In contrast to AChE BuChE Table exhibited a measurable time
In contrast to AChE, BuChE (Table 2) exhibited a measurable time-dependent inactivation only with ap-1 transcription factor 2 among the four acetophenones in this study. Values of Ki obtained from steady-state inhibition of BuChE by all four acetophenones were, as noted in the Results, 10- to 200-fold greater (indicating lower affinity) than those for the same compounds with AChE (Table 1, Table 2). The three acetophenones that showed time-dependent inhibition of AChE gave association rate constants (ka) in excess of 107M−1min−1 (Table 1). The ka values for 1 and 2 are within the range of those reported for eel, torpedo, and mouse AChEs.29, 30, 47 Only compound 2 provided a measurable ka value with BuChE; compounds 1, 3 and 4 attained steady state equilibrium with BuChE so rapidly that no reaction rate constants could be measured here. The ka value of 13×107M−1min−1 for 2 with BuChE (Table 2) was five-fold more than the ka for 2 with AChE (Table 1), consistent with the larger and more accessible active site gorge in BuChE. The observation of a nonlinear dependence of kobs on the inhibitor concentration for all of the time-dependent reactions in Table 1, Table 2 indicated that the successive equilibria for acetophenone binding in Scheme 5 and Eqs. (3), (4), (5), (6) were more appropriate here than the single equilibrium in Scheme 4 and Eq. 2. Scheme 5 and Eqs. (3), (4), (5), (6) were invoked previously to account for the binding of compound 1 to eel and torpedo AChE, but it is noteworthy that in that report Scheme 4 and Eq. 2 were sufficient to account for the binding of compound 2 to eel or torpedo AChE. The hemiketal complex formed by acetophenones with cholinesterases is analogous to the tetrahedral transition state intermediate formed during the hydrolysis of ester substrates like acetylcholine, and enzyme stabilization of this complex promotes catalytic hydrolysis. Since all the acetophenone compounds described here are halogenated methyl ketones, the formation of the hemiketal is likely to be qualitatively similar for all, and this mechanism alone cannot account for the differences in the observed affinities of the derivatives with AChE and BuChE. However, there is a strong correlation between the meta-substituent on a trifluoroacetophenone and the potency of its inhibition of AChE. The inhibitor m-(N,N,N-trimethylammonium)trifluoroacetophenone (TMTFA) has the highest reported affinity of any trifluoroacetophenone with AChE.29, 30 Its Ki with mouse AChE is 5fM after correction for hydration and 0.3nM before correction, values that are nearly 1000-fold higher after correction but only 6-fold higher before correction than those for compound 2 with mouse AChE.29, 47 The high in vitro affinity of TMTFA can be attributed largely to a stabilizing cation–π interaction between the positively charged nitrogen of the trimethylammonio group with the indole ring of a tryptophan residue (Trp86 in mouse AChE) and, to a lesser extent, to a more electrophilic carbonyl carbon resulting from this electron-withdrawing group. This electrophilic effect could account for the 6- to 8-fold higher affinities of the trifluoroacetophenones 1 and 2 for AChE relative to the corresponding chlorodifluoroacetophenones 3 and 4, which have less electrophilic carbonyl carbons. In contrast, there are no differences in the affinities of 1 relative to 3 and 2 relative to 4 for BuChE, where hemiketal formation occurs to a lesser extent. Alternatively, these differences may reflect a smaller acyl pocket in the active site of AChE relative to that of BuChE. Table 1, Table 2 indicate that the m-tert-butylacetophenones 2 and 4 are 15- to 50-fold more potent reversible inhibitors of both AChE and BuChE than the corresponding m-dimethylaminoacetophenones 1 and 3. It is possible that the lower affinity of the m-dimethylamino derivatives may result from repulsion between the lone pair on the neutral nitrogen atom in the m-dimethylamino group of the compound and the indole nitrogen of the corresponding tryptophan residue of human cholinesterases. The absence of such repulsion for compounds 2 and 4 and the larger size of the tert-butyl moiety may favour inhibitor stabilization within the active site gorge and help to account for their higher affinities relative to the m-dimethylamino acetophenone compounds (1 or 3) in our studies.