Polyol-induced activation by excess substrate of the D70G butyrylcholinesterase mutant

doi:10.1016/S0167-4838(98)00253-2

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology

Volume 1429, Issue 2, 11 January 1999, Pages 422-430

Biochimica et Biophy...

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Abstract

Wild-type human butyrylcholinesterase (BuChE) has a non-Michaelian behaviour showing substrate activation with butyrylthiocholine (BTC) as the substrate. The D70G mutant has a catalytic constant identical to that of the wild-type enzyme, but a 10-fold lower affinity for BTC compared to wild-type enzyme, and it does not exhibit activation by excess BTC under conventional conditions. In the present work it was found that addition of polyols or sugars changed the kinetic behaviour of the D70G mutant with BTC. In the presence of 40% sucrose, the D70G mutant enzyme displayed marked activation by excess substrate. Because D70 is hydrogen bonded to Y332, mutants of Y332 were studied. Mutant Y332F had a behaviour similar to that of wild-type BuChE, whereas mutants Y332A, Y332A/D70G and D70G had negligible substrate activation. The behaviour of wild-type, Y332F, Y332A and Y332A/D70G did not change in the presence of high concentrations of sugar. Substrate activation has been explained by binding of a second substrate molecule in the peripheral site at D70. The D70G mutant should be incapable of substrate activation, if D70 were the only residue involved in substrate activation. The ability of the D70G mutant to display substrate activation by medium engineering suggests that other residues are involved in initial substrate binding and activation by excess substrate. Osmolyte-induced change in conformation and/or hydration status of Y332 and other solvent-exposed residues may account for the non-Michaelian behaviour of the D70G mutant.

Introduction

Cholinesterases (ChE) are ubiquitous serine hydrolases divided into two classes according to their catalytic and inhibition specificities and tissue localisation: acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1.1.8). AChE is primarily involved in cholinergic transmission [1], but there is no physiological function assigned to BuChE. Both enzymes are the main targets of organophosphate poisons [2]. The physiological and toxicological importance of ChEs as well as their ability to catalyse substrate hydrolysis at rates approaching the diffusion-controlled limit [3], [4] have stimulated research on these enzymes. In particular, study of the effects of environmental variables, medium and ligands on the catalytic properties of ChEs has proven to be useful for understanding mechanisms of ChE catalysis and for the search for new drugs against organophosphate poisoning.

The active site serine of ChEs, S198 in human BuChE, is located at the bottom of a narrow (4 Å) and deep (20 Å) gorge [5], [6]. In human BuChE, residues D70 [7], [8] and Y332 [9] have been found to be components of the peripheral anionic site (PAS); these two residues are hydrogen-bonded [5], [6] (Fig. 1). The D70 residue (D72 in Torpedo AChE) as a key element of PAS is important for control of substrate activation, inhibition and aging of cholinesterases [7], [8], [10], [11], [12], [13], [14], [15], [16]. Under conventional conditions, wild-type BuChE shows substrate activation with BTC at pH 7.0; this has been interpreted in terms of binding of a second substrate molecule [17], [18], [7]. Mutation D70G causes loss of activation of BuChE by excess butyrylthiocholine (BTC) [7], [8]. The Ω-loop is thought to play a key role in binding of substrate and in allosteric regulation of AChE catalysis [7], [8], [9], [10], [11]. It was suggested that the catalytic behaviour of BuChE is related to the conformational dynamics of the Ω-loop [7], [8]. The Ω-loop, a flexible single-turn segment, formed by residues C65 to C92 in human BuChE, has a definite structure through a network of hydrogen bonds; it lies on the surface of the enzyme molecule where it forms a 1 residue thick wall between the surface and the active site gorge [10]. The Ω-loop has been shown to be important for the proper orientation of residues D70 and W82 in human BuChE [16].

The starting idea of the present work was that given the Ω-loop is accessible to solvent molecules, it is expected to be sensitive to environmental conditions. Thus, changes in the Ω-loop motion were expected to be modulated by engineering of the medium, in particular by high concentrations of low-molecular-mass additives which have been found to alter hydration/conformational state and conformational plasticity of proteins [19], [20], [21], [22]. In this paper, the effect of polyols on the kinetics of substrate hydrolysis of wild-type BuChE and its D70G mutant are compared. It is shown that polyols induced change in the catalytic behaviour of the D70G mutant, restoring activation by excess substrate. Study on Y332 mutants suggests that osmolyte-induced change in orientation and/or hydration of residue Y332 and other solvent exposed residues may account for the non-Michaelian behaviour of the D70G mutant. Binding of substrate on the PAS of D70G mutant in a low water activity environment may thus affect the Ω-loop flexibility or facilitate the sliding of the second substrate molecule down the gorge, both mechanisms causing acceleration of substrate hydrolysis.

Section snippets

Expression of recombinant wild-type and mutant BuChE

The methods to make mutants and to express wild-type BuChE and mutants (D70G, Y332F, Y332A and Y332A/D70G) in mammalian cells have been described previously [7], [8], [23]. Wild-type and D70G mutant enzymes were partly purified from cell culture medium by affinity chromatography on procainamide sepharose [23].

