Uncompetitive Type Of Inhibition Research Articles

Assessment of Anticholinesterase Activity of Gelidiella acerosa: Implications for Its Therapeutic Potential against Alzheimer's Disease.

The effect of various solvent extracts of Gelidiella acerosa on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities was investigated. AChE and BuChE inhibitory activities were analyzed by spectrophotometric method. Phytochemical screening of the compounds present in the solvent extracts was done qualitatively. Characterization of the compounds present in the benzene extract of G. acerosa was done by GC-MS analysis. The results showed that, at 487.80 μg/mL, benzene extract showed significant (P < 0.05) inhibitory activity against both AChE and BuChE with the percentage of inhibition 54.18 ± 5.65 % (IC50 = 434.61 ± 26.53 μg/mL) and 78.43 ± 0% (IC50 = 163.01 ± 85.35 μg/mL), respectively. The mode of inhibition exhibited by benzene extract against the AChE and BuChE was found to be competitive and uncompetitive type of inhibition, respectively. Preliminary phytochemical analysis coupled with GC-MS illustrates that the benzene extract possesses high amount of terpenoids, which could be the reason for potential cholinesterase inhibitory activity.

Alpha-amylase inhibition kinetics by caulerpenyne

Many algae have important secretions which are generally used for defensive purposes. These secretions take attentions of a lot of researchers who are wondering if these metabolites can be used for medical researches or not. Among these metabolites, caulerpenyne (CYN) which is the main metabolite of Caulerpa species, have had an important place in Caulerpa researches since the results related to its determined properties such as cytotoxic, antiviral, antiproliferative and apoptotic effects have been proven by many scientific reports. In the present study, the inhibitory effect of CYN isolated from C. prolifera on alpha-amylase was investigated. The inhibition experiments were done with CYN by spectrophotometric determination method. In order to evaluate the type of inhibition Lineweaver–Burk plot was produced. The results obtained from enzyme kinetic studies exhibited an un-competitive type of inhibition, which is characterized by the difference of Vmax and KM from those of the free enzyme, of alpha-amylase in the presence of CYN. The present study showed that Caulerpa species can be a potential target for producing diabetic drugs in the light of the results obtained for CYN.

Ethidium bromide inhibits rat brain acetylcholinesterase activity in vitro

Ethidium bromide (EtBr), a fluorescent dark red compound and stain for double-stranded DNA and RNA was used to study acetylcholinesterase (AChE) activity in vitro together with kinetic parameters of this enzyme in the striatum (ST), hippocampus (HP), cerebral cortex (CC) and cerebellum (CB) of adult rats. AChE activity in vitro in the ST, HP, CC and CB was significantly reduced ( p < 0.05) in the presence of EtBr at concentrations of 0.00625, 0.0125, 0.025, 0.05 and 0.1 mM. For the analysis of the kinetic three concentrations of EtBr were tested (0.00625, 0.025 and 0.1 mM). An uncompetitive inhibition type was observed in the ST, HP and CC, whereas in the CB the inhibition type was mixed. These data indicate that EtBr should be considered a strong inhibitor of AChE activity demonstrating that there is an interaction between this compound and the cholinergic system.

Plasmodium falciparum glutathione S‐transferase—Structural and mechanistic studies on ligand binding and enzyme inhibition

Glutathione S-transferase of the malarial parasite Plasmodium falciparum (PfGST) represents a novel class of GST isoenzymes. Since the architecture of the PfGST substrate binding site differs significantly from its human counterparts and there is only this one isoenzyme present in the parasite, PfGST is considered a highly attractive target for antimalarial drug development. Here we report the mechanistic, kinetic, and structural characterization of PfGST as well as its interaction with different ligands. Our data indicate that in solution PfGST is present as a tetramer that dissociates into dimers in the presence of glutathione (GSH). Fluorescence spectroscopy shows that in the presence of GSH GST serves as ligandin for parasitotoxic ferriprotoporphyrin IX with a high- and a low-affinity binding site. This is supported by a clear uncompetitive inhibition type. Site-directed mutagenesis studies demonstrate that neither Cys 86 nor Cys 101 contribute to the peroxidase activity of the enzyme, which is thus performed GSH-dependently at the active site. Tyr 9 is responsible for the deprotonation of GSH and Lys 15, but also Gln 71 are involved in GSH binding. We furthermore report the 2.4 A resolution X-ray structure of PfGST cocrystallized with the inhibitor S-hexylglutathione. In comparison with a previously reported structure obtained by crystal soaking, differences occur at the C-terminal end of helix alpha4 and at the S-hexylmoiety of the inhibitor. We furthermore show that, in contrast to previous reports, the antimalarial drug artemisinin is not metabolized by PfGST.

