p.D70G/Y332D Asp70Gly/Tyr332Asp (p.D98G/Y360D Asp98Gly/Tyr360Asp in primary sequence with 28 amino-acids signal peptide) Peripheral Anionic site;interraction 70(98)/332(360)
Kinetic parameters
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none
References:
Title: Concentration-dependent reversible activation-inhibition of human butyrylcholinesterase by tetraethylammonium ion Stojan J, Golicnik M, Froment MT, Estour F, Masson P Ref: European Journal of Biochemistry, 269:1154, 2002 : PubMed
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 BCHE (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 BCHE, by selected mutants and by horse BCHE 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.
Title: Interaction between the peripheral site residues of human butyrylcholinesterase, D70 and Y332, in binding and hydrolysis of substrates Masson P, Xie W, Froment MT, Levitsky V, Fortier PL, Albaret C, Lockridge O Ref: Biochimica & Biophysica Acta, 1433:281, 1999 : PubMed
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.
