No abstract

We have reported that benzene-1,2-, 1,3-, and 1,4-di-N-substituted carbamates (1-15) are characterized as the conformationally constrained inhibitors of acetylcholinesterase and mimic gauche, eclipsed, and anti-conformations of acetylcholine, respectively (J Biochem Mol Toxicol 2007;21:348-353). We further report the inhibition of butyrylcholinesterase by these inhibitors. Carbamates 1-15 are also characterized as the pseudosubstrate inhibitors of butyrylcholinesterase as in the acetylcholinesterase catalysis. Benzene-1,4-di-N-n-hexylcarbamate (12) and benzene-1,4-di-N-n-octylcarbamate (13) are the two most potent inhibitors of butyrylcholinesterase among inhibitors 1-15. These two para compounds, with the angle of 180 degrees between two C(benzene)--O bonds, mimic the preferable anti C--O/C--N conformers for the choline ethylene backbone of butyrylcholine during the butyrylcholinesterase catalysis. The second n-hexylcarbamyl or n-octylcarbamyl moiety of inhibitors 12 and 13 is proposed to bind tightly to the peripheral anionic site of butyrylcholinesterase from molecular modeling. Butyrylcholinesterase prefers para-carbamates to ortho- and meta-carbamates, whereas acetylcholinesterase prefers para- and meta-carbamates to ortho-carbamates. This result implies that the anionic site of butyrylcholinesterase is relatively smaller than that of acetylcholinesterase because meta-carbamates, which may bind to the anionic sites of both enzymes, are not potent inhibitors of butyrylcholinesterase.

The goal of this work is to study kinetics and quantitative structure-activity relationships for steady states of Pseudomonas sp. lipase-catalyzed hydrolysis of both diesters and monoesters of ethylene glycol. Based on the steady-state kinetics of the enzyme-catalyzed hydrolysis of the diesters of ethylene glycol, the diesters and the monoesters react simultaneously as soon as monoester has started to build up in the reaction medium. In other words, the apparent K m values of the diesters are the K m values of the diesters (K mA ) plus the K m values of the monoesters (K mB ), and all V max values are about the same. Moreover, the pHstat titration curve of the enzyme-catalyzed hydrolysis of the diesters of ethylene glycol is initially hyperbolic, then there is a sharp falloff in the hydrolysis rate. The abrupt stoppage of the reaction (relaxation stage) may be due to the existence of two phases in the reaction medium, that is, the product (ethylene glycol) and the substrates (the diesters of ethylene glycol) are not miscible. Furthermore, quantitative structure-activity relationships for varied acyl groups of mono-and diesters of ethylene glycol are studied. The fact that both pK mA and pK mB values are linearly correlated with the hydrophobicity constant (π) but not with the electronic substituent constants (σ*) indicates that the affinity of these substrates for the enzyme depends only on the hydrophobicity of substrates. FIG. 4. Plots of (A) pK mA , (B) log (V maxA /K mA ), (C) pK mB , and (D) log (V maxB /K mB ) values for PSL-catalyzed hydrolysis of diesters 1-6 and monoesters 7-12 against electronic substituent constants σ*. For abbreviation see Figure 1. FIG. 5. Plot of the log k relax. values for PSL-catalyzed hydrolysis of diesters 1-6 against π. For abbreviation see Figure 1. 206 S.-Y. CHIOU ET AL. JAOCS, Vol. 83, no. 3 (2006) FIG. 6.Proposed mechanism for PSL-catalyzed hydrolysis of diesters 1-6 to ethylene glycerol. The first step (K mA ) is formation of the enzyme-diester tetrahedral complex. The second step (k catA ) involves formation of monoester 7-10 as the first product of the reaction and then formation of the second product, FA. The third step is diffusion of the monoesters 7-10 into the active site of the enzyme. The fourth step (K mB ) involves the formation of the enzyme-monoester tetrahedral complex. The fifth step (k caB ) involves the formation of ethylene glycol. ψ, slope for log k vs. π plot; ACS, acyl chain binding site; for other abbreviation see Figure 1.