Picolinic Acids as Inhibitors of Dopamine β‐Monooxygenase: QSAR and Putative Binding Site

Dopamine beta-monooxygenase (DBM, EC 1.14.17.1) catalyzes the oxidation of dopamine into (R)-noradrenaline. DBM inhibitors may act as antihypertensive drugs. A series of 22 picolinic acids substituted in 4- and 5-position was previously synthesized and tested for inhibition of DBM from bovine adrenal medulla. The QSAR of these compounds were investigated by Hansch analysis and comparative molecular field analysis (CoMFA). The correlation of pI(50) values with electronic (nucleophilic substituent constant sigma(p) (-), oxygen net charges and highest occupied molecular orbital energy calculated by AMPAC-AM1), hydrophobic (pi values of R(4)) and steric descriptors (molar refraction and Sterimol parameters of R(5)) indicated that a more negatively charged carboxylate moiety, more lipophilic R(4) groups as well as wider bulk and higher molar refraction of 5-substituents increase DBM inhibition. The CoMFA approach generally reproduced these QSAR in terms of steric and electrostatic field variables, the latter restricted to the carboxylate area. To predict a putative binding site, dopamine and fusaric acid were docked into a partial homology model of DBM derived from a crystal structure of peptidylglycine alpha-hydroxylating monooxygenase (EC 1.14.17.3). The inhibitor is suggested to interact by its carboxylate group with the copper site CuB and the protonated amino group of dopamine according to the uncompetitive type of inhibition. R(4) points to a tyrosine side chain. R(5) protrudes into the fringe of the catalytic crevice. It may "freeze" to the solvated surface of polar amino acids and additionally contact an isoleucine residue. Taken together, the model explains the QSAR results by corresponding types of interaction.

5-Substituted Tetrazoles as Bioisosteres of Carboxylic Acids. Bioisosterism and Mechanistic Studies on Glutathione Reductase Inhibitors as Antimalarials

Plasmodium parasites are exposed to elevated fluxes of reactive oxygen species during intraerythrocytic life. The most important antioxidative systems are based on the glutathione reductases of the malarial parasite Plasmodium falciparum and the host erythrocyte. The development of menadione chemistry has led to the selection of the carboxylic acid 6-[2'-(3'-methyl)-1',4'-naphthoquinolyl] hexanoic acid M(5) as an inhibitor of the parasitic enzyme. As reported here, revisiting the mechanism of M(5) action revealed an uncompetitive inhibition type with respect to both NADPH and glutathione disulfide. Masking the polarity of the acidic function of M(5) by ester or amide bonds improved antiplasmodial activity. Bioisosteric replacement of the carboxylic function by tetrazole to increase bioavailability and to maintain comparable acidity led to improved antimalarial properties as well, but only with the cyanoethyl-protected tetrazoles. Using computed ab initio quantum methods, detailed analyses of the electronic profiles and the molecular properties evidenced the similarity of M(5) and the bioisoteric tetrazole T(4). The potential binding site of these molecules is discussed in light of the recently solved crystallographic structure of P. falciparum enzyme.

Mushroom Tyrosinase Inhibition by Two Potent Uncompetitive Inhibitors

Two new bi-pyridine compounds, [1,4'] Bipiperidinyl-1'-yl-naphthan-2-yl-methanone (I) and [1,4'] Bipiperidinyl-1'-yl-4-methylphenyl-methane (II) were synthesized and examined for inhibition of the catecholase activity of mushroom tyrosinase in 10 mM phosphate buffer pH 6.8, at 293 K using UV spectrophotometry. Inhibition kinetics indicated that they were uncompetitive inhibitors and the value of the inhibition constants were 5.87 and 1.31 μM for I and II, respectively, which showed high potency. Fluorescent studies confirmed the uncompetitive type of inhibition for these two inhibitors. The inhibition mechanism presumably comes from the presence of a particular hydrophobe site which can accommodate these inhibitors. This site could be formed due to a probable conformational change that was induced by binding of substrate with the enzyme.

