On Mechanism of Interaction of Organophosphorus Inhibitors with Cholinesterases of Different Origin

A study is carried out as a development of A.P. Brestkin's concept of mechanism of irreversible inhibition of cholinesterases (ChE) by organophosphorus inhibitors (OPI) with taking into account reversibility of the first stage of this reaction, which has made it possible to determine individual constants of separate stages of the process. For the first time, a comparative study is performed on horse blood serum BuChE, human erythrocyte AChE, and ChE of optical ganglia of Pacific squid Todarodes pacificus. Besides, the OPI set is enlarged essentially due to use of some highly specific inhibitors of each of the enzymes. To evaluate the cholinesterase activity, chromogenic indophenol esters are used as substrates. For each of the studied ChE, differences in sensitivity to the studied OPI are realized only in values of the kinetic constant of formation of the enzyme-inhibitor complex (k ₅), whereas the rate constants of dissociation of this complex to initial components (ChE and OPI) (k _–5) and of process of its transformation into phosphorylated ChE (k ₆) are close to each other by the values, values of these constants k _–5 and k ₆ for different enzymes also being similar. Some statements about the molecular mechanism of the cholinesterase catalysis are formulated. It is suggested that the revealed elements of similarity of different ChE are realized in the work of the catalytic machine of active centers of the enzymes.     

Comparative Study of Catalytic Properties of Monoamine Oxidases of the Liver of the Squid Todarodes pacificus and of the Liver of the Wistar Rat

A comparative study of substrate specificity of monoamine oxidase (MAO) in mitochondria of liver of the Pacific squid Todarodes pacificus and of Wistar rats is carried out. It is revealed that the squid liver MAO, unlike the rat liver MAO, is capable of deaminating not only tyramine, serotonin, and benzylamine, but also histamine. The squid liver MAO activity in relation to all studied substrates is approximately 10 times lower, while the sorption ability, several tens times lower, than the rat liver MAO. Semicarbazide, a classic inhibitor of diamine oxidase, at a concentration 1 × 10^–2 M did not inhibit the catalytic activity of both studied enzymes. The specificity of action of an irreversible inhibitor, proflavine, is established, which was seen at deamination of various substrates by the squid liver MAO to the greater degree, than by the rat liver MAO. The values of the bimolecular rate constant of the irreversible inhibition (k _II) by proflavine were 2.5–20-fold higher (depending on substrate) in the case of the squid liver MAO, than of the rat liver MAO. A suggestion is put forward about the probable presence of several centers of substrate binding in the enzyme of the studied marine invertebrate, like in the mammalian enzyme.     

Design,synthesis and biological activity of novel tacrine-isatin Schiff base hybrid derivatives

A series of novel tacrine-isatin Schiff base hybrid derivatives (7a-p) were designed, synthesized and evaluated as multi-target candidates against Alzheimer’s disease (AD). The biological assays indicated that most of these compounds displayed potent inhibitory activity toward acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) and specific selectivity for AChE over BuChE. It was also found that they act as excellent metal chelators. The compounds 7k and 7m were found to be good inhibitors of AChE-induced amyloid-beta (Aβ) aggregation. Most of the compounds inhibited AChE with the IC₅₀ values, ranging from 0.42 nM to 79.66 nM. Amongst them, 7k, 7m and 7p, all with a 6 carbon linker between tacrine and isatin Schiff base exhibited the strongest inhibitory activity against AChE with IC₅₀ values of 0.42 nM, 0.62 nM and 0.95 nM, respectively. They were 92-, 62- and 41-fold more active than tacrine (IC₅₀ = 38.72 nM) toward AChE. Most of the compounds also showed a potent BuChE inhibition among which 7d with an IC₅₀ value of 0.11 nM for BuChE is the most potent one (56-fold more potent than that of tacrine (IC₅₀ = 6.21 nM)). In addition, most compounds exhibited the highest metal chelating property. Kinetic and molecular modeling studies revealed that 7k is a mixed-type inhibitor, capable of binding to catalytic and peripheral site of AChE. Our findings make this hybrid scaffold an excellent candidate to modify current drugs in treating Alzheimer’s disease (AD).     

