Inhibitory effects of salts on the nicotinamide adenine dinucleotide specific isocitrate dehydrogenase from Blastocladiella emersonii.

The effect of different salts on the NAD-specific isocitrate dehydrogenase from Blastocladiella emersonii has been studied. The results show that the salt inhibition depends on the size of the anions and that the ionic strength is of minor importance.The salts inhibit the enzyme in a competitive manner with regard to isocitrate. The isocitrate concentration giving half saturation increased by the same factor whether or not the activator AMP was present. The finding that higher salt concentrations are needed to inhibit the enzyme in the presence of AMP is due to the fact that in this case isocitrate is more tightly bound.Stopped-flow experiments demonstrated that when the enzyme was incubated with isocitrate and metal ions prior to initiation of the reaction by addition of NAD, the salt inhibition needed several seconds to be fully expressed. Moreover, a lag occurred before NADH was formed when the enzyme was mixed with its substrates and cofactor. The data suggest that the hysteretic properties of the enzyme are due to isomerization of the enzyme molecules, and that specific binding sites are involved in the salt inhibition.     

Sodium cholate-induced changes in the conformation and activity of rat pancreatic cholesterol esterase

Pancreatic cholesterol esterase (CEase) regulates dietary cholesterol absorption and is activated in the presence of trihydroxy bile salts while remaining inactive monohydroxy bile salts. CEase from rat pancreas has been purified by ammonium sulfate precipitation, hydroxylapatite chromatography, and gel filtration on Sephacryl S-200/S-300 columns connected in series, and its homogeneity and Mr (55,418 +/- 288) have been determined by sedimentation equilibrium centrifugation. The effects of tri-, di-, and monohydroxy bile salts on the conformation of the purified enzyme in buffer solution and in an in vitro assay system were studied by circular dichroism spectropolarimetry. The CD spectrum of the enzyme in solution shows a curve shape suggestive of an alpha-helicity, but low mean residue ellipticity (MRE) values may indicate an important beta-turn contribution. Sodium cholate, a trihydroxy bile salt, induces a decrease in the negative MRE values of the enzyme in solution at bile salt concentrations of 70-100 nM, with no further spectral changes at concentrations as high as 1 mM. Sodium cholate concentrations higher than 1 microM also induce an increase in the enzyme's negative MRE values under activity assay conditions, which reverts toward its original value once the reaction reaches equilibrium. These latter changes are interpreted as induced by substrate binding to the enzyme followed by partial substrate depletion after the reaction reaches equilibrium. Sodium deoxycholate, a dihydroxy bile salt, induces unstable transient increases and decreases in the MRE values of CEase in buffer solution and under activity assay conditions. These changes are bile salt concentration-dependent and may reflect self-association of the protein. Sodium taurolithocholate, a monohydroxy bile salt, does not affect the CD spectrum of CEase, and neither the di- or the monohydroxy bile salt activates the enzyme.     

Molecular recognition by acetylcholinesterase at the peripheral anionic site: structure-activity relationships for inhibitions by aryl carbamates

Substituted phenyl-N-butyl carbamates (1-9) are potent irreversible inhibitors of Electrophorus electricus acetylcholinesterase. Carbamates 1-9 act as the peripheral anionic site-directed irreversible inhibitors of acetylcholinesterase by the stop-time assay in the presence of a competitive inhibitor, edrophonium. Linear relationships between the logarithms of the dissociation constant of the enzyme inhibitor adduct (Ki), the inactivation constant of the enzyme-inhibitor adduct (k2), and the bimolecular inhibition constant (k(i)) for the inhibition of Electrophorus electricus acetylcholinesterase by carbamates 1-9 and the Hammett substituent constant (sigma), are observed, and the reaction constants (ps) are -1.36, 0.35 and -1.01, respectively. Therefore, the above reaction may form a positive charged enzyme-inhibitor intermediate at the peripheral anionic site of the enzyme and may follow the irreversible inactivation by a conformational change of the enzyme.     

Photoaffinity labelling of cholinesterases. Discrimination between active and peripheral sites.