Inhibition of HIV-1 reverse transcriptase and protease by phlorotannins from the brown alga Ecklonia cava.

The bioassay-directed isolation of a marine brown alga, Ecklonia cava, afforded four phlorotannin derivatives, eckol (1), 8,8'-bieckol (2), 8,4"'-dieckol (3), and phlorofucofuroeckol A (4). Among these compounds, 2 and 3 exhibited an inhibitory effect on human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease. Specifically, they inhibited the RT more potently than the protease. The inhibitory activity of compound 2 (IC(50), 0.51 microM) against HIV-1 RT was comparable to that of nevirapine (IC(50), 0.28 microM), a reference compound. An enzyme kinetic assay showed that this compound inhibited the RNA-dependent DNA synthesis activity of HIV-1 RT noncompetitively against dUTP/dTTP with a K(i) value of 0.78 microM. With respect to the homopolymeric template/primer, (rA)n(dT)15, 8,8'-bieckol (2) displayed an uncompetitive type of inhibition (K(i), 0.23 microM).

Inhibition of Aromatic Amino Acid Decarboxylase by a Group of New Potential Nonsteroidal Anti-Inflammatory Drugs with Antileukotrienic Effects

Possible in vitro inhibition of aromatic amino acid decarboxylase (AAD, EC 4.1.1.28) by a group of phenylsulfonylbenzoic acid derivatives VUFB 19363, 19369, 19370, 19371, and 19760 as new potential anti-inflammatory compounds was studied using the substrate L-tyrosine. Enzyme inhibition by 2.7 x 10(-5) M concentration of compound VUFB 19363 (Quinlukast) was 17%, AAD inhibition at 3.75 x 10(-5) M concentration of compounds VUFB 19369, 19370, 19371, and 19760 ranged between 9-23%. There were distinct differences between individual compounds. Evaluation of inhibition kinetics suggested full reversibility with VUFB 19369 and the uncompetitive type of inhibition in the case of compound VUFB 19363. Considering the anti-inflammatory activity of the compounds studied, the weak AAD inhibition found is rather favourable for their prospective pharmacological effect.

Hypochlorous acid-induced oxidative damage of human red blood cells: effects of tert-butyl hydroperoxide and nitrite on the HOCl reaction with erythrocytes

Hypochlorous acid, one of the most powerful biological oxidants, is believed to be important in the pathogenesis of some diseases. The purpose of this study was to further characterise the membrane and intracellular events which resulted in HOCl-induced oxidative impairments and haemolysis of human erythrocytes and interaction of different oxidative agents, which accumulated during respiratory burst, in the process of RBS oxidation. The sequence of cellular events after red blood cell exposure to HOCl: cell morphological transformations, oxidation of cellular constituents, enzyme modifications, and haemolysis have been evaluated. It was shown that HOCl-treated cells underwent colloid-osmotic haemolysis, preceded by rapid morphological transformations and membrane structural transitions. The activation energy of the process of haemolysis (after removal of the excess of oxidative agent) was estimated to be 146±22 kJ/mol at temperatures above the break point of Arrhenius plot (31–32 °C). This value corresponds to the activation energy of the process of protein denaturation. Modification of erythrocytes by HOCl inhibited membrane acetylcholinesterase (uncompetitive type of inhibition), depleted intracellular glutathione, activated intracellular glutathione peroxidase, but did not induce membrane lipid peroxidation. The presence of other oxidants, nitrite or tert-butyl hydroperoxide ( t-BHP), promoted the oxidative damage induced by HOCl and led to new oxidative reactions.

Inhibition of UDP-glucuronosyltransferases in rat liver microsomes by natural mutagens and carcinogens.

