Pea alcohol dehydrogenase: Substrate specificity and binding of coenzyme to enzyme

1979 ◽  
Vol 44 (2) ◽  
pp. 631-636 ◽  
Author(s):  
Marie Stiborová ◽  
Roman Lapka ◽  
Noemi Nováková ◽  
Sylva Leblová
Keyword(s):  
Alcohol Dehydrogenase ◽  
Substrate Specificity ◽  
Competitive Inhibitor ◽  
Substrate Oxidation ◽  
Enzyme Protein ◽  
Ph Optimum ◽  
Substrate Reduction ◽  
Noncompetitive Inhibitor ◽  
Ph Dependent ◽  
Broad Specificity

Pea alcohol dehydrogenase (EC 1.1.1.1) shows a broad specificity with respect to aldehydes and alcohols. The pH-optimum of substrate oxidation is 8.7 and of substrate reduction 7.0. The enzyme is inhibited by ATP, adenosine, and adenine. The inhibition is competitive with respect to NAD. The inhibition by ATP is pH-dependent. The competitive character of the inhibition by adenine and its derivatives with respect to NAD indicates the importance of the adenine moiety of the coenzyme for its binding to the enzyme. Phenanthroline is a competitive inhibitor with respect to NAD, a mixed inhibitor with respect to ethanol and a noncompetitive inhibitor with respect to acetaldehyde. Experiments carried out simultaneously with ATP and phenanthroline show that the adenine moiety of NAD does not bind via the zinc atom to the enzyme protein.

Characterization of alcohol dehydrogenase isolated from germinating bean Vicia faba seeds

1989 ◽  
Vol 54 (9) ◽  
pp. 2519-2527 ◽  
Author(s):  
Sylva Leblová ◽  
Mustafa El Ahmad
Keyword(s):  
Amino Acids ◽  
Alcohol Dehydrogenase ◽  
Pyridoxal Phosphate ◽  
Deae Cellulose ◽  
Kinetic Studies ◽  
Carbon Chain ◽  
Substrate Oxidation ◽  
Gel Chromatography ◽  
Ph Optimum ◽  
Substrate Reduction

We have isolated alcohol dehydrogenase (ADH, E.C. 1.1.1.1) from beans germinating 3 days by ammonium sulfate precipitation of the sodium phosphate extract of the homogenate of seeds, followed by chromatography on DEAE-cellulose and gel chromatography on Sephadex G-200 and G-100; both chromatographic operations were repeated twice. The activity of ADH increased 960 times after these procedures. The enzyme whose Mr is 60 000 consists of two identical subunits of Mr = 30 000. Allyl alcohol is oxidized and acetaldehyde reduced at the highest rate of all the alcohols and aldehydes tested. The reaction rate decreases with the increasing length of the carbon chain of the substrate. In contrast, the rate of oxidation of alcohols with a double bond in their molecules increased. The pH-optimum of substrate oxidation (pH 8.5) is different from the pH-optimum of substrate reduction (pH 7.5). From kinetic studies of the effect of pH on Vmax and Km the pK-value of amino acids participating on substrate oxidation is 9.2 and 8.5 whereas amino acids with pK 8.3 and 6.8 participate on substrate reduction. We have verified the participation of the SH-groups in experiments with the inactivation of ADH by iodoacetate: the inactivation was weaker after the enzyme had been preincubated with NAD or AMP, adenosine or nicotinamide. Likewise pyridoxal phosphate inactivates bean ADH by modifying its ε-amino group of lysine. The degree of inactivation depends also on the pH and the ionic strength of the medium. The protective effect of NAD or its analogs shows that lysine is present in the active center of the enzyme in the coenzyme-binding site.

