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Cationic triarylmethane dyes (TAM(+))s which are used as colorants in industry and as frequent tools and reagents in analytical, cell biological and biomedical research have been recently characterized as reversible inhibitors of human butyrylcholinesterase. In this study, the inhibitory effects of two TAM(+)s, malachite green (MG) and methyl green (MeG) on five human BChE mutants (A277V, P285L, H77L, A328F and F329A) were studied spectrophotometrically at 25 degrees C in 50 mM MOPS buffer pH 8, using butyrylthiocholine as substrate. The kinetic results obtained with mutant enzymes were compared to those obtained with recombinant wild type BChE. MG and MeG were found to act as competitive/linear mixed inhibitors of recombinant wild type BChE and all BChE mutants except the F329A mutant. Both dyes caused complex nonlinear inhibition of F329A mutant, pointing to multisite binding. K-i values for MG and MeG, estimated by nonlinear regression analysis, were 3.8 and 27 mu M, respectively, as compared to the 50- to 150-fold lower values observed with recombinant wild type BChE. The observed significant differences in kinetic pattern and K-i values between recombinant wild type BChE and F329A mutant suggest that phenylalanine at position 329 in human BChE is a critical residue in MG and MeG binding to enzyme. (C) 2011 Elsevier Inc. All rights reserved.

The kinetic effects of a selection of triarylmethane, phenoxazine and phenothiazine dyes (pararosaniline (PR), malachite green (MG), methyl green (MeG); meldola blue (MB), nile blue (NB), nile red (NR); methylene blue (MethB)) and of ethopropazine on horse serum butyrylcholinesterase were studied spectrophotometrically at 25 degrees C in 50 mM MOPS buffer, pH 8, using butyrylthiocholine as substrate. PR, MeG, MB and ethopropazine acted as linear mixed type inhibitors of the enzyme, with respective K(i) values of 4.5 +/- 0.50 mu M, 0.41 +/- 0.007 mu M, 0.44 +/- 0.086 mu M and 0.050 +/- 0.0074 mu M. MG, NB, MethB and NR caused complex, nonlinear inhibition pointing to cooperative binding at two sites. Intrinsic K ' values ([I](2)(0.5) extrapolated to [S]=0) for MG, NB, NR and MethB were 0.20 +/- 0.096 mu M, 0.0018 +/- 0.0015 mu M, 0.92 +/- 0.23 mu M and 0.23 +/- 0.08 mu M. NB stood out as a potent inhibitor effective at nM levels. Comparison of inhibitory effects on horse and human serum butyrylcholinesterases suggested that the two enzymes must have distinct microstructural features. (c) 2008 Elsevier Inc. All rights reserved.

Two cationic phenoxazine dyes, meldola blue (MB) and nile blue (NB), and the structurally related phenothiazine, methylene blue (MethB), were found to act as complex inhibitors of human plasma cholinesterase (butyrylcholinesterase, BChE). Studied at 25 °C, in 100 mM MOPS buffer (pH 8.0), with butyrylthiocholine as substrate, the kinetic pattern of inhibition indicated cooperative I binding at 2 sites. Intrinsic K′ values ( ≡ [ I ] 0.5 2 extrapolated to [S] = 0) for MB, NB and MethB were 0.64 ± 0.05, 0.085 ± 0.026 and 0.42 ± 0.04 μM, respectively. Under the same experimental conditions the dyes acted as single-occupancy, hyperbolic-mixed inhibitors of electric eel acetylcholinesterase (AChE), with Ki = 0.035 ± 0.010, 0.026 ± 0.0034 and 0.017 ± 0.0063 μM (for MB, NB, MethB); α (coefficient of competitive interaction) = 1.8–2.4 and β (coefficient of noncompetitive interaction) = 0.15–0.28. The complexity of the BChE inhibitory effect of phenoxazine/phenothiazine dyes contrasted with that of conventional ChE inhibitors which cause single-occupancy (n = 1), competitive or mixed inhibition in both AChE and BChE and signaled novel modes of ligand interaction at (or remote from) the active site gorge of the latter enzyme.