Two para-dialkylaminobenzenediazonium salts, the dimethylamino (A) and dibutylamino (B) derivatives, are presented as structural probes for acetylcholinesterase and butyrylcholinesterase. While being reversible competitive inhibitors in the dark, A and B behave, upon irradiation and through the formation of arylcation species, as irreversible labels of ammonium-binding sites of both enzymes. The observed variations of the different inactivation rate constants point to a different structural environment for acetylcholinesterase-binding and butyrylcholinesterase-binding sites. Moreover, in the case of acetylcholinesterase, protection experiments with specific ligands (edrophonium and propidium) showed that the dimethylamino salt A exclusively labels the hydrolytic anionic site, whereas the dibutylamino salt B also labels the peripheral site. Specificities and stoechiometries of the incorporations were determined and, in the case of acetylcholinesterase, the irradiated protein was submitted to chemical degradation. Peptide maps were obtained by gel-permeation chromatography and HPLC, giving access to labelled peptides which belong either to the active or to the peripheral site.     

Effects of salts on the conformation and catalytic properties of d-amino acid aminotransferase

The effects of salts on the biochemical properties of D-amino acid aminotransferase from Bacillus sp. YM-1 have been studied to elucidate both the inhibitory effects of salts on the activity and the protective effects of salts on the substrate-induced inactivation. The results from UV-visible spectroscopy studies on the reaction of the enzyme with D-serine revealed that salt significantly reduced the rate of the formation of the quinonoid intermediate and its accumulation. The kinetic and spectroscopy studies of the reaction with alpha-[(2)H]-DL-serine in different concentrations of NaCl provided evidence that the rate-limiting step was changed from the deprotonation of the external aldimine to another step(s), presumably to the hydrolysis of the ketimine. Gel filtration chromatography data in the presence of NaCl showed that the enzyme volume was reduced sharply with the increasing NaCl concentration, up to 100 mM. An additional increase of the NaCl concentration did not affect the elution volume, which suggests that the enzyme has a limited number of salt-binding groups. These results provide detailed mechanistic evidence for the way salts inhibit the catalytic activity of Damino acid aminotransferase     

Catalytic properties of acetylcholinesterase, modified by N,N-dimethyl-2-phenylaziridinium. An alkane sulfonation reaction

By means of affinity labelling with N,N-dimethyl-2-phenylaziridinium ion (DPA) two forms of acetylcholinesterase were synthesized that contained one or two molecules of the label covalently attached to the enzyme. The reaction of native and covalently modified acetylcholinesterases with n-alkane sulfonyl chlorides CnH2n + 1SO2Cl at n = 1 -4 was used to characterize the reactivity and properties of the enzymes. It was found that labelling of acetylcholinesterase with one molecule of DPA did not affect the enzyme's reactivity. Acetylcholinesterase containing two labels (the second one presumably located at the anionic centre of the enzyme) displayed enhanced and more specific reactivity towards alkane sulfonyl chlorides. It was found that the phenomenon of acceleration caused by affinity modification is analogous to the influence of n-tetraalkylammonium ions on the same reaction. Therefore, the mechanism of regulation of the properties of the esteric centre, caused by affinity labelling of the enzyme at the anionic centre, is the same as in the case of n-tetralkylammonium ions.     

Stimulatory effect of ammonium sulfate at high concentrations on the aminoacylation of tRNA and tRNA-like molecules

The influence of various salts on the aminoacylation of tRNA(Val) and the tRNA-like structure from turnip yellow mosaic virus RNA by yeast valyl-tRNA synthetase has been studied. As expected, increasing the concentration of salts inhibits the enzymatic reaction. However, in the presence of high concentration of ammonium sulfate, and only this salt, the inhibitory effect is suppressed. Under such conditions, the aminoacylation becomes comparable to that measured in the absence of salt. It was shown that ammonium sulfate affects both the catalytic rate of the reaction and the affinity between valyl-tRNA synthetase and the RNAs. Because the affinity between the partners in the complex is increased when the concentration of the salt is high, it is suggested that hydrophobic effects are involved in tRNA/synthetase interactions.     

