind to AChE in a manner that structurally resembles transition states in the acylation stage of catalysis. Substituent variation in the inhibitors allows one to gauge the importance for AChE function of molecular recognition in the quaternary ammonium binding locus of the active site. This locus is frequently termed the "anionic site" and consists of E199, W84, and perhaps Y130 and F330. Correlations of pKi versus hydrophobicity constant are linear for alkyl and trifluoromethyl substituents but fail for nitrogen-containing substituents. However, three-dimensional correlations of pKi versus sigma m and molar refractivity of substituents indicate that dispersion interactions in the anionic locus contribute approximately 10(5)-fold (delta delta G = 7 kcal mol-1) to the above-mentioned 10(7)-fold range of inhibitor potencies. The remaining approximately 100-fold arises from the inductive electronic effects of substituents on the stability of the tetrahedral adduct that forms between the ketone carbonyl of inhibitors and S200 in the esteratic locus of the active site. Values of k(on), the second-order rate constant for binding of time-dependent inhibitors, monitor a diffusion-controlled process. Moreover, k(on) for the quaternary ammonio inhibitor is 20-70-fold higher than for inhibitors that have uncharged meta substituents, which likely reflects the effect of the electrical field of AChE on ligand and substrate binding.
Department of Chemistry, University of Iowa, Iowa City 52242.
H K
HK
Nair
J
J
Seravalli
T
T
Arbuckle
D M
DM
Quinn
D000098:Acetophenones / Q000378:metabolism / Q000494:pharmacology; D000110:Acetylcholinesterase / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D004593:Electrophorus; D007659:Ketones / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D013816:Thermodynamics; D014101:Torpedo
D000098:Acetophenones; D002800:Cholinesterase Inhibitors; D007659:Ketones; C088432:3-(N,N-dimethylamino)trifluoroacetophenone; D000110:Acetylcholinesterase