STUDIES ON THE KINETICS OF ACETYLCHOLINESTERASE IN THE RESISIANT AND SUSCEPTIBLE STRAINS OF HOUSEFLY (MUSCA DOMESTICA)

Abstract Acetylcholinesterase (AChE) in the susceptible (S) and the resistant (R) strains of housefly (Musca domestica) was investigated using kinetic analysis. The V_max values of AChE for hydrolyzing acetylthiocholine (ATCh) and butyrylthiocholine (BTCh) were 4578.50 and 1716.08nmol/min/mg* protein in the R strain, and were 1884.75 and 864.72 nmol/min/mg. protein in the Sstrain, respectively. The V_max ratios of R to S enzyme were 2.43 for ATCh and 1.98 for BTCh. The K_m values of AChE for ATCh and BTCh were 0.069 and 0.034 mmol/L in the S strain, and 0.156, 0.059 mmol/L in the R strain, respectively. The K_m ratios of R to S enzyme were 2.26 for ATCh and 1.74 for BTCh. The k_i ratios of S to R enzyme for three insecticides propoxur, methomyl and paraoxon were 46.04, 4.17 and 2. 86, respectively. In addition, k_cat and k_cat/K_m for measuring turnover and catalytic efficiency of AChE were determined using eserine as titrant. The k_cat values of AChE from the R strain for both ATCh and BTCh were higher than those values from the S strain. But the values of k_cat/K_m were in contrary to the k_cat values with R enzyme compared to S enzyme. The AChE catalytic properties and sensitivity to the inhibition by three insecticides in the R and S strains of housefly were discussed based on contribution of V_max, K_m, k_i, k_cat and k_cat/K_m. All these data implied that AChE from the R strain might be qualitatively altered. We also observed an intriguing phenomenon that inhibitors could enhance the activity of AChE from the resistant strain. This “flight reaction” of the powerful enzyme might be correlated with the developing resistance of housefly to organophosphate or carbamate insecticides.     

Some kinetic properties of the β-d-glucosidase (cellobiase) in a commercial cellulase product from Penicillium funiculosum and its relevance in the hydrolysis of cellulose

Some kinetic parameters of the β-d-glucosidase (cellobiase, β-d-glucoside glucohydrolase, EC 3.2.1.21) component of Sturge Enzymes CP cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] from Penicillium funiculosum have been determined. The Michaelis constants (K_m) for 4-nitrophenyl β-d-glucopyranoside (4NPG) and cellobiose are 0.4 and 2.1 mM, respectively, at pH 4.0 and 50°C. d-Glucose is shown to be a competitive inhibitor with inhibitor constants (K_i) of 1.7 mM when 4NPG is the substrate and 1 mM when cellobiose is the substrate. Cellobiose, at high concentrations, exhibits a substrate inhibition effect on the enzyme. d-Glucono-1,5-lactone is shown to be a potent inhibitor (K_i = 8 μM; 4NPG as substrate) while d-fructose exhibits little inhibition. Cellulose hydrolysis progress curves using Avicel or Solka Floc as substrates and a range of commercial cellulase preparations show that CP cellulase gives the best performance, which can be attributed to the activity of the β-d-glucosidase in this preparation in maintaining the cellobiose at low concentrations during cellulose hydrolysis.     

Substituents effects on activity of kynureninase from Homo sapiens and Pseudomonas fluorescens

A series of substituted kynurenines (3-bromo-dl, 3-chloro-dl, 3-fluoro-dl, 3-methyl-dl, 5-bromo-l, 5-chloro-l, 3,5-dibromo-l and 5-bromo-3-chloro-dl) have been synthesized and tested for their substrate activity with human and Pseudomonas fluorescens kynureninase. All of the substituted kynurenines examined have substrate activity with both human as well as P. fluorescens kynureninase. For the human enzyme, 3- and 5-substituted kynurenines have k_cat and k_cat/K_m values higher than l-kynurenine, but less than that of the physiological substrate, 3-hydroxykynurenine. However, 3,5-dibromo- and 5-bromo-3-chlorokynurenine have k_cat and k_cat/K_m values close to that of 3-hydroxykynurenine with human kynureninase. The effects of the 3-halo substituents on the reactivity with human kynureninase may be due to electronic effects and/or halogen bonding. In contrast, for the bacterial enzyme, 3-methyl, 3-halo and 3,5-dihalokynurenines are much poorer substrates, while 3-fluoro, 5-bromo, and 5-chlorokynurenine have k_cat and k_cat/K_m values comparable to that of its physiological substrate, l-kynurenine. Thus, 5-bromo and 5-chloro-l-kynurenine are good substrates for both human as well as bacterial enzyme, indicating that both enzymes have space for substituents in the active site near C-5. The increased activity of the 5-halokynurenines may be due to van der Waals contacts or hydrophobic effects. These results may be useful in the design of potent and/or selective inhibitors of human and bacterial kynureninase.     