Eight toxic compounds of natural origin present in the human diet or used as drugs were tested as inhibitors of UDP-glucuronosyltransferase (UGT) activity in rat liver microsomes with 1-naphthol (1-NA), phenolphthalein (PPh) and 4-nitrophenol (4-NP) as substrates. Strong inhibitory effects were observed with tannic acid (tannin) and the antifungal drug griseofulvin (GF): at a concentration of 1 mM, the two compounds completely suppressed the glucuronidation of 1-NA and PPh, respectively. A concentration of 0.1 mM still proved to be highly inhibitory, and even at a concentration as low as 50 microM, tannin produced nearly 50% inhibition of 1-NA conjugation. The UGT isoforms converting 4-NP were less sensitive to the tested compounds (with the exception of GF). Kinetic studies with tannin revealed an uncompetitive type of inhibition toward 1-NA, with an apparent Ki value of 20 microM. The inhibition by GF was non-competitive with respect to PPh and was of a mixed type toward UDP-glucuronic acid, with apparent Ki values of 40 microM and 30 microM, respectively. Tannin and GF did not act as substrates for rat microsomal UGT.

Inhibition of tyrosinase activity by a polyphenol esterase using selected phenolic substrates

The inhibition of tyrosinase activity by polyphenol esterase (PPE) fractions, recovered from the biomass of Aspergillus niger, was investigated using a wide range of mono- and di-phenols as substrates. An inhibitory effect on tyrosinase activity was exhibited by the partially purified PPE fraction FI with 3,4-dihydroxyphenylacetic acid (DHPAA), l-3,4-dihydroxyphenylalanine ( l-DOPA), 4-methylcatechol, catechol and caffeic acid as substrates. No inhibitory effect on tyrosinase activity was detected using fraction FI with 4-hydroxyphenylpyruvic acid and m- and p-cresol, measured spectrophotometrically. The purification of fraction FI by ion-exchange chromatography resulted in a more purified one (FII), which markedly increased the inactivation of tyrosinase activity using all substrates indicated above. Purified PPE exhibited, using spectrophotometric analysis, an uncompetitive type of inhibition on tyrosinase activity with caffeic acid and 4-methylcatechol as substrates and a mixed one with DHPAA, catechol, l-DOPA, 4-hydroxyphenylpyruvic acid and m- and p-cresol. The results also indicated that, using the polarographic method, fraction FII reduced the rate of oxygen consumption with diphenols as substrates but not with monophenols.

Effect ofs-triazine and phenoxyalkanoic acid herbicides on UDP-glucuronosyltransferase in rat liver microsomes

Twelve herbicides, representatives of two chemical groups, substituted phenoxyalkanoic acids and s-triazines, were tested for their inhibitory effect on the glucuronidation of 4-nitrophenol (4-NP), phenolphthalein (PPh) and 4-methylumbelliferone (4-MU) by rat liver microsomes. One millimole MCPA, ametryn and cyanazine significantly decreased PPh UDP-glucuronosyltransferase (UGT) activity, while propazine was found to be a most potent inhibitor of 4-NP glucuronidation. Concentrations of 0.1 mM dichlorprop and cyanazine were still inhibitory against PPh-UGT. The inhibition of 4-MU glucuronidation by the herbicides was low and not specific. As a whole, s-triazine derivatives were more inhibitory than the substituted phenoxyalkanoic acids. Kinetic studies with propazine revealed a non-competitive type of inhibition towards the acceptor substrate 4-NP, with an apparent Ki value of 0.540 mM. With ametryn, an uncompetitive type of inhibition against PPh and a mixed type of inhibition towards UDPGA were found, with apparent Ki values of 0.330 mM and 0.380 mM, respectively. © 1999 Society of Chemical Industry

Effects of lead and selected pyrethroids/piperonyl butoxide alone and in combination on Na, K‐ATPase