The action of a potent tricyclic cholinesterase inhibitor ethopropazine on the hydrolysis of acetylthiocholine and butyrylthiocholine by purified horse serum butyrylcholinesterase (EC 3.1.1.8) was investigated at 25 and 37 degrees C. The enzyme activities were measured on a stopped-flow apparatus and the analysis of experimental data was done by applying a six-parameter model for substrate hydrolysis. The model, which was introduced to explain the kinetics of Drosophila melanogaster acetylcholinesterase [Stojan et al. (1998) FEBS Lett. 440, 85-88], is defined with two dissociation constants and four rate constants and can describe both cooperative phenomena, apparent activation at low substrate concentrations and substrate inhibition by excess of substrate. For the analysis of the data in the presence of ethopropazine at two temperatures, we have enlarged the reaction scheme to allow primarily its competition with the substrate at the peripheral site, but the competition at the acylation site was not excluded. The proposed reaction scheme revealed, upon analysis, competitive effects of ethopropazine at both sites; at 25 degrees C, three enzyme-inhibitor dissociation constants could be evaluated; at 37 degrees C, only two constants could be evaluated. Although the model considers both cooperative phenomena, it appears that decreased enzyme sensitivity at higher temperature, predominantly for the ligands at the peripheral binding site, makes the determination of some expected enzyme substrate and/or inhibitor complexes technically impossible. The same reason might also account for one of the paradoxes in cholinesterases: activities at 25 degrees C at low substrate concentrations are higher than at 37 degrees C. Positioning of ethopropazine in the active-site gorge by molecular dynamics simulations shows that A328, W82, D70, and Y332 amino acid residues stabilize binding of the inhibitor.

Benactyzine and drofenine are widely used anticholinergic drugs. Benactyzine is used to treat organophosphate poisoning and drofenine acts on smooth muscle to stop muscle spasms. Both of these drugs are esters. After they enter the bloodstream, they will interact with butyrylcholinesterase (BChE; acylcholine acyl hydrolase: EC 3.1.1.8), which has an ability to hydrolyze a wide variety of esters. Therefore, the kinetic analysis of their inhibitory effects on human serum BChE was examined using butyrylthiocholine as substrate. Both drugs were competitive inhibitors of BChE and the Ki values of benactyzine and drofenine were calculated to be 0.010 ± 0.001 and 0.003 ± 0.000 mM, respectively, using the Systat (version 5.03, 1991) nonlinear regression analysis software package. According to these parameters, drofenine is a more potent competitive inhibitor of BChE than benactyzine.

Butyrylcholinesterase in human plasma and acetylcholinesterase in human red blood cells have aryl acylamidase activity toward o-nitroacetanilide, hydrolyzing the amide bond to produce o-nitroaniline and acetate. People with a genetic variant of butyrylcholinesterase that had no detectable activity with butyrylthiocholine, nevertheless had aryl acylamidase activity in their plasma. To determine the source of this aryl acylamidase activity we tested fatty acid free human albumin for activity. We found that albumin had aryl acylacylamidase activity and that this activity was inhibited by diisopropylfluorophosphate. Since the esterase activity of albumin is also inhibited by diisopropylfluorophosphate, and since it is known that diisopropylfluorophosphate covalently binds to Tyr 411 of human albumin, we conclude that the active site for aryl acylamidase activity of albumin is Tyr 411. Albumin accounts for about 10% of the aryl acylamidase activity in human plasma.

