Probing the structure of the nicotinic acetylcholine receptor with 4-benzoylbenzoylcholine, a novel photoaffinity competitive antagonist.
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[(3)H]4-Benzoylbenzoylcholine (Bz(2)choline) was synthesized as a photoaffinity probe for the Torpedo nicotinic acetylcholine receptor (nAChR). [(3)H]Bz(2)choline acts as an nAChR competitive antagonist and binds at equilibrium with the same affinity (K(D) = 1.4 microm) to both agonist sites. Irradiation at 320 nm of nAChR-rich membranes equilibrated with [(3)H]Bz(2)choline results in the covalent incorporation of [(3)H]Bz(2)choline into the nAChR gamma- and delta-subunits that is inhibitable by agonist, with little specific incorporation in the alpha-subunits. To identify the sites of photoincorporation, gamma- and delta-subunits, isolated from nAChR-rich membranes photolabeled with [(3)H]Bz(2)choline, were digested enzymatically, and the labeled fragments were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high performance liquid chromatography. For the gamma-subunit, Staphylococcus aureus V8 protease produced a specifically labeled peptide beginning at gammaVal-102, whereas for the delta-subunit, endoproteinase Asp-N produced a labeled peptide beginning at deltaAsp-99. Amino-terminal sequence analysis identified the homologous residues gammaLeu-109 and deltaLeu-111 as the primary sites of [(3)H]Bz(2)choline photoincorporation. This is the first identification by affinity labeling of non-reactive amino acids within the acetylcholine-binding sites, and these results establish that when choline esters of benzoic acid are bound to the nAChR agonist sites, the para substituent is selectively oriented toward and in proximity to amino acids gammaLeu-109/deltaLeu-111. (+info)
Rate-determining step of butyrylcholinesterase-catalyzed hydrolysis of benzoylcholine and benzoylthiocholine. Volumetric study of wild-type and D70G mutant behavior.
(2/8)
The rate-limiting step for hydrolysis of the positively charged oxoester benzoylcholine (BzCh) by human butyrylcholinesterase (BuChE) is deacylation (k(3)), whereas it is acylation (k(2)) for hydrolysis of the homologous thioester benzoylthiocholine (BzSCh). Steady-state hydrolysis of BzCh and BzSCh by wild-type BuChE and its peripheral anionic site mutant D70G was investigated at different hydrostatic pressures, which allowed determination of volume changes associated with substrate binding, and the activation volumes for the chemical steps. A differential nonlinear pressure-dependence of the catalytic parameters for hydrolysis of both substrates by both enzymes was shown. Nonlinearity of the plots may be explained in terms of compressibility changes or rate-limiting changes. To distinguish between these two possibilities, enzyme phosphorylation by diisopropylfluorophosphate (DFP) in the presence of substrate (BzSCh) under pressure was studied. There was no pressure dependence of volume changes for DFP binding or for phosphorylation of either wild-type or D70G. Analysis of the pressure dependence for steady-state hydrolysis of substrates, and for phosphorylation by DFP provided evidence that no enzyme compressibility changes occurred during the catalyzed reactions. Thus, the nonlinear pressure dependence of substrate hydrolysis reflects changes in the rate-limiting step with pressure. Change in rate-determining step occurred at a pressure of 100 MPa for hydrolysis of BzCh by wild-type and at 75 MPa for D70G. For hydrolysis of BzSCh the change occurred at higher pressures because k(2) << k(3) at atmospheric pressure for this substrate. Elementary volume change contributions upon initial binding, productive binding, acylation and deacylation were calculated from the pressure differentiation of kinetic constants. This analysis shed light on the molecular events taking place along the hydrolysis pathways of BzCh and BzSCh by wild-type BuChE and the D70G mutant. In addition, volume change differences between wild-type and D70G provided new evidence that residue D70 in the peripheral site controls hydration of the active site gorge and the dynamics of the water molecule network during catalysis. Finally, a steady-state kinetic study of the oxyanion hole mutant (G117H) showed that substitution of the ethereal sulfur for oxygen in the substrate alters the final adjustment of substrate in the active site and stabilization of the acylation transition state. (+info)
Hydrolysis of oxo- and thio-esters by human butyrylcholinesterase.
(3/8)
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 pi-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. (+info)
Cholinesterases from flounder muscle. Purification and characterization of glycosyl-phosphatidylinositol-anchored and collagen-tailed forms differing in substrate specificity.
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Flounder (Platichthys flesus) muscle contains two types of cholinesterases, that differ in molecular form and in substrate specificity. Both enzymes were purified by affinity chromatography. About 8% of cholinesterase activity could be attributed to collagen-tailed asymmetric acetylcholinesterase sedimenting at 17S, 13S and 9S, which showed catalytic properties of a true acetylcholinesterase. 92% of cholinesterase activity corresponded to an amphiphilic dimeric enzyme sedimenting at 6S in the presence of Triton X-100. Treatment with phospholipase C yielded a hydrophilic form and uncovered an epitope called the cross-reacting determinant, which is found in the hydrophilic form of a number of glycosyl-phosphatidylinositol-anchored proteins. This enzyme showed catalytic properties intermediate to those of acetylcholinesterase and butyrylcholinesterase. It hydrolyzed acetylthiocholine, propionylthiocholine, butyrylthiocholine and benzoylthiocholine. The Km and the maximal velocity decreased with the length and hydrophobicity of the acyl chain. At high substrate concentrations the enzyme was inhibited. The p(IC50) values for BW284C51 and ethopropazine were between those found for acetylcholinesterase and butylcholinesterase. For purified detergent-soluble cholinesterase a specific activity of 8000 IU/mg protein, a turnover number of 2.8 x 10(7) h-1, and 1 active site/subunit were determined. (+info)
Comparison of a commercially available assay system with two reference methods for the determination of plasma cholinesterase variants.