Phosphoenolpyruvate Carboxylase of Thiobacillus thiooxidans. Kinetic and Metabolic Control Properties

1972 ◽  
Vol 50 (2) ◽  
pp. 158-165 ◽  
Author(s):  
R. L. Howden ◽  
H. Lees ◽  
Isamu Suzuki
Keyword(s):  
Enzyme Activity ◽  
Competitive Inhibitor ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Pep Carboxylase ◽  
Thiobacillus Thiooxidans ◽  
Powerful Activator ◽  
Michaelis Constants ◽  
Noncompetitive Inhibitor ◽  
Decreasing Ph

Phosphoenolpyruvate (PEP) carboxylase (orthophosphate:oxalacetate carboxy-lyase (phosphorylating), EC 4.1.1.31) was purified 19-fold from Thiobacillus thiooxidans. The level of enzyme activity was dependent on culture age. No enzyme activity could be obtained from frozen cells.The pH optimum of the enzyme was determined to be around 8.0. Apparent Michaelis constants were determined for the substrates:phosphoenolpyruvate (1.4, 1.5 mM), bicarbonate (0.4, 1.1 mM), and magnesium (1.1, 0.8 mM) at pH 7.0 and 8.0, respectively. Acetyl-CoA was found to be a powerful activator of this enzyme, with the degree of activation increasing with decreasing pH. The concentration of acetyl-CoA to obtain half-maximal activation, however, remained fairly constant and low, namely 1.2 and 1.0 μM at pH 7.0 and 8.0, respectively. L-Aspartate and L-malate were strong inhibitors of enzyme activity. In the presence of aspartate at pH 7.0 the double reciprocal activity plots for PEP became nonlinear, a characteristic of negative cooperativity. These plots became linear with the addition of acetyl-CoA with aspartate now acting as a noncompetitive inhibitor with respect to PEP. At pH 8.0, the same plots were linear with aspartate acting as a competitive inhibitor of PEP. All the other effectors of PEP carboxylase from Salmonella typhimurium and Escherichia coli were found to be ineffective towards the enzyme from T. thiooxidans.

Iodoacetate inactivation of rape alcohol dehydrogenase

1980 ◽  
Vol 45 (5) ◽  
pp. 1601-1607 ◽  
Author(s):  
Marie Stiborová ◽  
Sylva Leblová
Keyword(s):  
Alcohol Dehydrogenase ◽  
Protein Molecule ◽  
Sulfhydryl Groups ◽  
Inactivation Rate ◽  
First Order ◽  
Ph Dependent ◽  
Kinetics Of

Iodoacetate inactivates rape alcohol dehydrogenase (ADH, EC 1.1.1.1). The inactivation rate follows the kinetics of the first order, is pH-dependent, and decreases below pH 7.5. Besides irreversible alkylation of the sulfhydryl groups of the enzyme iodoacetate also forms a reversible complex with rape ADH. The coenzyme (NAD) and its analogs (ATP, ADP, AMP) competitively protect the enzyme against alkylation; o-phenanthroline also protects the enzyme against alkylation yet noncompetitively with respect to iodoacetate. Imidazole and o-phenanthroline compete with one another for binding to the protein molecule of rape ADH. Whereas o-phenanthroline decreases the inactivation rate imidazole increases the rate of iodoacetate inactivation.

scholarly journals Inhibition of Butyrylcholinesterase and Human Monoamine Oxidase-B by the Coumarin Glycyrol and Liquiritigenin Isolated from Glycyrrhiza uralensis

Molecules ◽  
2020 ◽  
Vol 25 (17) ◽  
pp. 3896
Author(s):  
Geum Seok Jeong ◽  
Myung-Gyun Kang ◽  
Joon Yeop Lee ◽  
Sang Ryong Lee ◽  
Daeui Park ◽  
...  
Keyword(s):  
Monoamine Oxidase ◽  
Binding Affinity ◽  
Competitive Inhibitor ◽  
Glycyrrhiza Uralensis ◽  
Form Hydrogen Bond ◽  
Docking Simulations ◽  
Mao B ◽  
Mao A ◽  
Ic50 Values ◽  
Noncompetitive Inhibitor

