Interaction between the peripheral site residues of human butyrylcholinesterase, D70 and Y332, in binding and hydrolysis of substrates. (1/19)

Human butyrylcholinesterase displays substrate activation with positively charged butyrylthiocholine (BTC) as the substrate. Peripheral anionic site (PAS) residues D70 and Y332 appear to be involved in the initial binding of charged substrates and in activation control. To determine the contribution of PAS residues to binding and hydrolysis of quaternary substrates and activation control, the single mutants D70G/Y and Y332F/A/D and the double mutants Y332A/D70G and Y332D/D70Y were studied. Steady-state hydrolysis of the charged substrates, BTC and succinyldithiocholine, and the neutral ester o-nitrophenyl butyrate was measured. In addition, inhibition of wild-type and mutant enzymes by tetramethylammonium was investigated, at low concentrations of BTC. Single and double mutants of D70 and Y332 showed little or no substrate activation, suggesting that both residues were important for activation control. The effects of double mutations on D70 and Y332 were complex. Double-mutant cycle analysis provided evidence for interaction between these residues. The category of interaction (either synergistic, additive, partially additive or antagonistic) was found to depend on the nature of the substrate and on measured binding or kinetic parameters. This complexity reflects both the cross-talk between residues involved in the sequential formation of productive Michaelian complexes and the effect of peripheral site residues on catalysis. It is concluded that double mutations on the PAS induce a conformational change in the active site gorge of butyrylcholinesterase that can alter both substrate binding and enzyme acylation.  (+info)

Differential effect of pressure and temperature on the catalytic behaviour of wild-type human butyrylcholinesterase and its D70G mutant. (2/19)

The combined action of temperature (10-35 degrees C) and pressure (0. 001-2 kbar) on the catalytic activity of wild-type human butyrylcholinesterase (BuChE) and its D70G mutant was investigated at pH 7.0 using butyrylthiocholine as the substrate. The residue D70, located at the mouth of the active site gorge, is an essential component of the peripheral substrate binding site of BuChE. Results showed a break in Arrhenius plots of wild-type BuChE (at Tt approximately 22 degrees C) whatever the pressure (dTt/dP = 1.6 +/- 1.5 degrees C.kbar-1), whereas no break was observed in Arrhenius plots of the D70G mutant. These results suggested a temperature-induced conformational change of the wild-type BuChE which did not occur for the D70G mutant. For the wild-type BuChE, at around a pressure of 1 kbar, an intermediate state, whose affinity for substrate was increased, appeared. This intermediate state was not seen for the mutant enzyme. The wild-type BuChE remained active up to a pressure of 2 kbar whatever the temperature, whereas the D70G mutant was found to be more sensitive to pressure inactivation (at pressures higher than 1.5 kbar the mutant enzyme lost its activity at temperatures lower than 25 degrees C). The results indicate that the residue D70 controls the conformational plasticity of the active site gorge of BuChE, and is involved in regulation of the catalytic activity as a function of temperature.  (+info)

Concentration-dependent reversible activation-inhibition of human butyrylcholinesterase by tetraethylammonium ion. (3/19)

Tetraalkylammonium (TAA) salts are well known reversible inhibitors of cholinesterases. However, at concentrations around 10 mm, they have been found to activate the hydrolysis of positively charged substrates, catalyzed by wild-type human butyrylcholinesterase (EC 3.1.1.8) [Erdoes, E.G., Foldes, F.F., Zsigmond, E.K., Baart, N. & Zwartz, J.A. (1958) Science 128, 92]. The present study was undertaken to determine whether the peripheral anionic site (PAS) of human BuChE (Y332, D70) and/or the catalytic substrate binding site (CS) (W82, A328) are involved in this phenomenon. For this purpose, the kinetics of butyrylthiocholine (BTC) hydrolysis by wild-type human BuChE, by selected mutants and by horse BuChE was carried out at 25 degreeC and pH 7.0 in the presence of tetraethylammonium (TEA). It appears that human enzymes with more intact structure of the PAS show more prominent activation phenomenon. The following explanation has been put forward: TEA competes with the substrate at the peripheral site thus inhibiting the substrate hydrolysis at the CS. As the inhibition by TEA is less effective than the substrate inhibition itself, it mimics activation. At the concentrations around 40 mm, well within the range of TEA competition at both substrate binding sites, it lowers the activity of all tested enzymes.  (+info)

High activity of human butyrylcholinesterase at low pH in the presence of excess butyrylthiocholine. (4/19)

Butyrylcholinesterase is a serine esterase, closely related to acetylcholinesterase. Both enzymes employ a catalytic triad mechanism for catalysis, similar to that used by serine proteases such as alpha-chymotrypsin. Enzymes of this type are generally considered to be inactive at pH values below 5, because the histidine member of the catalytic triad becomes protonated. We have found that butyrylcholinesterase retains activity at pH +info)

An evaluation of the inhibition of human butyrylcholinesterase and acetylcholinesterase by the organophosphate chlorpyrifos oxon. (5/19)

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ENZO: a web tool for derivation and evaluation of kinetic models of enzyme catalyzed reactions. (6/19)

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The proline-rich tetramerization peptides in equine serum butyrylcholinesterase. (7/19)

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Molecular and kinetic properties of two acetylcholinesterases from the western honey bee, Apis mellifera. (8/19)

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