MOLECULAR RECOGNITION IN ACETYLCHOLINESTERASE CATALYSIS - FREE-ENERGYCORRELATIONS FOR SUBSTRATE TURNOVER AND INHIBITION BY TRIFLUORO KETONE TRANSITION-STATE ANALOGS

Ten meta-substituted aryl trifluoromethyl ketones (m-XC(6)H(4)COCF(3); X = H, CH3, CF3, C2H5, isopropyl, t-butyl, NH2, NMe(2), N(+)Me(3), NO2 ) have been evaluated as inhibitors of acetylcholinesterases from Elec trophorus electricus and Torpedo californica. Trifluoro ketones that h ave small meta substituents (X = H, CH3, CF3, C2H5, NH2, NO2) are rapi d reversible inhibitors, whereas the remaining compounds in this study show time-dependent inhibition. Dissociation constants (K-i values) f or these compounds span a range of similar to 10(7)-fold, with trifluo roacetophenone (X = H) being the least potent and m-(N,N,N-trimethylam monio)trifluoroacetophenone (X = Me(3)N(+)) being the most potent inhi bitor. For the latter compound K-i values are 1.5 and 15 fM for inhibi tions of the respective acetylcholinesterases (Nair, H. K., Lee, K., & Quinn, D. M. (1993) J. Am. Chem. Sec. 115, 9939-9941). Linear correla tions of log(k(cat)/K-m) for substrate turnover versus pK(i) of inhibi tors have slopes of similar to 0.6, which suggest that aryl trifluoro ketones bind to AChE in a manner that structurally resembles transitio n states in the acylation stage of catalysis. Substituent variation in the inhibitors allows one to gauge the importance for AChE function o f molecular recognition in the quaternary ammonium binding locus of th e active site. This locus is frequently termed the ''anionic site'' an d consists of E199, W84, and perhaps Y130 and F330. Correlations of pK (i) versus hydrophobicity constant are linear for alkyl and trifluorom ethyl substituents but fail for nitrogen-containing substituents. Howe ver, three-dimensional correlations of pK(i) versus sigma(m) and molar refractivity of substituents indicate that dispersion interactions in the anionic locus contribute N 10(5)-fold (Delta Delta G = 7 kcal mol (-1)) to the above-mentioned 10(7)-fold range of inhibitor potencies. The remaining similar to 100-fold arises from the inductive electronic effects of substituents on the stability of the tetrahedral adduct th at forms between the ketone carbonyl of inhibitors and S200 in the est eratic locus of the active site. Values of k(on), the second-order rat e constant for binding of time-dependent inhibitors, monitor a diffusi on-controlled process. Moreover, k(on) for the quaternary ammonio inhi bitor is 20-70-fold higher than for inhibitors that have uncharged met a substituents, which likely reflects the effect of the electrical fie ld of AChE on ligand and substrate binding.