The inhibitory effects of the cationic triarylmethane (TAM+) dyes, pararosaniline (PR+), malachite green (MG+), and methyl green (MeG+) on human plasma cholinesterase (BChE) were studied at 25 degrees C in 100 mM Mops, pH 8.0, with butyrylthiocholine as substrate. PR+ and MG+ caused linear mixed inhibition of enzyme activity. The respective inhibitory parameters were K(i) = 1.9 +/- 0.23 microM, alpha = 13 +/- 48, beta = 0 and K(i) = 0.28 +/- 0.037 microM, alpha = 23 +/- 7.4, beta = 0. MeG+ acted as a competitive inhibitor with K(i) = 0.12 +/- 0.017 microM (alpha, infinity, beta, not applicable). The K(i) values were within the same range reported for a number of ChE inhibitors including propidium ion, donepezil, and the phenothiazines, suggesting that TAM+s are active site ligands. On the other hand, the alpha values failed to correlate with values previously reported for a number of ChE inhibitors. It appears that mixed inhibition is the combined result of more than one type of binding and S-I interference. The impact of ligands at the choline-specific and peripheral anionic sites (or, possibly, accessory structural domains) on BChE activity needs to be studied in further detail.

Human plasma cholinesterase was found to be inhibited by 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide in a biphasic manner. The faster phase of the inhibition led to loss of approximately 50% of the activity (measured at pH 7.0, 30 °C, using 2.5 m m butyrylthiocholine) and was irreversible. Inhibition in the slower phase was reversible by 0.25 m hydroxylamine. The protective effect of 1 m m propranolol indicated that the target residue in both phases was localized at the active site. Lineweaver-Burk plots for butyrylthiocholine were obtained at different times during the course of inactivation. It was found that for both native and partially inactivated enzymes the plots could be analyzed in terms of two activities showing hyperbolic saturation with the substrate, with K m values of 0.055 ± 0.015 and 2.0 ± 0.2 m m . The carbodiimide affected the maximal velocities of the component activities, leaving the K m 's unchanged. The low- K m component was lost in the first phase of the inactivation. The loss of the high- K m component paralleled the second phase. It was concluded that the active sites in the tetrameric enzyme form two classes, differing in their affinity for butyrylthiocholine and their susceptibility to inhibition by the active site-directed carbodiimide.

The inhibition of horse plasma cholinesterase by propranolol showed characteristics which depended upon the identity of the substrate used. With butyrylthiocholine as substrate, the inhibition showed a first-order dependence on inhibitor concentration, and was characterized by a K i of 8 μ m (pH 7.4, 20 °C). With p -nitrophenylbutyrate as substrate, a biphasic v −1 versus [I] relationship was obtained. The biphasic curve could be resolved into two components, with apparent K i 's of 9 μ m and 1.3 m m . Use of butyrylthiocholine as alternative substrate resulted in partial inhibition of p -nitrophenylbutyrate hydrolysis. Inhibition of butyrylthiocholine hydrolysis by p -nitrophenylbutyrate could be accounted for by pure competitive inhibition at two sites. The results were interpreted in terms of a four-site, low-symmetry model, in which two active sites could process both substrates, and the remaining sites could process only p -nitrophenylbutyrate.

One of the previously described five purified monoglyceride-cleaving carboxylesterases from rat liver microsomes proved to be a carnitine ester hydrolase. This esterase, with an isoelectric point of 5.2, is most active with medium-chain acyl- l -carnitines (C 12 -C 14 ). The esterase is also remarkably active with 1,3-diglycerides, especially 1,3-dioctanoylglycerol, that are hydrolyzed faster than the corresponding 1-monoglycerides and triglycerides. Only one of the other four purified carboxylesterases has moderate acylcarnitine-hydrolyzing activity. An altered procedure for the separation of the two microsomal acylcarnitine-cleaving enzymes is described. Both enzymes hydrolyze carnitine esters optimally at pH 8 and both are inactive with acetylcarnitine, palmitoyl-CoA, and butyrylthiocholine. The possible natural functions of the hydrolases are discussed. Besides their detoxifying action on natural membrane-lysing detergents (like carnitine esters and lysophospholipids), these enzymes could be involved in the transport of carnitine out of the liver.