Design,synthesis and pharmacological evaluation of chalcone derivatives as acetylcholinesterase inhibitors

A novel series of chalcone derivatives (4a–8d) were designed, synthesized, and evaluated for the inhibition activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The log P values of the compounds were shown to range from 1.49 to 2.19, which suggested that they were possible to pass blood brain barriers in vivo. The most promising compound 4a (IC₅₀: 4.68 μmol/L) was 2-fold more potent than Rivastigmine against AChE (IC₅₀: 10.54 μmol/L) and showed a high selectivity for AChE over BuChE (ratio: 4.35). Enzyme kinetic study suggested that the inhibition mechanism of compound 4a was a mixed-type inhibition. Meanwhile, the result of molecular docking showed its potent inhibition of AChE and high selectivity for AChE over BuChE.     

Inhibition kinetics of human serum butyrylcholinesterase by Cd2+, Zn2+ and Al3+: comparison of the effects of metal ions on cholinesterases

Butyrylcholinesterase (BChE, EC 3.1.1.8) has been purified about 6600-fold from human serum with a procedure including ammonium sulfate fractionation (55–70%) with acid step at pH 4.5 and procainamide–Sepharose 4B affinity chromatography. The purified enzyme exhibited negative cooperativity with respect to butyrylthiocholine (BTCh) binding at pH 7.5. K_S was found to be 0.128±0.012 mM. Inhibition kinetics of the enzyme by Cd²⁺, Zn²⁺ and Al³⁺ were studied in detail. The 1/v vs 1/[BTCh] plots in the absence (control plot) and in the presence of different concentrations of cations intersected above 1/[BTCh]-axis. The data were analyzed by means of a nonlinear curve fitting program. The results demonstrated that all of the three cations are the linear mixed-type inhibitors of BChE. Ca²⁺ and Mg²⁺ had no effect on the enzyme activity in the experimental conditions. But when the enzyme was inhibited by 0.5 mM Cd²⁺ or Zn²⁺, Ca²⁺ and Mg²⁺ partially reactivated the inhibited allosteric form of BChE. Results were compared with data obtained from brain BChE purified from sheep.     

Acyl Pocket of the Cholinesterase Active Center and Dialkylphosphates: Study of Interaction by Statistical Methods

The statistical analysis is performed of changes of the bimolecular rate constant value (log k _II) of inhibition of human AChE, mouse AChE, AChE of flies Musca domestica and Calliphora vicina, and horse BuChE by dialkylphosphates with the general formula (AlkO) ₂P(O)X at elongation of alkyl radicals and change of their branching in comparison with three physical-chemical characteristics (hydrophobicity, polarity, and volume of the side chain) of 6 amino acid residues in acyl and alkoxyl pockets variable in the studied ChE (Nos. 282, 287, 288, 290, 330, 335 in Torpedo ray AChE sequence). It has been shown that depending on structure of alkyl radicals, the rate of ChE interaction with OPI is determined by sterical hindrances to sorption (residues 282, 287, 290, 335), hydrophobic interactions (288) or polarity of microenvironment (287). This dependence in most cases is statistically significant; however, rather low values of the correlation coefficient indicate influence of structure of the OPI leaving part. The decrease of the statistical significance with elongation of alkyl radicals seems to be due to an increase of the number of possible conformational states of the OPI molecule.     

Metal Ion Selectivity and Substrate Inhibition in the Metal Ion Chelation Catalyzed by Human Ferrochelatase

Protoporphyrin IX ferrochelatase (EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX. Ferrochelatase shows specificity, in vitro, for multiple metal ion substrates and exhibits substrate inhibition in the case of zinc, copper, cobalt, and nickel. Zinc is the most biologically significant of these; when iron is depleted, zinc porphyrins are formed physiologically. Examining the k_cat/K_m^app ratios for zinc and iron reveals that, in vitro, zinc is the preferred substrate at all concentrations of porphyrin. This is not the observed biological specificity, where zinc porphyrins are abnormal; these data argue for the existence of a specific iron delivery mechanism in vivo. We demonstrate that zinc acts as an uncompetitive substrate inhibitor, suggesting that ferrochelatase acts via an ordered pathway. Steady-state characterization demonstrates that the apparent k_cat depends on zinc and shows substrate inhibition. Although porphyrin substrate is not inhibitory, zinc inhibition is enhanced by increasing porphyrin concentration. This indicates that zinc inhibits by binding to an enzyme-product complex (EZnD_IX) and is likely to be the second substrate in an ordered mechanism. Our analysis shows that substrate inhibition by zinc is not a mechanism that can promote specificity for iron over zinc, but is instead one that will reduce the production of all metalloporphyrins in the presence of high concentrations of zinc.     