Inhibition of acetylcholinesterase from Electrophorus electricus (L.) by tricyclic antidepressants

The effects of tricyclic antidepressants drugs (TCA) amitriptyline, imipramine and nortriptyline, on purified Electrophorus electricus (L.) acetylcholinesterase (AChE; acetylcholine hydrolase, EC 3.1.1.7) were studied using kinetic methods and specific fluorescent probe propidium. The antidepressants inhibited AChE activity by a non-competitive mechanism. Inhibition constants range from 200 to 400 microM. Dimethylated amitriptyline and imipramine were more potent inhibitors than the monomethylated nortriptyline. Fluorescence measurements using bis-quaternary ligand propidium were used to monitor ligand-binding properties of these cationic antidepressants to the AChE peripheral anionic site (PAS). This ligand exhibited an eight-fold fluorescence enhancement upon binding to the peripheral anionic site of AChE from E. electricus (L.) with K(D)=7 x 10(-7)M. It was observed that TCA drugs displaced propidium from the enzyme. On the basis of the displacement experiments antidepressant dissociation constants were determined. Similar values for the inhibition constants suggest that these drugs have similar affinity to the peripheral anionic site. The results also indicate that the catalytic active center of AChE does not participate in the interaction of enzyme with tricyclic antidepressants. These studies suggest that the binding site for tricyclic antidepressants is located at the peripheral anionic site of E. electricus (L.) acetylcholinesterase.     

Chemical modification of electric eel acetylcholinesterase by tetranitromethane

The reaction of acetylcholinesterase (acetylcholinehydrolase, EC 3.1.1.7) with tetranitromethane has been studied. The reaction caused a decrease in enzyme activity as measured with the substrate acetylthiocholine under conditions where hydrolysis of the neutral substrate indophenyl acetate was unaltered. The inactivation of acetylcholinesterase by tetranitromethane was greatly accelerated by the quaternary oximes pyridine-2-aldoxime methyl nitrate or toxogonin, though not by other quaternary inhibitors tested and not by an aliphatic oxime. The enhanced inactivation by tetranitromethane in the presence of pyridine-2-aldoxime methyl nitrate was blocked by the enzyme inhibitor decamethonium.The oxime-induced inactivation of acetylcholinesterase by tetranitromethane was accompanied by significant changes in the immunological properties of the enzyme as demonstrated by complement fixation. The reaction also resulted in the disappearance of tyrosine and appearance of nitrotyrosine.     

Effect of salts on Azotobacter vinelandii nitrogenase activities. Inhibition of iron chelation and substrate reduction

The effect of salts on the catalytic activity of the molybdenum-containing nitrogenase complex from Azotobacter vinelandii has been investigated. NaCl was found to inhibit the reduction of the substrates, protons, acetylene, and dinitrogen by a common mechanism. The pattern of inhibition is sigmoidal, indicating a highly cooperative interaction involving multiple inhibitor sites. Sixteen other salts that were investigated also exhibited this pattern of inhibition. NaCl functions as a dead-end inhibitor without altering the number of MgATP hydrolyzed/electron transferred to substrate. The level of expressed inhibition is sensitive to MgATP concentration, the molar ratio of the MoFe-protein (Av1) to the Fe-protein (Av2), and total protein concentration. In addition, NaCl is an inhibitor of the MgATP-dependent, iron chelation of Av2. Although the inhibition is exhibited over the same salt concentration range as that for inhibition of substrate reduction, the pattern of inhibition is hyperbolic. A model based upon simple equilibrium interactions among the enzyme species, nucleotides, and inhibitor has been developed which quantitatively accounts for the observed effects of salt. In this model, the formation of the active complex between Av1 and Av2 is abolished by salts. Likewise, the apparent affinity of Av2 for MgATP is reduced. An additional prediction based upon the model is that the affinity between Av2 and Av1 is independent of nucleotide binding.     

