Acetylcholinesterase inhibition by pitofenone: a spasmolytic compound.

Pitofenone, a spasmolytic compound, inhibited the acetylcholinesterase activity from bovine erythrocytes and from electric eel. It is a potent inhibitor of this enzyme from the two sources, with Ki values of 36 and 45 microM, respectively. Of the five compounds structurally related to pitofenone, only those containing a piperidine moiety show acetylcholinesterase inhibition. All these inhibitions are reversible, linear, and noncompetitive in nature. A qualitative correlation between the anticholinesterase and the corresponding antimuscarinic activity for some of these compounds was apparent. Good separation of these two effects would be a desirable feature for newer muscarinic antagonists.     

Acetylcholinesterase. Two types of modifications confer resistance to insecticide.

Quantitative and qualitative changes in acetylcholinesterase confer resistance to insecticides. We have constructed several Drosophila melanogaster strains producing various amounts of enzyme by P-mediated transformation. Toxicological analysis of these strains demonstrates that resistance to organophosphorus insecticides is correlated with the amount of acetylcholinesterase in the central nervous system. Resistance may also be qualitatively determined. Comparison of the Drosophila acetylcholinesterase gene between a resistant strain caught in the wild and a wild type susceptible strain only revealed one nucleotide transition resulting in the replacement of a phenylalanine by a tyrosine. Flies mutant for acetylcholinesterase and rescued with a minigene mutagenized for this same transition produced an altered enzyme which renders flies resistant to pesticides.     

Kinetics of the two-sited enzyme. I. Activation and inhibition by substrate

Equations have been derived and plotted to describe apparent modifier effects of a single substrate which is randomly bound, in rapid binding equilibria, at two sites of an enzyme. Three special cases have been considered: independent, non-equivalent catalytic sites; equivalent, interacting catalytic sites; one catalytic site and one modifier site. In each case, the curvature of Lineweaver-Burk plots has been determined by evaluating the limits of the derivatives, d(1/υ₀)/d(1/S) and d(S/υ₀)/dS. The direction of curvature has been correlated with modifier effects by distinguishing between activating and inhibiting effects on maximal velocities (V), or on dissociation constants of enzyme-substrate complexes (K). Upward curvature, with a minimum in the plot, corresponds to V-inhibition. Upward curvature without a minimum corresponds to various combinations of activating effects. Downward curvature represents either K-inhibition, with or without simultaneous V-activation, or no interaction at all.     

Steady state kinetics of an enzyme reaction with one substrate and one modifier

This study examines the steady state kinetics of a reaction involving an enzyme, a substrate and a modifier when the reaction follows Michaelis-Menten kinetics. Conditions for Michaelis-Menten kinetics are deduced, and it is shown that an analogue of detailed balance determines the complexity of the rate equations in these cases. A scheme to distinguish many cases of Michaelis-Menten kinetics is presented. It is shown that steady state kinetics are, in general, insufficient to specify the mechanism of a reaction, since different effects of a modifier can give identical steady state kinetic data.     

Features of thiamine diphosphate-dependent enzyme inhibition by oxodihydrothiochrome and its phosphorylated derivatives

Anticoenzyme action of new derivatives of thiamine: oxodihydrothiochrome and its mono- and diphosphoric esters has been studied in the experiments on mice. It is shown that the given compounds exert an inhibiting action on transketolase and pyruvate dehydrogenase and do not change activity of 2-oxoglutarate dehydrogenase in the animal organism. Antivitamin effect of the studied inhibitors is observed with the lower doses and in the earlier terms as compared with the other known inhibitors of thiamine-diphosphate-dependent enzymes. The preparations inhibit activity of the yeast pyruvate-decarboxylase by the mixed (with respect to thiamine-diphosphate) type (Ki for oxodihydrothiochrome and its mono- and diphosphoric esters: 2.3 x 10(-3), 7.2 x 10(-4), 5.6 x 10(-5) M, respectively). Possible mechanisms of the action of the mentioned compounds as thiamine antimetabolites are discussed.     

Acetylcholinesterase from rat red cells and cholinesterase ofPseudomonas aeruginosa: Different types of inhibition by atropine

Summary The inhibition by atropine of cholinesterase from Pseudomonas aeruginosa has been studied in parallel with the membrane bound acetylcholinesterase from rat red cells. Acetylcholinesterase of rat red cells, like other animal cholinesterases, was competitively inhibited while the cholinesterase fromPseudomonas aeruginosa was partially non competitively inhibited by atropine. These results clearly indicated a different behavior of cholinesterase fromPseudomonas aeruginosa in comparison with the enzyme ofPseudomonas fluorescens and other animal cholinesterases.     