In vitro effects of Pb2+, the pyrethroid insecticides cypermethrin, fenvalerate and the syner‐gist piperonyl butoxide on sodium‐potassium‐activated adenosine triphosphatase (Na,K‐ATPase) from dog kidney were determined. Pb2+ with an estimated IC50 value of 5.2 μM was found to be a potent inhibitor of Na,K‐ATPase activity, whereas Na,K‐ATPase was less sensitive to the pyrethroids tested and piperonyl butoxide. Investigation with circular dichroism (CD) spec‐troscopy showed that inhibition occurs through conformational changes of the α‐subunit of the enzyme. The kinetic characteristics of inhibition of Na,K‐ATPase with varying substrate (ATP) concentrations as well as with varying Na+ concentrations exhibited a competitive type of inhibition with Pb2+ in the μM range. With Pb2+ alone in the enzyme assay no conformational changes of the protein could be observed which confirmed the assumption that Pb2+ can bind to the Na+ binding site of the α‐subunit. Uncompetitive type of inhibition occurred with varied K+ concentrations demonstrating that this cation binding site is not affected directly by Pb2+. Complete reversal of Pb2+ by DTT confirms that a possible target for interaction of this heavy metal ion with Na, K‐ATPase are specific SH groups. Synergistic effects could only be determined with higher Pb2+ concentrations of 3, 5 and 7 μM plus piperonyl butoxide while all other combinations with this heavy metal plus organic substances where of the additive type. With CD spectroscopy also only additive effects were observed. These results demonstrate that higher concentrations of piperonyl butoxide favor the binding of Pb2+ to the Na+ binding site by conformational changes of the protein.

Effect of propranolol on rat brain synaptosomal Na +K +-ATPase, Mg 2+-ATPase and Ca 2+-ATPase

The beta blocker drug propranolol (PPL) significantly inhibited Na +K +-ATPase, Mg 2+ -ATPase and Ca 2+-ATPase activities in a concentration dependent manner in rat brain synaptosomes. The concentrations required for 50% inhibition (IC 50) in the activity of these enzymes were 1.5–1.8 mM. The double-reciprocal plot of ATP-stimulated Na +K +-ATPase activity in the presence of PPL showed apparent decrease in K m and V max and the inhibition was of an uncompetitive type with respect to ATP. The nature of inhibition by PPL of Na +-activated Na +K +-ATPase activity was of a mixed type showing an increase in K m and decrease in V max. Potassium activation kinetics of Na +K +-ATPase displayed uncompetitive type of inhibition with PPL since K m and V max were decreased. Magnesium activation of Mg 2+-ATPase showed decrease in V max with no apparent change in K m in the presence of PPL. The drug inhibited synaptosomal Ca 2+-ATPase in an uncompetitive manner. The observed inhibition of synaptosomal ATPases indicates possible alterations in the synaptic transmission by the beta blocker drug PPL.

Evaluation of four types of inhibition for modelling the influence of carbon dioxide on oxygen consumption of fruits and vegetables

Abstract High carbon dioxide (CO 2 ) concentrations can reduce the oxygen (O 2 ) consumption rate of a number of fruits and vegetables. This reduction can be modelled by four types of inhibition in an enzyme kinetics model: (1) the competitive type; (2) the uncompetitive type; (3) a combination of both previous types; and (4) the non-competitive type. These different types of inhibition were tested for describing the CO 2 influence on O 2 consumption using experimental data supplemented with data from literature. The gas exchange rates of apples (cv. Golden Delicious and Elstar), asparagus, broccoli, mungbean sprouts and cut chicory were measured under a wide range of O 2 and CO 2 concentrations. With the range of CO 2 concentrations used, no influence was found on gas exchange rates of apples. There was a clear influence of high CO 2 on the gas exchange rates of the other produce. A good estimation of C 2 consumption could be obtained with the inhibition models. This supports the use of Michaelis-Menten kinetics for modelling O 2 consumption. Depending on the product the statistical analysis gave good results for the competitive and the uncompetitive type of inhibition. Based on gas exchange data only, no distinction between the competitive and uncompetitive type of inhibition could be made. It suggests the simultaneous existence of both types of inhibition. The idea is that the combined inhibition equation is most closely related to what is actually occurring in plant tissues. However, for reasons of simplicity the non-competitive type of inhibition is preferred, showing similar results to the combined inhibition, and giving good results for all the products tested.