Eight compounds were isolated from the roots of Glycyrrhiza uralensis and tested for cholinesterase (ChE) and monoamine oxidase (MAO) inhibitory activities. The coumarin glycyrol (GC) effectively inhibited butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) with IC50 values of 7.22 and 14.77 µM, respectively, and also moderately inhibited MAO-B (29.48 µM). Six of the other seven compounds only weakly inhibited AChE and BChE, whereas liquiritin apioside moderately inhibited AChE (IC50 = 36.68 µM). Liquiritigenin (LG) potently inhibited MAO-B (IC50 = 0.098 µM) and MAO-A (IC50 = 0.27 µM), and liquiritin, a glycoside of LG, weakly inhibited MAO-B (>40 µM). GC was a reversible, noncompetitive inhibitor of BChE with a Ki value of 4.47 µM, and LG was a reversible competitive inhibitor of MAO-B with a Ki value of 0.024 µM. Docking simulations showed that the binding affinity of GC for BChE (−7.8 kcal/mol) was greater than its affinity for AChE (−7.1 kcal/mol), and suggested that GC interacted with BChE at Thr284 and Val288 by hydrogen bonds (distances: 2.42 and 1.92 Å, respectively) beyond the ligand binding site of BChE, but that GC did not form hydrogen bond with AChE. The binding affinity of LG for MAO-B (−8.8 kcal/mol) was greater than its affinity for MAO-A (−7.9 kcal/mol). These findings suggest GC and LG should be considered promising compounds for the treatment of Alzheimer’s disease with multi-targeting activities.

Investigation of Structural Determinants for the Substrate Specificity in the Zinc-Dependent Alcohol Dehydrogenase CPCR2 fromCandida parapsilosis

ChemBioChem ◽  
2015 ◽  
Vol 16 (10) ◽  
pp. 1512-1519 ◽  
Author(s):  
Christoph Loderer ◽  
Gaurao V. Dhoke ◽  
Mehdi D. Davari ◽  
Wolfgang Kroutil ◽  
Ulrich Schwaneberg ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Substrate Specificity ◽  
Structural Determinants

Wedelolactone protects against cisplatin-induced nephrotoxicity in mice via inhibition of organic cation transporter 2

2021 ◽  
pp. 096032712110479
Author(s):  
Guangju Wang ◽  
Yajuan Bi ◽  
Hui Xiong ◽  
Tongwei Bo ◽  
Lifeng Han ◽  
...  
Keyword(s):  
Natural Compound ◽  
Organic Cation ◽  
Kidney Injury ◽  
Organic Cation Transporter ◽  
Competitive Inhibitor ◽  
Hek293 Cells ◽  
Cytotoxicity Studies ◽  
Organic Cation Transporter 2 ◽  
Uptake Assay ◽  
Noncompetitive Inhibitor

The balance of cisplatin uptake and efflux, mediated mainly by organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 (MATE1), respectively, determines the renal accumulation and nephrotoxicity of cisplatin. Using transporter-mediated cellular uptake assay, we identified wedelolactone (WEL), a medicinal plant-derived natural compound, is a competitive inhibitor of OCT2 and a noncompetitive inhibitor of MATE1. Wedelolactone showed a selectivity to inhibit OCT2 rather than MATE1. Cytotoxicity studies revealed that wedelolactone alleviated cisplatin-induced cytotoxicity in OCT2-overexpressing HEK293 cells, whereas it did not alter the cytotoxicity of cisplatin in various cancer cell lines. Additionally, wedelolactone altered cisplatin pharmacokinetics, reduced kidney accumulation of cisplatin, and ameliorated cisplatin-induced acute kidney injury in the Institute of Cancer Research mice. In conclusion, these findings suggest a translational potential of WEL as a natural therapy for preventing cisplatin-induced nephrotoxicity and highlight the need for drug–drug interaction investigations of WEL with other treatments which are substrates of OCT2 and/or MATE1.

Carboxymethylhydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate catabolic pathway of Pseudomonas putida

1986 ◽  
Vol 64 (12) ◽  
pp. 1288-1293 ◽  
Author(s):  
Josefa M. Alonso ◽  
Amando Garrido-Pertierra
Keyword(s):  
Pseudomonas Putida ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Competitive Inhibitor ◽  
Catabolic Pathway ◽  
Ph Optimum ◽  
Semialdehyde Dehydrogenase ◽  
Standard Assay ◽  
Assay Conditions