Human renin inhibition by a diazoacyl reagent: Relationship of the enzyme to other proteinases

Human renin is inactivated by a diazoacyl compound (diazoacetylglycine ethyl ester; N₂CHCO-Gly-OEt) in the presence of Cu(II). The mechanism of the inactivation is presumably identical to that which has been determined for pepsin and several other proteinases: esterification of the β-carboxyl of an aspartic acid residue at the active site of the enzyme. Renin's inhibition by the diazoacyl reagent, its specificity toward a hydrophobic sequence, and its inhibition by pepstatin, all suggest a close relationship to the acid proteinases, especially pepsin and cathepsin D. However, renin, a neutral proteinase, would be better classified together with other diazoacyl-inhibited enzymes by active site rather than pH optimum. The term “aspartic proteinase” is suggested for this group of enzymes.     

Conformational adaptability of the active site of beta-galactosidase. Interaction of the enzyme with some substrate analogous effectors.

The action of different effectors, glycosides, and alcohols on the reactions catalyzed by beta-galactosidase is analyzed in this paper. Effectors as large as tri- and tetrasaccharides have no effect on the enzyme activity, suggesting that the binding site has rather small size. Most of the beta-galactosides produce a competitive inhibition. The other compounds assayed behave either as noncompetitive inhibitors, and they are deadened inhibitors, or as uncompetitive inhibitors which exhibit a better affinity for the chemical intermediate than for free enzyme; nearly all of them give transfer products. The analysis of the data indicates that the active center of beta-galactosidase is made up of two subsites: a galactose and a glucose subsite. This latter site is in a more favorable conformation in the galactosylenzyme than in free enzyme; possibly it might even by generated by the galactose binding. Conformational rearrangements of the active center deduced from the inhibition data have been directly observed by differential spectroscopy. The conformational adaptability of the enzyme and its consequence for the functional properties of beta-galactosidase are discussed.     

Acetylcholinesterase: inhibition by tetranitromethane and arsenite. Binding of arsenite by tyrosine residues

Tetranitromethane inhibits acetylcholinesterase with respect to the hydrolysis of both acetylthiocholine and indophenyl acetate. The loss of activity with indophenyl acetate, a poor substrate, is preceded by an increase in enzyme activity. Only 12 of the 21 tyrosine residues/monomer of enzyme are susceptible to nitration. Loss of activity with respect to indophenyl acetate occurs well after no further nitration of tyrosines occurs and must be due to the modification of other residues. Incubation of the enzyme with arsenite before nitration results in the nitration of only 10 tyrosines. This experiment reveals that the structural basis for the binding of arsenite is the formation of a diester with two tyrosine residues.     

The formation and stabilization of an adaptive enzyme in the absence of its substrate

It is shown that various substrates accelerate the disappearance of an adaptive enzyme when its own substrate has been removed from the medium. The order of effectiveness of such substrates appears to be connected with their chemical similarity to the adaptive substrate. It is shown that two conditions which are able to inhibit the formation of adaptive enzymes-anaerobiosis and the presence of sodium azide-are equally able to prevent the disappearance of an adaptive enzyme after the removal of its substrate. Finally, it is shown that rapidly growing cultures, under optimal conditions for synthetic activity, are able to maintain and even appreciably to increase their initial content of an adaptive enzyme, in the absence of its specific substrate and in the presence of a normally competitive substrate. In the light of these results, the three major theories of enzyme formation hitherto proposed are evaluated.     

Acetylcholinesterase inhibition by eserine: rate constants of reaction. Part II

The inhibition of eel acetylcholinesterase by physostigmine at 20 degrees and 25 degrees C have been investigated. In our evaluation the unimolecular reactivation rate constant, k3, the carbamylation rate constant, k2, and the binding constant, Ka, are the first simultaneously determined. The mechanism of this reaction is discussed.     

Reversible inhibition in bimolecular rapid-equilibrium random-order enzyme systems. The effect of substrate–substrate and inhibitor–substrate interactions

A model is presented that accounts for all types of reversible inhibition by a single inhibitor molecule in bimolecular rapid-equilibrium random-order enzyme systems. The characterization of inhibition mechanisms by graphical methods is examined, and a system of nomenclature is suggested.     