The characterization of Lucilia cuprina acetylcholinesterase as a drug target, and the identification of novel inhibitors by high throughput screening

Acetylcholinesterase (AChE, EC3.1.1.7.) is the molecular target for the carbamate and organophosphate pesticides that are used to combat parasitic arthropods. In this paper we report the functional heterologous expression of AChE from Lucilia cuprina (the sheep blowfly) in HEK293 cells. We show that the expressed enzyme is cell-surface-exposed and possesses a glycosyl-phosphatidylinositol membrane anchor. The substrates acetyl-, propionyl- and butyrylthiocholine (AcTC, PropTC, ButTC), and also 11 further thiocholine and homo-thiocholine derivatives were chemically synthesized to evaluate and compare their substrate properties in L. cuprina AChE and recombinant human AChE. The Michaelis-Menten constants K_M for AcTC, PropTC and ButTC were found to be 3-7-fold lower for the L. cuprina AChE than for the human AChE. Additionally, 2-methoxyacetyl-thiocholine and isobutyryl-thiocholine were better substrates for the insect enzyme than for the human AChE. The AcTC, PropTC and ButTC specificities and the Michaelis-Menten constants for recombinant L. cuprina AChE were similar to those determined for AChE extracted from L. cuprina heads, which are a particularly rich source of this enzyme. The median inhibition concentrations (IC₅₀ values) were determined for 21 organophosphates, 23 carbamates and also 9 known non-covalent AChE inhibitors. Interestingly, 11 compounds were 100- to >4000-fold more active on the insect enzyme than on the human enzyme. The substrate and inhibitor selectivity data collectively indicate that there are structural differences between L. cuprina and human AChE in or near the active sites, suggesting that it may be possible to identify novel, specific L. cuprina AChE inhibitors. To this end, a high throughput screen with 107,893 compounds was performed on the L. cuprina head AChE. This led to the identification of 195 non-carbamate, non-organophosphate inhibitors with IC₅₀ values below 10 ??M. Analysis of the most potent hit compounds identified 19 previously unknown inhibitors with IC₅₀ values below 200 nM, which were up to 335-fold more potent on the L. cuprina enzyme than on the human AChE. Some of these compounds may serve as leads for lead optimization programs to generate fly-specific pesticides.     

Kinetic analysis of the “substrate protective effect” in cholinesterases of different origin

The authors’ own and literature data are summarized on interaction of 17 irreversible organophosphorus inhibitors with different types of cholinesterases (ChE): erythrocyte acetylcholinesterase (AChE), serum butyrylcholinesterase (BuChE), and cholinesterase of the Pacific squidTodarodes pacificus, in the presence of 9 substrates. The kinetic analysis of the “substrate protective effect” based on A.P. Brestkin’s equation is performed, and the current interpretation of individual components of this process is done. An essential effect of the inhibitor structure on individual phases of the reaction is revealed. Among choline substrates, only formylcholine did not show a protective effect. The inability of an uncationic substrate, phenylacetate, to regulate ChE reactivity is confirmed. Among the studied ChE, the highest substrate protective effect was revealed in the Pacific squid ChE.     

Substrate-inhibitory analysis of the cholinesterase in the freshwater oligochaete Lumbriculus variegatus (O.F. Műller, 1773; Lumbriculidae,Oligochaeta)

The substrate-inhibitory analysis has shown that single “atypical” cholinesterase (ChE) presents in tissues of freshwater oligochaete Lumbriculus variegatus (O.F. M?ller). This enzyme differs both from “typical” acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Specific activity of oligochaete ChE ranges 55–100 μmol ATCh g⁻¹ tissue min⁻¹ or 0.7–1 μmol ATCh mg⁻¹ protein min⁻¹, ratio of maximal rates (V) of substrate hydrolysises is 100:72:71:83 for acetyl-, propionyl-, butyryl- and acetyl-β-metylthiocholine respectively. Values of Michaelis constant (K_m) for these substrates are (1.9–2.5) × 10⁻⁴ M. The bimolecular enzyme inhibition rate constants (k_II) for organophosphorus inhibitors paraoxon, DDVP, and iso-OMPA are 10⁷, 10⁶ и 10³ mol⁻¹ | min⁻¹. ATCh and BuTCh exhibit the effect of substrate inhibition of ChE activity, while PrTCh and MeTCh do not.     

