Thermal inactivation of butyrylcholinesterase and acetylcholinesterase (original) (raw)
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The rate of thermal inactivation of Torpedo acetylcholinesterase is not reduced in the C231 S mutant
FEBS Letters, 1996
The rate of thermal inactivation of Torpedo AChE at pH 8.5 was increased by the sullhydryl reagent 5,5'-dithiobis-(Znitrohenzoic acid) (DTNB). At 30°C or 37'C, inactivation rates with 0.3 mM DTNB increased about 5-fold for the wild-type enzyme and for two site-specific mutants, D72S and V129R. The reversible active site inhibitor, amhenonium, completely stabilized the wild type enzyme and partially stabilized the D72S mutant. However, amhenonium did not protect against the destabilization introduced by DTNB, which still accelerated inactivation of D72S 5-fold. When the only free sulfhydryl group in AChE was removed by replacing cysteine 231 with serine, increased rates of thermal inactivation were observed. The inactivation rate incrased by a factor of 2 to 3 for the single mutant (C231S) and by a factor of 5 for the double mutant V129RK231S. Even in the C231S mutants, DTNB still had an additional effect. It increased the inactivation rate for C231S and V129RK231 by a factor of about 1.5 to 3 beyond the rates seen in the absence of DTNB. Therefore, at least part of the destabilization seen with DTNB in enzymes that retain C231 does not involve reaction of DTNB with C231.
High activity of human butyrylcholinesterase at low pH in the presence of excess butyrylthiocholine
European Journal of Biochemistry, 2003
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 a-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 £ 5, under conditions of excess substrate activation. This low-pH activity appears with wildtype butyrylcholinesterase as well as with all mutants we examined: A328G, A328I, A328F, A328Y, A328W, E197Q, L286W, V288W and Y332A (residue A328 is at the bottom of the active-site gorge, near the p-cation-binding site; E197 is next to the active-site serine S198; L286 and V288 form the acyl-binding pocket; and Y332 is a component of the peripheral anionic site). For example, the k cat value at pH 5.0 for activity in the presence of excess substrate was 32 900 ± 4400 min )1 for wild-type, 55 200 ± 1600 min )1 for A328F, and 28 700 ± 700 min )1 for A328W. This activity is titratable, with pK a values of 6.0-6.6, suggesting that the catalytic histidine is protonated at pH 5. The existence of activity when the catalytic histidine is protonated indicates that the catalytic-triad mechanism of butyrylcholinesterase does not operate for catalysis at low pH. The mechanism explaining the catalytic behaviour of butyrylcholinesterase at low pH in the presence of excess substrate remains to be elucidated.
2009
Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) are enzymes that belong to the superfamily of α/β-hydrolase fold proteins. While they share many characteristics, they also possess many important differences. For example, whereas they have about 54% amino acid sequence identity, the active site gorge of acetylcholinesterase is considerably smaller than that of butyrylcholinesterase. Moreover, both have been shown to display simple and complex kinetic mechanisms, depending on the particular substrate examined, the substrate concentration, and incubation conditions. In the current study, incubation of butyrylthiocholine in a concentration range of 0.005 mM-3.0 mM, with 317 pM human butyrylcholinesterase in vitro resulted in rates of production of thiocholine that were accurately described by simple Michaelis-Menten kinetics, with a K m of 0.10 mM. Similarly, the inhibition of butyrylcholinesterase in vitro by the organophosphate chlorpyrifos oxon was described by simple Michaelis-Menten kinetics, with a k i of 3,048 nM −1 h −1 , and a K D of 2.02 nM. In contrast to inhibition of butyrylcholinesterase, inhibition of human acetylcholinesterase by chlorpyrifos oxon in vitro followed concentration-dependent inhibition kinetics, with the k i increasing as the inhibitor concentration decreased. Chlorpyrifos oxon concentrations of 10 nM and 0.3 nM gave k i s of 1.2 nM −1 h −1 and 19.3 nM −1 h −1 , respectively. Although the mechanism of concentration-dependent inhibition kinetics is not known, the much smaller, more restrictive active site gorge of acetylcholinesterase almost certainly plays a role. Similarly, the much larger active site gorge of butyrylcholinesterase likely contributes to its much greater reactivity towards chlorpyrifos oxon, compared to acetylcholinesterase.
Effect of mutations within the peripheral anionic site on the stability of acetylcholinesterase
Molecular pharmacology, 1999
Torpedo acetylcholinesterase is irreversibly inactivated by modifying a buried free cysteine, Cys231, with sulfhydryl reagents. The stability of the enzyme, as monitored by measuring the rate of inactivation, was reduced by mutating a leucine, Leu282, to a smaller amino acid residue. Leu282 is located within the "peripheral" anionic site, at the entrance to the active-site gorge. Thus, loss of activity was due to the increased reactivity of Cys231. This was paralleled by an increased susceptibility to thermal denaturation, which was shown to be due to a large decrease in the activation enthalpy. Similar results were obtained when either of two other residues in contact with Leu282 in Torpedo acetylcholinesterase, Trp279 and Ser291, was replaced by an amino acid with a smaller side chain. We studied the effects of various ligands specific for either the active or peripheral sites on both thermal inactivation and on inactivation by 4,4'-dithiodipyridine. The wild-type an...
