Molecular Recognition in Acetylcholinesterase Catalysis: Free-Energy Correlations for Substrate Turnover and Inhibition by Trifluoro Ketone Transition-State Analogs

Haridasan K. Nair, Javier Seravalli, Tomira Arbuckle, Daniel M. Quinn

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Abstract

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, N02) 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 ∼107-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 ∼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 El99, 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 σm and molar refractivity of substituents indicate that dispersion interactions in the anionic locus contribute ∼ 105-fold (ΔΔG = 7 kcal mol−1) to the above-mentioned 107-fold range of inhibitor potencies. The remaining ∼ 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 kon, the second-order rate constant for binding of time-dependent inhibitors, monitor a diffusion-controlled process. Moreover, kon 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.

Original languageEnglish (US)
Pages (from-to)8566-8576
Number of pages11
JournalBiochemistry
Volume33
Issue number28
DOIs
StatePublished - Jul 1 1994

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Molecular recognition
Acetylcholinesterase
Ketones
Catalysis
Free energy
Substrates
Catalytic Domain
Electrophorus
Torpedo
Acylation
Time and motion study
Cholinesterase Inhibitors
Hydrophobicity
Hydrophobic and Hydrophilic Interactions
Ammonium Compounds
Gages
Rate constants
Refractive index
Nitrogen
Ligands

ASJC Scopus subject areas

  • Biochemistry

Cite this

Molecular Recognition in Acetylcholinesterase Catalysis : Free-Energy Correlations for Substrate Turnover and Inhibition by Trifluoro Ketone Transition-State Analogs. / Nair, Haridasan K.; Seravalli, Javier; Arbuckle, Tomira; Quinn, Daniel M.

In: Biochemistry, Vol. 33, No. 28, 01.07.1994, p. 8566-8576.

Research output: Contribution to journalArticle

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