Synthesis and biological evaluation of berberine–thiophenyl hybrids as multi-functional agents: Inhibition of acetylcholinesterase,butyrylcholinesterase, and Aβ aggregation and antioxidant activity

A series of berberine–thiophenyl hybrids were designed, synthesised, and evaluated as inhibitors of acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and β-amyloid (Aβ) aggregation and as antioxidants. Among these hybrids, compounds 4f and 4i, berberine linked with o-methylthiophenyl and o-chlorothiophenyl by a 2-carbon spacer, were observed to be potent inhibitors of AChE, with IC₅₀ values of 0.077 and 0.042 μM, respectively. Of the tested compounds, 4i was also the most potent inhibitor of BuChE, with an IC₅₀ value of 0.662 μM. Kinetic studies and molecular modelling simulations of the AChE-inhibitor complex indicated that a mixed-competitive binding mode existed for these berberine derivatives. The biological studies also demonstrated that these hybrids displayed interesting activities, including Aβ aggregation inhibition and antioxidant properties.     

A remarkable activity of human leukotriene A4 hydrolase (LTA4H) toward unnatural amino acids

Leukotriene A₄ hydrolase (LTA4H––EC 3.3.2.6) is a bifunctional zinc metalloenzyme, which processes LTA₄ through an epoxide hydrolase activity and is also able to trim one amino acid at a time from N-terminal peptidic substrates via its aminopeptidase activity. In this report, we have utilized a library of 130 individual proteinogenic and unnatural amino acid fluorogenic substrates to determine the aminopeptidase specificity of this enzyme. We have found that the best proteinogenic amino acid recognized by LTA4H is arginine. However, we have also observed several unnatural amino acids, which were significantly better in terms of cleavage rate (k _cat/K _m values). Among them, the benzyl ester of aspartic acid exhibited a k _cat/K _m value that was more than two orders of magnitude higher (1.75 × 10⁵ M^?1 s^?1) as compared to l-Arg (1.5 × 10³ M^?1 s^?1). This information can be used for design of potent inhibitors of this enzyme, but may also suggest yet undiscovered functions or specificities of LTA4H.     

Cholinesterases of aphids—II: Anticholinesterase potency and toxicity of different organophosphorous inhibitors for spring grain aphid Schizaphis gramina rond

Anticholinesterase potency and toxicity for spring grain aphids of 52 organophosphorous inhibitors (OPI) of different structures were investigated. As regards sensitivity to OPI, the acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) of the spring grain aphid are shown to differ significantly from the “typical” cholinesterases, i.e. from human erythrocyte AChE and horse serum BuChE, which is presumably associated with differences in the structures of the active surface of these enzymes. The extremely high sensitivity of spring grain aphid AChE to cationic OPI containing the onium atom in the leaving part of the molecule indicates the fundamental importance of the anionic site in the active centre of the enzyme. By contrast, spring grain aphid BuChE has low sensitivity to OPI, typical of the cationic type, which suggests that it has no anionic site. Correlation was established between the toxicity and anticholinesterase potency of OPI which contain no cationic groups or bulky hydrophobic radicals.     

Kinetic properties and specificity of trimeric Plasmodium falciparum and human dUTPases

Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, and plays important roles in nucleotide metabolism and DNA replication. Hydrolysis of other nucleotides similar in structure to dUTP would be physiologically negative and therefore high substrate specificity is essential. Binding and hydrolysis of nucleotides different to dUTP by the dUTPases from Plasmodium falciparum (PfdUTPase) and human (hdUTPase) was evaluated by applying isothermal titration calorimetry (ITC). The ribo and deoxyribonucleoside triphosphates dGTP, dATP, dCTP, dTTP, UTP, FdUTP and IdUTP have been analysed. dUTP and FdUTP were the most specific substrates for both enzymes. The specificity constants (k_cat/K_m) for the remaining ones, except for the IdUTP, were very similar for both enzymes, although PfdUTPase showed a slightly higher specificity for dCTP and UTP and the human enzyme for dTTP and dCTP. PfdUTPase was very efficient in using FdUTP as substrate indicating that small size substituents in the 5′ position are well tolerated. In addition product inhibition was assessed by binding studies with the nucleoside monophosphate derivatives and thermodynamic parameters were established. When FdUTP hydrolysis was monitored, Plasmodium dUTPase was more sensitive to end-product inhibition by FdUMP than the human enzyme. Taken together these results highlight further significant differences between the human and Plasmodium enzymes that may be exploitable in selective inhibitor design.     