Two types of allograft-induced cytotoxic macrophage, one against allografts and the other against syngeneic or allogeneic tumor cells

In the 1990s, based on the results of studies using beta(2)M, CD4 or CD8 knockout mice, several groups reported that the main effector cells responsible for skin or organ allograft rejection were non-T, non-NK cells. Similarly, we demonstrated that in an animal model of transplantation of BALB/c (H-2(d)) skin onto or Meth A (H-2(d)) tumor cells into C57BL/6 (H-2(b)) mice, AIM, which expressed iNOS, IL-12, and IL-18, were the main effector cells and also that they were cytotoxic against syngeneic tumor cells. Here, we examined whether the same population of macrophages could react with two distinct types of target cell. When BALB/c skin or Meth A tumor cells were transplanted into C57BL/6 mice, cytotoxic activity against the allograft was induced in the transplantation site on days 5-14 and was recovered in non-adherent cells after a 20-min incubation in a serum-coated dish, suggesting the induction of a type of AIM (AIM-1) in the transplantation site. The AIM-1-expressing receptors for H-2D(d)K(d) antigens had no cytotoxic activity against syngeneic tumor cells. In contrast, AIM-2, which were recovered in the fraction adherent to the serum-coated dish, exhibited cytotoxic activities against various types of tumor cells, whereas they were inactive toward BALB/c skin. AIM expressed iNOS (AIM-1 < AIM-2), IL-12 (AIM-1 > AIM-2), and IL-18 (AIM-2 alone) mRNAs. These results indicate that after allografting, two distinct types of cytotoxic AIM were induced in the transplantation site, one against the allografted skin or tumor (AIM-1) and the other against allogeneic or syngeneic tumor cells (AIM-2).     

Transient kinetic studies of malic enzyme. A conformational change associated with substrate inhibition by malate.

The oxidative decarboxylation of l-malate catalyzed by malic enzyme has been studied by stopped-flow spectrophotometry and by initial rate measurements with large concentrations of NADP⁺, malate, and Mn²⁺. The results show that hybride transfer is fast, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{< 0.7}$ ms. The formation of enzyme-bound NADPH in an amount equivalent to about half of the enzyme active center concentration is followed by turnover at a rate which is initially faster than the steady-state rate, under conditions such that substrate inhibition by malate is observed in the steady state. The steady-state rate is reached after about 0.5 s. It is suggested that a conformational change in the abortive complex of enzyme, manganese, NADPH, and malate is responsible for the malate inhibition and for the slow approach to the true steady state. The relief of malate inhibition by increasing Mn²⁺ concentrations is described, and the results are described in relation to other evidence of nonidentical binding sites for, or negatively cooperative binding of, substrate and activator and possible half-of-the-sites reactivity.     

Novel inhibition of porcine pepsin by a substituted piperidine. Preference for one of the enzyme conformers

Pepsin inhibition by 3-alkoxy-4-arylpiperidine (substituted piperidine; (3R,4R)-3-(4-bromobenzyloxy)-4-[4-(2-naphthalen-1-yl-2-oxo-ethoxy)phenyl]piperidine) has been studied using steady-state kinetic and pre-equilibrium binding methods. Data were compared with pepstatin A, a well known competitive inhibitor of pepsin. Steady-state analysis reveals that the substituted piperidine likewise behaves as a competitive inhibitor. Pre-equilibrium binding studies indicate that the substituted piperidine can displace a fluorescently labeled statine inhibitor from the enzyme active site. Simulation of the stopped-flow fluorescence transients provided estimates of the K(d) values of 1.4 +/- 0.2 microm and 39 +/- 2 nm for the piperidine and the fluorescently labeled statine, respectively. The effects of combinations of these two inhibitors resulted in a series of parallel lines when plotted by the method of Yonetani and Theorell (Yonetani, T., and Theorell, H. (1964) Arch. Biochem. Biophys. 106, 234-251), suggesting that the two inhibitors bind in a mutually exclusive fashion to pepsin. Fitting of the entire data set to the appropriate equation yielded an alpha factor of 8 +/- 1. The magnitude of this factor ( infinity > alpha > 1) can be explained by a conformational distinction between the enzyme species that bind each inhibitor. The effects of pH on the inhibition constants for pepstatin A and the substituted piperidine also suggest that the inhibitors bind to distinct conformational forms of the enzyme. No inhibition by the piperidine was observed at acidic pH, while pepstatin A inhibition is maximal at low pH values. Inhibition by the piperidine was maximal when a group with pK 4.8 +/- 0.2 was deprotonated and another group with pK 5.9 +/- 0.2 was protonated. Most likely these two groups are the catalytic aspartates with perturbed ionization properties as a result of a significant and unique conformational change. Taken together, these data suggest that the enzyme can readily interconvert between two conformers, one capable of binding substrate and pepstatin A and the other capable of binding the substituted piperidine.     