Effects of salts on the function and conformational stability of shikimate kinase

The unfolding of shikimate kinase (SK) from Erwinia chrysanthemi by urea and its subsequent refolding on dilution of the denaturing agent has been studied in detail [Eur. J. Biochem. 269 (2002) 2124]. Comparison of the effects of urea on the enzyme with those of guanidinium chloride (GdmCl) and NaCl indicated that chloride ions significantly weakened the binding of shikimate. This finding prompted a more detailed examination of the effects of salts on the structure, function and stability of the enzyme; the effects of NaCl and Na(2)SO(4) were investigated in detail. These salts have very small effects on the secondary structure as judged by far UV CD circular dichroism (CD), although the near UV CD spectra suggest that some limited changes in the environment of aromatic amino acids may occur. Both salts inhibit SK activity and analysis of the kinetic and substrate binding parameters point to a complex mechanism for the inhibition. Inclusion of salts leads to a marked stabilisation against unfolding of the enzyme by urea. When the enzyme is unfolded by incubation in 4 M urea, addition of NaCl or Na(2)SO(4) leads to a relatively slow refolding of the enzyme as judged by the regain of native-like CD and fluorescence. In addition, the refolded enzyme can bind shikimate, though more weakly than the native enzyme. However, the refolded enzyme does not appear to be capable of binding nucleotides, nor does it possess detectable catalytic activity. The refolding process brought about by addition of salt in the presence of 4 M urea is not associated with any change in the fluorescence of the probe 8-anilino-1-naphthalenesulfonic acid (ANS), indicating that an intermediate formed by hydrophobic collapse is unlikely to be significantly populated. The results point to both specific and general effects of salts on SK. These are discussed in the light of the structural information available on the enzyme.     

Amyloid-cholinesterase interactions. Implications for Alzheimer's disease

Acetylcholinesterase is an enzyme associated with senile plaques. Biochemical studies have indicated that acetylcholinesterase induces amyloid fibril formation by interaction throughout the peripherical anionic site of the enzyme forming highly toxic acetylcholinesterase-amyloid-beta peptide (Abeta) complexes. The pro-aggregating acetylcholinesterase effect is associated with the intrinsic amyloidogenic properties of the corresponding Abeta peptide. The neurotoxicity induced by acetylcholinesterase-Abeta complexes is higher than the that induced by the Abeta peptide alone, both in vitro and in vivo. The fact that acetylcholinesterase accelerates amyloid formation and the effect is sensitive to peripherical anionic site blockers of the enzyme, suggests that specific and new acetylcholinesterase inhibitors may well provide an attractive possibility for treating Alzheimer's disease. Recent studies also indicate that acetylcholinesterase induces the aggregation of prion protein with a similar dependence on the peripherical anionic site.     

Effect of surface-active agents on peroxidase activity. IV. Effect of lipids and fatty acids from milk

The effect of surfactants, lipids and fatty acid salts isolated from cow milk on the activity of heme-containing horseradish peroxidase in solution was studied. As the surfactant concentration increases, the rate of the enzymic reaction successively decreases, increases, and again decreases, down to zero in the case of the fatty acid salts. The initial deceleration of the reaction rate results from the enzyme inhibition. The subsequent increase is caused by an improved accessibility for the substrate and the enhanced activity of the catalytic site of the enzyme due to its immobilization in the surfactant aggregates. A shielding of the protein by these aggregates can explain the secondary deceleration of the enzymic reaction rate. The general character of the dependence is similar and does not depend on the surfactant structure for a series of fatty acid salts and phospholipids; however, it is quite different in the case of cholesterol and sphingomyelin.     

The steady-state kinetics of yeast phosphoglycerate kinase. Anomalous kinetic plots and the effects of salts on activity.

1. A re-investigation of the kinetics of yeast phosphoglycerate kinase in the direction of 1,3-bisphosphoglycerate formation has been carried out, covering a 1000-fold range in substrate concentrations. A variety of improved spectrophotometric and fluorimetric assay procedures have been used. 2. Kinetic plots proved to be non-linear for each variable substrate. A variety of checks have been carried out to show that this is not due to artifacts in the assay procedures or heterogeneity of the enzyme preparation. 3. The effects of a variety of salts on the activity of the enzyme have been examined. Most salts, especially those with multivalent anions, can cause activation of the enzyme, but inhibit at high concentration. 4. The salt effect is shown to be principally due to anions rather than cations, and not to ionic strength changes. Sulphate, as one of the most effective anions has been used in most comparisons. 5. Salt activation is steepest when the substrate concentrations are low; maximum activation has been about 5-fold with 0.2 mM MgATP and 0.2 mM 3-phosphoglycerate. Inhibition at the higher salt concentrations is strongest at the same substrate concentrations as when activation is steepest, indicating a link between the two effects. 6. The presence of 20 mM or more Na2SO4 converted non-linear kinetic plots to linear ones. A study of the kinetics in the presence of 40 mM Na2SO4 was interpreted in terms of a random sequential binding mechanism, with sulphate acting as a competitive inhibitor. 7. Possible explanations for these anomalous results are discussed in terms of several mechanisms which have been shown to apply in other systems.     