Kinetics of papaya pectinesterase

Purified papaya ( Carica papaya L. var. exotica) pectinesterase (EC 3.1.1.11) was investigated for its activity as a function of NaCl, pH and temperature, and determination of its kinetic parameters. The activity was linear up to 20 min with an enzyme concentration of up to 6.14 μg. Optimum activity was obtained with 0.25 m NaCl concentration and the optimum pH was found to be 8. The energy of activation of enzyme was 5690 cal mol −1. A Q 10 of 1.29 was observed in the temperature range of 30–50 °C and the optimum temperature for the enzyme activity was 65 °C. The K m value for citrus pectin was 0.11 mg/ml, corresponding to a V max value of 730 μmole/min/mg protein. The turnover number was calculated as 23 360 mole/(mole.min). Enzyme activity was found to be inhibited by the addition of polygalacturonic acid, alginic acid and sucrose in the reaction mixture and their K i values were calculated as 0.019 mg/ml, 0.17 mg/ml and 29%, respectively. Polygalacturonic acid was found to act as a competitive inhibitor whereas alginic acid and sucrose showed a competitive-non-competitive and uncompetitive type of inhibition, respectively.

Competition of bile acids on the sulfobromophthalein uptake in basolateral rat liver plasma membrane vesicles

The effect of different bile acids (BA) on the hepatic uptake of sulfobromophthalein (BSP) was investigated in liver plasma membrane vesicles enriched in basolateral fraction. BSP uptake was measured either in the absence (electroneutral component) or in the presence of a membrane potential (electrogenic component) induced by the addition of valinomycin in the presence of an inwardlydirected potassium gradient. BSP uptake was also measured in the presence of different BA [cholate (C), taurocholate (TC), ursodeoxycholate (UDC) and tauroursodeoxycholate (TUDC)]. Electrogenic BSP uptake was not affected by BA. Conversely, the electroneutral portion of the BSP uptake was inhibited with an inhibition constant ( K i , μM) of 230 ± 40 for C, 103 ± 33 for TC, 99 ± 34 for UDC and 120 ± 39 for TUDC, respectively (means ± SD, N = 4). The Dixon and Cornish-Bowden plot of the data revealed an uncompetitive type of inhibition for each BA. These data indicate that the electroneutral, but not the electrogenic, BSP transport system is modulated by BA.

Analysis of free fatty acid effect on methotrexate binding to albumin

The binding of methotrexate to human serum albumin and the inhibitory effect of serum free fatty acids (FFA) have been studied by equilibrium dialysis with radiolabeled methotrexate. Methotrexate was bound to albumin via a single site (1.03 ± 0.02) with a low affinity (1350 ± 60 M −1). The effect of FFA on binding by albumin of methotrexate was analysed according to the classical inhibition models with computation of the free inhibitor concentration and was ascribed to an uncompetitive type of inhibition. These results were in agreement with the observed serum binding of methotrexate (45–50%) and allowed the simulation of the effect of various concentrations of FFA on methotrexate albumin binding in human serum.

Inhibition of anthocyanin synthesis and phenylalanine ammonialyase activity by Co2+ in leaf disks of Terminalia catappa

Treatment with sucrose induced anthocyanin synthesis and phenylalanine ammonialyase (PAL, EC 4. 3. 1. 5) activity in leaf disks of Indian almond (Terminalia catappa L. Duthie). Co2+, an inhibitor of ethylene biosynthesis, inhibited anthocyanin synthesis and PAL activity when given together with sucrose. Ethephon (an exogenous source of ethylene) given along with sucrose, promoted anthocyanin synthesis and PAL activity, but in the presence of Co2+ its effectiveness decreased. In an attempt to understand the inhibitory action of Co2+ in the presence of ethephon, the effect of Co2+ on PAL activity was studied in vitro. A kinetic study showed an uncompetitive type of inhibition of PAL by Co2+, which was not time dependent. Addition of 2‐mercaptoethanol, cysteine or glutathione overcame the in vitro effect of Co2+, and 2‐mercaptoethanol also restored the activity of PAL extracted from Co2+‐treated leaf disks. It is suggested that sulfhydryl group(s) might be involved in the inactivation of PAL by Co2+. The effects of N‐ethylmaleimide (NEM) and HgCl2 (other sulfhydryl reagents) were also studied. Both NEM and Hg2+ competitively inhibited PAL activity in vitro, and the inhibition could be reversed by sulfhydryl compounds.