Activity and molecular dynamics relationship within the family of human cholinesterases
Physical chemistry chemical physics : PCCP, 2012
The temperature dependence of the dynamics of recombinant human acetylcholinesterase (hAChE) and plasma human butyrylcholinesterase (hBChE) is examined using elastic incoherent neutron scattering. These two enzymes belong to the same family and present 50% amino acid sequence identity. However, significantly higher flexibility and catalytic activity of hAChE when compared to the ones of hBChE are measured. At the same time, the average height of the potential barrier to the motions is increased in the hBChE, e.g. more thermal energy is needed to cross it in the latter case, which might be the origin of the increase in activation energy and the reduction in the catalytic rate of hBChE observed experimentally. These results suggest that the motions on the picosecond timescale may act as a lubricant for those associated with activity occurring on a slower millisecond timescale.
European Journal of Biochemistry, 1993
The secondary structure of the acetylcholinesterase and its temperature behaviour have been investigated using Fourier-transform infrared (FTIR) spectroscopy. The data are compared to the structure obtained by X-ray analysis of the crystalline enzyme. The secondary structure was determined using the spectral features observed in the amide-I band (H,O buffer) and amide-I' band (D,O buffer) at 1600-1700 cm-I, taking advantage of resolution-enhancement techniques along with least-squares band-fitting procedures. The relative amounts of different secondary-structure elements, 34-36% for a-helices, 19-25% for p-sheets, 15-16% for turns and 13-17% for irregular structures, were estimated. These data, obtained with the enzyme in solution, correlate well with X-ray data of the crystalline protein [Sussman, J. L., Hard, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. & Silman, I. (1991) Science 253, 872-8791. These results are also in good agreement with those obtained by computing the t +v and q5 angles of the peptide backbone using the Kabsch and Sanders method [Kabsch, W. & Sanders, C. (1983) Biopolymers 22, 2577-26371. In conjunction with the X-ray data, two bands in the FTIR spectra were assigned to different populations of long and short a-helices. Until now this phenomenon has only been described by theoretical calculations Biopolymers 15, 637-6481. The relationship between the thermally induced loss of enzyme activity and secondary-structure changes has also been investigated. The decrease in enzyme activity to zero at 30-40 "C was accompanied only by minor changes in the secondary structure. At 55-6OoC, denaturation of AChE occurs. In this temperature range, all bands assigned to the various secondary-structure elements abruptly disappear in a co-operative and irreversible manner, whereas the &aggregation bands (at 1622 cm-' and the corresponding high-frequency band) increase in intensity at the same rate.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 2001
The influence of inorganic salts on the inhibition of acetylcholinesterase by charged organophosphorous inhibitors has been studied. It has been shown that the salt effect on the reaction of acetylcholinesterase with anionic bis(p-nitrophenyl) phosphate is determined by the influence of added salts on the activity coefficient of the inhibitor. In contrast to the salt effects on the reaction of acetylcholinesterase with cationic compounds, it does not include contribution from the enzyme charges. The smaller salt effect in the case of anionic inhibitor can be explained assuming that the anionic inhibitor does not form a non-covalent complex with the enzyme before the phosphorylation step of the reaction. Comparison of salt effects on the substrate turnover showed that in the case of cholinesterases from natural sources they are larger than in the case of enzymes expressed in recombinant cell clones. The enhanced salt effects may result from post-translational modification of the enzyme. ß
Invertebrate Neuroscience, 2008
We have studied the thermal inactivation at 37°C of wild type and mutant ChE2 (C310A, F312I, C466A, C310A/F312I, and C310A/C466A) from amphioxus (Branchiostoma floridae) expressed in vitro in COS-7 monkey cells under three sets of conditions: 30°C for 48 h, 30°for 24 h and C37°C for 24 h, and 37°C for 48 h. We found biphasic denaturation curves for all enzymes and conditions, except wild type and C310A ChE2 expressed at 30°C for 48 h. Generally, single mutants are more unstable than wild type, and the double mutants are even more unstable. We propose a model involving stable and unstable conformations of the enzymes to explain these results, and we discuss the implications of the model. We also found a correlation between the melting temperature of the ChEs and the rates at which they denature at 37°C, with the denaturation of the unstable conformation dominating the relationship. Reversible cholinergic inhibitors protect the ChEs from thermal denaturation, and in some cases produce monophasic denaturation curves; we also propose a model to explain this stabilization.