Isolation and characterization of the polyphenoloxidase from senescent leaves of black poplar

The polyphenoloxidase (PPO) from black poplar senescent leaves has been purified to almost complete homogeneity by a combination of ammonium sulphate precipitation, Sephadex G75 filtration and DEAE-cellulose chromatography. The purified enzyme has a MW of 60 000 and is probably a Cu⁺ enzyme. Peroxidase (PO) activity co-purifies with PPO and has the same MW as it. The two enzymes differ in pH optimum and in response to the effect of ionic strength. Natural phenols are either substrates, inhibitors or activators of black poplar PPO. This enzyme is an o-diphenoloxidase which binds substrates with K_m in the millimolar range. With caffeic and chlorogenic acids inhibition by excess substrate is observed. Benzoic acid phenols and cinnamic acid phenols are either competitive or non-competitive inhibitors of PPO. Hydroquinone is a highly potent non-competitive inhibitor of the enzyme (K_i  90 μM). Ferulic acid is a potent activator of the PPO-catalysed oxidation of catechol (K_a  0.34 mM, ν_sat/ν_o  7.7).     

Kinetics of Human Serum Butyrylcholinesterase Inhibition by a Novel Experimental Alzheimer Therapeutic,Dihydrobenzodioxepine Cymserine

Cholinergic loss is the single most replicated neurotransmitter deficiency in Alzheimer’s disease (AD) and has led to the use of acetylcholinesterase inhibitors (AChE-Is) and unselective cholinesterase inhibitors (ChE-Is) as the mainstay of treatment. AChE-Is and ChE-Is, however, induce dose-limiting adverse effects. Recent studies indicate that selective butyrylcholinesterase inhibitors (BuChE-Is) elevate acetylcholine (ACh) in brain, augment long-term potentiation, and improve cognitive performance in rodents without the classic adverse actions of AChE-Is and ChE-Is. BuChE-Is thereby represent a new strategy to ameliorate AD, particularly since AChE activity is depleted in AD brain, in line with ACh levels, whereas BuChE activity is elevated. Our studies have focused on the design and development of cymserine analogues to induce selective time-dependent brain BuChE inhibition, and on the application of innovative and quantitative enzyme kinetic analyses to aid selection of drug candidates. The quantitative interaction of the novel inhibitor, dihydrobenzodioxepine cymserine (DHBDC), with human BuChE was characterized. DHBDC demonstrated potent concentration-dependent binding with BuChE. The IC₅₀ and specific new kinetic constants, such as K_T50, P_PC, K_T1/2 and R_I, were determined at dual substrate concentrations of 0.10 and 0.60 mM butyrylthiocholine and reaction times, and are likely attainable in humans. Other classical kinetic parameters such as K_ia, K_ma, V_ma and V_mi were also determined. In synopsis, DHBDC proved to be a highly potent competitive inhibitor of human BuChE in comparison to its structural analogue, cymserine, and represents an interesting drug candidate for AD. Dedicated to Professor Moussa Youdim, Dept. Pharmacology, Technion, Haifa, Israel, in celebrating 45 years in scientific research focused on drug discovery and 30 years at Technion.     

Propionylcholinesterase from Allolobophora caliginosa

1. Propionylcholinesterase from 100,000 g supernatant of Allolobophora caliginosa is purified by affinity chromatography on a procainamide containing gel.2. Purified enzyme is single protein band on PAGE. SDS-electrophoresis resolved subunits of 28 and 62 kD.3. On the basis of k_cal/K_m propionylthiocholine and acetylthiocholine are the best substrates. Propionylthiocholine shows the highest k_cat.4. Tetramethylammonium, tetraethylammonium, procainamide, trimethyl-(p-aminophenyl)ammonium, and d-tubocurarine are competitive inhibitors. Mixed-type inhibition is shown by tetrapropylammonium and tetrabutylammonium.5. Inhibition by atropine is depending on both substrate and inhibitor concentrations.