Influence of NaCl on the kinetic behaviour of mammalian muscle acetylcholinesterase

Acetylcholinesterase was solubilized from rabbit white muscle by means of dilute buffer and Triton X-100 (0.5%). About 50% of total activity was brought into solution with buffer, the rest being solubilized by extracting the tissue with buffer and Triton X-100. The enzyme activity recovered in the supernatants was 170% of that found in the homogenate in the absence of Triton X-100 indicating that, to some extent, the enzyme could be found in an occluded form in muscle. At suboptimum substrate concentration the Triton-solubilized acetylcholinesterase displayed a negative cooperativity, this phenomenon being greatly modified in the presence of NaCl. As the salt concentration increased (0-400 mM) the enzyme activity decreased, the Km values being linearly-dependent on the NaCl concentration in the assay medium. We propose a kinetic pattern to explain both the negative cooperativity produced by the substrate and the effect of NaCl on the kinetic behaviour on this enzyme. Our data are consistent with the hypothesis of binding of substrate to both the catalytic anionic site and a peripheral anionic site, the salt showing the capacity to compete with the substrate for these two binding sites.     

Evidence for an ordered reaction mechanism for bile salt: 3'phosphoadenosine-5'-phosphosulfate: sulfotransferase from rhesus monkey liver that catalyzes the sulfation of the hepatotoxin glycolithocholate

The in vivo formation of the sulfate ester of glycolithocholate is a critical step in the elimination of this hepatotoxic bile salt. Rhesus monkeys fed chenodeoxycholate or ursodeoxycholate, the precursors of lithocholate, develop frank cirrhosis in association with accumulation of nonsulfated glycolithocholate in bile. An enzyme catalyzing the formation of glycolithocholate-3-sulfate has been isolated from hepatic cytosol of adult female rhesus monkeys and has been purified 146-fold. When reduced it appears as a 30 kD band on an SDS-polyacrylamide gradient gel. It has a pH optimum of 7.0 and is stimulated by low concentrations of Mg2+ (up to 2 mM), but does not have an absolute requirement for this metal ion. The kinetics of this enzyme have been investigated to ascertain whether its reaction mechanism can account for the poor in vivo rate of glycolithocholate sulfation. Inhibitor studies with an oxidized metabolite of lithocholate, 3-keto-5 beta-cholanoate, showed that the latter is a competitive inhibitor of glycolithocholate and is noncompetitive with the active form of sulfate, 3'phosphoadenosine-5'-phosphosulfate. The monophosphonucleotide 3'-AMP is a competitive inhibitor of 3'phosphoadenosine-5'-phosphosulfate, and is noncompetitive with glycolithocholate. These observations are consistent with a sequentially ordered Bi Bi reaction mechanism in which the bile salt is the first substrate to bind to the enzyme. Such a reaction mechanism for bile salt:3'phosphoadenosine-5'-phosphosulfate:sulfotransferase would be, therefore, the first time in which the sulfate acceptor (the bile salt) is the initial substrate to bind to a sulfotransferase. These studies have shown that although rhesus monkeys have a liver enzyme capable of forming the sulfate ester of glycolithocholate, its reaction mechanism and the potent inhibition caused by simple metabolites, such as 3-keto-5 beta-cholanoate, may serve to under-express the activity of the enzyme in vivo.     