Kinetic measurements

BuChE-catalysed hydrolysis of BTC was measured by the Ellman method [24] in 0.1 M potassium phosphate buffer (pH 7.0) at 25°C. BTC (Sigma) concentrations ranged from 0.025 to 50 mM. o-NPB

Effects of polyols on BTC hydrolysis by wild-type BuChE

The hydrolysis of the positively charged substrate BTC by wild-type BuChE in the range of polyol concentrations tested (0 to 40% (w/v) was non-Michaelian showing substrate activation as in conventional conditions (Fig. 2). However, catalytic parameters were slightly changed. Dependencies of steady-state kinetic parameters on molar concentration of hydroxyl groups of sucrose and glycerol are presented in Fig. 3. Increasing the concentration of sucrose hydroxyls up to 7.5 M (32% sucrose) and

The effects of polyols and sugars are not related to viscosity

The catalytic constants, k_cat, of both wild-type and D70G mutant BuChE for hydrolysis of BTC and o-NPB were changed by the presence of additives in the same manner. Only glycerol increased the k_cat whereas other polyols and sugars reduced the k_cat. All additives caused an increase in K_m of wild-type for both substrates and also caused an increase in Km of the D70G mutant for o-NPB. The K_m for BTC hydrolysis by the D70G mutant was slightly increased by glycerol, but it was decreased by sugars

Conclusion

A sharp change in the kinetics of hydrolysis of BTC by the D70G mutant of human BuChE, caused by sugars and polyols (except the smallest polyol glycerol) was found. No difference in kinetic behaviour of wild-type and D70G mutant BuChE with non-charged substrate was observed. Polyols/sugars increased the affinity for BTC of the D70G mutant by threefold and induced activation by excess substrate. The catalytic parameters did not correlate with a viscosity effect. Activation by excess substrate of

Acknowledgements

This work was supported by SDP/STTC (ex-DRET) Grant No. 97/12 to P.M. and by Grant DAMD17-97-1-7349 from the U.S. Army Medical Research and Material Command to O.L. V.L. had a fellowship from DRET/CIES (No. 93/811.00.067).

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Catalytic parameters of human butyrylcholinesterase (BuChE) for hydrolysis of homologous pairs of oxo-esters and thio-esters were compared. Substrates were positively charged (benzoylcholine versus benzoylthiocholine) and neutral (phenylacetate versus phenylthioacetate). In addition to wild-type BuChE, enzymes containing mutations were used. Single mutants at positions: G117, a key residue in the oxyanion hole, and D70, the main component of the peripheral anionic site were tested. Double mutants containing G117H and mutations on residues of the oxyanion hole (G115, A199), or the π-cation binding site (W82), or residue E197 that is involved in stabilization of tetrahedral intermediates were also studied. A mathematical analysis was used to compare data for BuChE-catalyzed hydrolysis of various pairs of oxo-esters and thio-esters and to determine the rate-limiting step of catalysis for each substrate. The interest and limitation of this method is discussed. Molecular docking was used to analyze how the mutations could have altered the binding of the oxo-ester or the thio-ester. Results indicate that substitution of the ethereal oxygen for sulfur in substrates may alter the adjustment of substrate in the active site and stabilization of the transition-state for acylation. This affects the k₂/k₃ ratio and, in turn, controls the rate-limiting step of the hydrolytic reaction. Stabilization of the transition state is modulated both by the alcohol and acyl moieties of substrate. Interaction of these groups with the ethereal hetero-atom can have a neutral, an additive or an antagonistic effect on transition state stabilization, depending on their molecular structure, size and enantiomeric configuration.
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Effects of mutations of active site residues and amino acids interacting with the Ω loop on substrate activation of butyrylcholinesterase
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The peripheral anionic site (PAS) of human butyrylcholinesterase is involved in the mechanism of substrate activation by positively charged substrates and ligands. Two substrate binding loci, D70 in the PAS and W82 in the active site, are connected by the Ω loop. To determine whether the Ω loop plays a role in the signal transduction between the PAS and the active site, residues involved in stabilization of the loop, N83, K339 and W430, were mutated. Mutations N83A and N83Q caused loss of substrate activation, suggesting that N83 which interacts with the D70 backbone may be an element of the transducing system. The K339M and W430A mutant enzymes retained substrate activation. Residues W82, E197, and A328 in the active site gorge have been reported to be involved in substrate activation. At butyrylthiocholine concentrations greater then 2 mM, W82A showed apparent substrate activation. Mutations E197Q and E197G strongly reduced substrate activation, while mutation E197D caused a moderate effect, suggesting that the carboxylate of residue E197 is involved in substrate activation. Mutations A328F and A328Y showed no substrate activation, whereas A328G retained substrate activation. Substrate activation can result from an allosteric effect due to binding of the second substrate molecule on the PAS. Mutation W430A was of special interest because this residue hydrogen bonds to W82 and Y332. W430A had strongly reduced affinity for tetramethylammonium. The bimolecular rate constant for reaction with diisopropyl fluorophosphate was reduced 10 000-fold, indicating severe alteration in the binding area in W430A. The k_cat values for butyrylthiocholine, o-nitrophenyl butyrate, and succinyldithiocholine were lower. This suggested that the mutation had caused misfolding of the active site gorge without altering the Ω loop conformation/dynamics. W430 as well as W231 and W82 appear to form the wall of the active site gorge. Mutation of any of these tryptophans disrupts the architecture of the active site.
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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.
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¹: Permanent address: M.V. Lomonosov Moscow State University, Chemistry Department, Enzymology Division, 119899 B-234 Moscow, Russia.

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Polyol-induced activation by excess substrate of the D70G butyrylcholinesterase mutant

Abstract

Introduction

Section snippets

Expression of recombinant wild-type and mutant BuChE

Kinetic measurements

Effects of polyols on BTC hydrolysis by wild-type BuChE

The effects of polyols and sugars are not related to viscosity

Conclusion

Acknowledgements

Prog. Neurobiol.

J. Biol. Chem.

Biochim. Biophys. Acta

Methods Enzymol.

Biophys. J.

Adv. Protein Chem.

Biochem. Pharmacol.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Biol. Chem.

Pharmacol. Ther.

Adv. Enzymol.

Chem. Rev.

Science

Proc. Natl. Acad. Sci. U.S.A.

Eur. J. Biochem.

Biochemistry

Biochemistry