5-Carboxymethyl-2-hydroxymuconic semialdehyde (CHMSA) dehydrogenase in the 4-hydroxyphenylacetate meta-cleavage pathway was purified from Pseudomonas putida by gel filtration, anion-exchange, and affinity chromatographies. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis analysis suggested an approximate tetrameric molecular weight of 200 000. The purified enzyme showed a pH optimum at 7.8. The temperature–activity relationship for the enzyme from 27 to 45 °C showed broken Arrhenius plots with an inflexion at 36–37 °C. Under standard assay conditions, the enzyme acted preferentially with NAD. It could also catalyze the reduction with NADP (which had a higher Km), at 18% of the rate observed for NAD. The following kinetic parameters were found: Km(NAD) = 20.0 ± 3.6 μM, Km(CHMSA) = 8.5 ± 1.8 μM, and Kd(enzyme–NAD complex) = 7.8 ± 2.0 μM. The product NADH acted as a competitive inhibitor against NAD.

scholarly journals Identification of glyA (Encoding Serine Hydroxymethyltransferase) and Its Use Together with the Exporter ThrE To Increase l-Threonine Accumulation by Corynebacterium glutamicum

2002 ◽  
Vol 68 (7) ◽  
pp. 3321-3327 ◽  
Author(s):  
Petra Simic ◽  
Juliane Willuhn ◽  
Hermann Sahm ◽  
Lothar Eggeling
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Corynebacterium Glutamicum ◽  
Serine Hydroxymethyltransferase ◽  
Acid Formation ◽  
Initial Activity ◽  
Intracellular Degradation ◽  
Cleavage Activity ◽  
Substrate Reduction

ABSTRACT l-Threonine can be made by the amino acid-producing bacterium Corynebacterium glutamicum. However, in the course of this process, some of the l-threonine is degraded to glycine. We detected an aldole cleavage activity of l-threonine in crude extracts with an activity of 2.2 nmol min−1 (mg of protein)−1. In order to discover the molecular reason for this activity, we cloned glyA, encoding serine hydroxymethyltransferase (SHMT). By using affinity-tagged glyA, SHMT was isolated and its substrate specificity was determined. The aldole cleavage activity of purified SHMT with l-threonine as the substrate was 1.3 μmol min−1 (mg of protein)−1, which was 4% of that with l-serine as substrate. Reduction of SHMT activity in vivo was obtained by placing the essential glyA gene in the chromosome under the control of P tac , making glyA expression isopropylthiogalactopyranoside dependent. In this way, the SHMT activity in an l-threonine producer was reduced to 8% of the initial activity, which led to a 41% reduction in glycine, while l-threonine was simultaneously increased by 49%. The intracellular availability of l-threonine to aldole cleavage was also reduced by overexpressing the l-threonine exporter thrE. In C. glutamicum DR-17, which overexpresses thrE, accumulation of 67 mM instead of 49 mM l-threonine was obtained. This shows that the potential for amino acid formation can be considerably improved by reducing its intracellular degradation and increasing its export.

scholarly journals Hydrolysis of a naturally occurring β-glucoside by a broad-specificity β-glucosidase from liver

1986 ◽  
Vol 237 (2) ◽  
pp. 469-476 ◽  
Author(s):  
K L LaMarco ◽  
R H Glew
Keyword(s):  
Guinea Pig ◽  
Competitive Inhibitor ◽  
Heat Denaturation ◽  
Pig Liver ◽  
Naturally Occurring ◽  
Aryl Glycosides ◽  
Beta Glucosidase ◽  
Hydrolysis Of ◽  
Guinea Pig Liver ◽  
Broad Specificity

We have isolated from guinea-pig liver a broad-specificity beta-glucosidase of unknown function that utilizes as its substrate non-physiological aryl glycosides (e.g. 4-methylumbelliferyl beta-D-glucopyranoside, p-nitrophenyl beta-D-glucopyranoside). The present paper documents that this enzyme can be inhibited by various naturally occurring glycosides, including L-picein, dhurrin and glucocheirolin. In addition, L-picein, which acts as a competitive inhibitor of the broad-specificity beta-glucosidase (Ki 0.65 mM), is also a substrate for this enzyme (Km 0.63 mM; Vmax. 277,000 units/mg). Heat-denaturation, kinetic competition studies, chromatographic properties and pH optima all argue strongly that the broad-specificity beta-glucosidase is responsible for the hydrolysis of both the non-physiological aryl glycosides and L-picein. This paper demonstrates that beta-glucosidase can catalyse the hydrolysis of a natural glycoside, and may provide a key to understanding the function of this enigmatic enzyme. A possible role in the metabolism of xenobiotic compounds is discussed.

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