Two-substrates packed-bed bioreactors: inhibition of enzyme and competitive binding of substrate and product to a ligand

An analytical model is developed to describe the performance of a packed-bed immobilized enzyme reactor in which parallel processes take place. In particular, two-substrate reaction, inhibition of the enzyme by one of the reaction products, and binding of one substrate and/or one product to an added ligand are taken into account. In addition, substrates and product diffusion into the porous catalyst are also considered. Using this model, numerical simulations were performed. The results point to the fact that, when all the above processes occur concomitantly, a variety of performance characteristics can be obtained, depending on the particular values of the related parameters. Moreover, under certain conditions, the reactor performance can be improved by controlled addition of ligand.List of Symbols A total concentration of ligand - C _1,i concentration of Substrate-1 in the pores of stage i - C _2,i concentration of Substrate-2 in its free form in the pores of stage i - _2,i concentration of the Substrate-2-Ligand Complex in the pores of stage i - total concentration of Substrate-2 in the pores of stage i - _i concentration of the Product-Ligand Complex in the pores of stage i - concentration of the free Product in the pores of stage i - total concentration of the Product in the pores of stage i - internal (pore) diffusion coefficient for the Substrate-Ligand Complex - D ₁ internal (pore) diffusion coefficient of Substrate-1 - D ₂ internal (pore) diffusion coefficient of Substrate-2 - effective (pore) diffusion coefficient for Substrate-2 - internal (pore) diffusion coefficient for the Product - internal (pore) diffusion coefficient for the Product-Ligand Complex - effective (pore) diffusion coefficient for the Product - K thermodynamic equilibrium constant for binding Substrate-2 to Ligand - K _m,1,K _m,2 Michaelis constants for Substrates-1 and 2, respectively - effective Michaelis constant for Substrate-2 - K _p thermodynamic equilibrium constant for binding the reaction Product to Ligand - effective equilibrium constant for binding Substrate-2 to Ligand - effective equilibrium constant for binding the reaction Product to Ligand. - K _b inhibition constant - K _q inhibition constant - effective inhibition constant - effective inhibition constant - k _a, k _d association and dissociation rate constants for Substrate-2 — Ligand complex - association and dissociation constants for Product —Ligand complex - n total number of elementary stages in the reactor - Q volumetric flow rate throughout the reactor - R _j,i reaction rate of Substrate-j in stage i, in terms of volumetric units - S _1,0 concentration of Substrate-1 in the reactor feed - total concentration of Substrate-2 in the reactor feed - S _1,i–1,S _1,i concentration of Substrate-1 in the bulk phase leaving stages i–1 and i, respectively - S _2,i concentration of Substrate-2 in its free form, in the bulk phase leaving stage i - _2,i–1, _2,i concentration of Substrate-2 in the bulk phase leaving stage i–1 and i, respectively - total concentration of Substrate-2 in the bulk phase leaving stages i–1 and i, respectively - _i concentration of the Product-Ligand Complex in the bulk phase of stage i - concentration of free Product in the bulk phase of stage i - total concentration of Product in the bulk phase of stage i - V total volume of the reactor - V _m maximal reaction rate in terms of volumetric units - y axial coordinate of the pores - y ₀ depth of the pores Greek Symbols ₁ dimensionless parameter - dimensionless parameter - dimensionless parameter - ₁ dimensionless parameter - dimensionless parameter - _1,i dimensionless concentration of Substrate-1 in pores of stage i - dimensionless total concentration of Substrate-2 (in both free and bound form) in pores of stage i - dimensionless total concentration of the reaction product in the pores of stage i - ₁ dimensionless parameter - dimensionless parameter - dimensionless parameter - dimensionless parameter - dimensionless parameter - dimensionless position along the pore - volumetric packing density of catalytic particles (dimensionless) - porosity of the catalytic particles (dimensionless) - _1,i dimensionless concentration of Substrate-1 in the bulk phase of stage i - dimensionless total concentration of Substrate-2 (in both free and bound form) in the bulk phase of stage i