Clonidine protection from soman and echothiophate toxicity in mice

The influence of clonidine on the toxicity produced by two irreversible, organophosphate cholinesterase inhibitors, soman and echothiophate, was studied in mice. At lethal doses, soman produced whole body tremor but no muscle fasciculation; at lethal doses, echothiophate produced muscle fasciculations but no whole body tremor. Pretreatment with clonidine protected against several toxic manifestations of soman, but had little effect on echothiophate toxicity. In addition to its documented effects on acetylcholine metabolism, clonidine was found to be a weak inhibitor of acetylcholinesterase. At certain concentrations, clonidine protected the enzyme from permanent inactivation by soman. These findings indicate that the toxicity of soman and echothiophate reflect primarily central and peripheral actions, respectively, and that clonidine has a much greater protective effect versus the centrally-acting agent. Moreover, direct interactions with acetylcholinesterase may contribute to clonidine protection from cholinesterase inhibitor toxicity.     

Effect of surfactants on peroxidase activity: IV. Effect of milk lipids and fatty acid salts

The effect of surfactants, lipids and fatty acid salts isolated from cow milk on the activity of hemecontaining horseradish peroxidase in solution was studied. As the surfactant concentration increases, the rate of the enzymic reaction successively decreases, increases, and again decreases, down to zero in the case of the fatty acid salts. The initial deceleration of the reaction rate results from the enzyme inhibition. The subsequent increase is caused by an improved accessibility for the substrate and the enhanced activity of the catalytic site of the enzyme due to its immobilization in the surfactant aggregates. A shielding of the protein by these aggregates can explain the secondary deceleration of the enzymic reaction rate. The general character of the dependence is similar and does not depend on the surfactant structure for a series of fatty acid salts and phospholipids; however, it is quite different in the case of cholesterol and sphingomyelin. For communication III, see [1].     

Novel allosteric sites on human erythrocyte acetylcholinesterase identified by two monoclonal antibodies.

Monoclonal antibodies against human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase EC 3.1.1.7) have been examined for inhibition of enzyme activity. Of sixteen antibodies analyzed, only one (C1B7) inhibited enzyme activity, indicating selection of an unusual susceptible site. The inhibitory activity of C1B7 was characterized and compared to another inhibitory antibody, AE-2, previously described by Fambrough et al. (Proc. Natl. Acad. Sci. USA 79, 1078, 1982). Maximal demonstrated inhibition was 84% for C1B7 and 72% for AE-2 and antibody inhibition of enzyme activity was equivalent for the reduced and alkylated acetylcholinesterase monomer and the intact dimer. The Ki (stoichiometry of the enzyme-antibody reaction estimated from enzyme kinetics) was 1.0 for C1B7 and 4.8 molecules of antibody per monomer of acetylcholinesterase for AE-2. The antibodies did not compete with one another for binding to acetylcholinesterase, indicating that they have different target epitopes on the enzyme. Antibody binding to the enzyme was not specifically affected by any of the anticholinesterase agents tested: (a) the irreversible esteratic site-directed inhibitor diisopropylfluorophosphate; (b) the reversible active site-directed inhibitors edrophonium, neostigmine, BW284c51, and carbachol; and (c) allosteric site-directed compounds propidium and gallamine. Kinetic analysis of their effects provide evidence that both antibodies decrease the catalytic rate of enzyme activity and have little or no effect on substrate binding.     

Rapid binding of a cationic active site inhibitor to wild type and mutant mouse acetylcholinesterase: Brownian dynamics simulation including diffusion in the active site gorge

It is known that anionic surface residues play a role in the long-range electrostatic attraction between acetylcholinesterase and cationic ligands. In our current investigation, we show that anionic residues also play an important role in the behavior of the ligand within the active site gorge of acetylcholinesterase. Negatively charged residues near the gorge opening not only attract positively charged ligands from solution to the enzyme, but can also restrict the motion of the ligand once it is inside of the gorge. We use Brownian dynamics techniques to calculate the rate constant k_on for wild type and mutant acetylcholinesterase with a positively charged ligand. These calculations are performed by allowing the ligand to diffuse within the active site gorge. This is an extension of previously reported work in which a ligand was allowed to diffuse only to the enzyme surface. By setting the reaction criteria for the ligand closer to the active site, better agreement with experimental data is obtained. Although a number of residues influence the movement of the ligand within the gorge, Asp74 is shown to play a particularly important role in this function. Asp74 traps the ligand within the gorge, and in this way helps to ensure a reaction. © 1998 John Wiley & Sons, Inc. Biopoly 46: 465–474, 1998