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For assaying plasma cholinesterase (EC 3.1.1.8) activity and phenotyping by means of dibucaine inhibition, we have compared a commercially available kit, in which butyrylthiocholine is used as substrate, with two reference methods, one using benzoylcholine and the other propionylthiocholine. With 50 different samples of three of the most common genetic variants, we could clearly differentiate the variants with benzoylcholine and dibucaine, whereas there was some overlap of the E1uE1u and E1uE1a phenotypes with the other two substrates at 30 degrees C. The phenotypes were better differentiated at 25 degrees C, and in our hands the use of butyrylthiocholine was preferable to propionylthiocholine for phenotyping with dibucaine. The affinity of the usual and atypical homozygotes for fluoride with butyrylthiocholine gave an inverted response to the affinity of these variants for the anion with benzoylcholine. We suggest that this may be explained by the role of the chromogen or its products in the assay procedure with the thiocholine substrate. (+info)
Rate assay for determination of serum pseudo-cholinesterase activity.
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A simple and reproducible method for the determination of serum pseudo-cholinesterase activity was developed by making use of a stable substrate, p-hydroxybenzoylcholine, with p-hydroxybenzoate hydroxylase as a linked enzyme. The method is based on spectrophotometric measurement of the decrease of NADPH. p-Hydroxybenzoate released from p-hydroxybenzoylcholine is hydroxylated by the action of p-hydroxybenzoate hydroxylase in the presence of NADPH and O2 to produce 3,4-dihydroxybenzoate and NADP+. This method is superior to the conventional methods in that this substrate is extremely stable up to pH 9.0, which is close to the optimum pH for the assay (pH 8.0). Serum interference was resolved by the use of p-hydroxybenzoate hydroxylase as a linked enzyme. The Km value of pseudocholinesterase for p-hydroxybenzoylcholine is 1 X 10(-5) M. The results of our method and Garry's method (Clin. Chem. 11, 91-96, 1965) correlated well (r = 0.962). The within-run and between-run C.V. values were 2.1 and 2.7, respectively. (+info)
Is serum cholinesterase activity a predictor of succinyl choline sensitivity? An assessment of four methods.
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Four methods for measuring serum cholinesterase activity have been applied to sera of normal individuals and of patients shown to be sensitive to short-acting muscle relaxants of the succinyldicholine type. They have been assessed according to their ability to differentiate between sensitive and insensitive individuals on the basis of enzyme activity measurements alone. The method described, based upon that of Dietz et al. [Clin. Chem. 19, 1309 (1973)], in which propionylthiocholine is used as substrate, is best for this purpose, being capable of identifying over 90% of affected individuals with no false positives. Acetylcholine and butyrylthiocholine are slightly inferior substrates in this respect, and benzoylcholine gives little useful information. (+info)
Kinetics of local anesthetic esters and the effects of adjuvant drugs on 2-chloroprocaine hydrolysis.
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A rapid, reliable method for the determination of 2-chloroprocaine in serum was developed. The method, using double-beam ultraviolet spectroscopy, provides rapid, accurate analysis of 2-chloroprocaine in the range of 5.5 to 111 microM (1.5--30 microgram/ml), as documented by comparison with the accepted gas chromatographic procedure. The contribution of 4-amino-2-chlorobenzoic acid, the principal metabolite of 2-chloroprocaine, to the total absorbance at 300 nm was examined and found to be negligible. Using the ultraviolet spectrophotometric method, values of the Michaelis-Menton constant (Km) and maximal reaction velocity (Vmax) for hydrolysis of procaine and 2-chloroprocaine by homozygous typical, heterozygous, and homozygous atypical plasma cholinesterase were determined. The Kms for the three genotypes were 5.0, 6.2, and 14.7 microM, respectively, for procaine, and 8.2, 17, and 103 microM, respectively for 2-chloroprocaine. The Vmaxs for the three genotyps were similar for all esters. Vmax for procaine was 18.6 +/- 0.9 nmol/min/ml serum, while Vmax for 2-chloroprocaine was 98.4 +/- 2.1 nmol/min/ml serum. At high concentrations, 2-chloroprocaine acts as an inhibitor of its hydrolysis. The inhibitory effects of lidocaine, bupivacaine, neostigmine, and succinyldicholine on 2-chloroprocaine hydrolysis for homozygous typical and atypical variants, respectively, were studied. Competitive inhibition was demonstrated for all four drugs. However, at clinically significant concentrations, only neostigmine and bupivacaine produced high degrees of inhibition. The competitive inhibition constants (K1) for the typical and atypical variants, respectively, were 3.3 +/- 0.3 microM and 15.1 +/- 4.8 microM for neostigmine, and 4.2 +/- 0.3 microM and 36.9 +/- 9.8 microM for bupivacaine. (+info)