Conversion of Pyruvate Under Natural and Artificial Anaerobiosis in Maize

1976 ◽  
Vol 3 (6) ◽  
pp. 755 ◽  
Author(s):  
S Leblova ◽  
J Zima ◽  
E Perglerova
Keyword(s):  
Molecular Weight ◽  
Alcohol Dehydrogenase ◽  
Chelating Agents ◽  
Competitive Inhibitor ◽  
Cyclohexanone Oxime ◽  
Alcohol Dehydrogenases ◽  
Sh Groups ◽  
Competitive Inhibitors ◽  
Optimum Ph ◽  
Probable Presence

Lactate is formed during the initial hours of seed swelling during natural anaerobiosis in maize. Ethanol is formed later, at a concentration greater by one order. With 7-day-old seedlings, first lactate and then ethanol are also formed after transfer of the plants to an atmosphere of nitrogen. Lactate and alcohol dehydrogenases are active in the germinating seed. The molecular weight of maize alcohol dehydrogenase (EC 1.1.1.1) is 62 000 � 5000. Inhibition by chelating agents and 'sulphydryl poisons' indicates the probable presence of metal and -SH groups. The enzyme oxidizes ethanol at an optimum pH of 8.7 with a Km of 1.8 x 10-2 M and reduces acetaldehyde at an optimum pH of 6.7 with a Km of 1.0 x 10-3M. It is inhibited by succinate, malate, lactate and acetate, non-competitively with respect to the substrate. Acetoxime is a competitive inhibitor and butyrylamide, acetamide and cyclohexanone oxime are non-competitive inhibitors.

Inhibition by Substrates of a Coniferyl Alcohol Dehydrogenase Purified from Sugarcane Stalks

2020 ◽  
Vol 15 (3) ◽  
pp. 206-214
Author(s):  
Borja Alarcón ◽  
Roberto de Armas ◽  
Carlos Vicente ◽  
María E. Legaz
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ph Value ◽  
Coniferyl Alcohol ◽  
Kinetic Properties ◽  
Optimum Temperature ◽  
Alcohol Dehydrogenases ◽  
Sinapyl Alcohol ◽  
Optimum Ph ◽  
Km Value ◽  
Similar Affinity

Aims and Objectives: This study aimed to characterize a coniferyl alcohol dehydrogenase from sugarcane stalks. Also, the purification of CAD from sugarcane stalks was also carried out to study kinetic properties and substrate specificity. Background: Sugarcane plants contain an alcohol dehydrogenase able to reduce both coniferyl and sinapyl aldehydes to their correspondent alcohols, although there are reasonable grounds for suspecting that these are two distinct enzymes. Methods: The enzyme, coniferyl alcohol dehydrogenase was 125-fold purified from sugarcane stalks. Its activity was estimated by HPLC by calculating the amount of product formed. Results: The enzyme showed an optimum pH value of 7.9, at an optimum temperature of 20-22°C and a molecular mass of 48 kDa. The Km value for coniferyl alcohol was 3.03 µM and the enzyme was shown to be inhibited by an excess of the substrate from 17 µM. This dehydrogenase showed a similar affinity to sinapyl alcohol (Km 1.78 µM). Conclusions: This paper provides circumstantial evidence about the existence of two different alcohol dehydrogenases, specific to each of the substrates.

scholarly journals Characterization of a Pseudomonas putidaAllylic Alcohol Dehydrogenase Induced by Growth on 2-Methyl-3-Buten-2-ol

1999 ◽  
Vol 65 (6) ◽  
pp. 2622-2630 ◽  
Author(s):  
Vincent F. Malone ◽  
Amy J. Chastain ◽  
John T. Ohlsson ◽  
Loelle S. Poneleit ◽  
Michele Nemecek-Marshall ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Benzyl Alcohol ◽  
Acinetobacter Calcoaceticus ◽  
Reduction Reaction ◽  
Terminal Sequence ◽  
Alcohol Dehydrogenases ◽  
Optimum Ph ◽  
Aromatic Alcohols ◽  
A Subunit

ABSTRACT We have been working to develop an enzymatic assay for the alcohol 2-methyl-3-buten-2-ol (232-MB), which is produced and emitted by certain pines. To this end we have isolated the soil bacteriumPseudomonas putida MB-1, which uses 232-MB as a sole carbon source. Strain MB-1 contains inducible 3-methyl-2-buten-1-ol (321-MB) and 3-methyl-2-buten-1-al dehydrogenases, suggesting that 232-MB is metabolized by isomerization to 321-MB followed by oxidation. 321-MB dehydrogenase was purified to near-homogeneity and found to be a tetramer (151 kDa) with a subunit mass of 37,700 Da. It catalyzes NAD+-dependent, reversible oxidation of 321-MB to 3-methyl-2-buten-1-al. The optimum pH for the oxidation reaction was 10.0, while that for the reduction reaction was 5.4. 321-MB dehydrogenase oxidized a wide variety of aliphatic and aromatic alcohols but exhibited the highest catalytic specificity with allylic or benzylic substrates, including 321-MB, 3-chloro-2-buten-1-ol, and 3-aminobenzyl alcohol. The N-terminal sequence of the enzyme contained a region of 64% identity with the TOL plasmid-encoded benzyl alcohol dehydrogenase of P. putida. The latter enzyme and the chromosomally encoded benzyl alcohol dehydrogenase ofAcinetobacter calcoaceticus were also found to catalyze 321-MB oxidation. These findings suggest that 321-MB dehydrogenase and other bacterial benzyl alcohol dehydrogenases are broad-specificity allylic and benzylic alcohol dehydrogenases that, in conjunction with a 232-MB isomerase, might be useful in an enzyme-linked assay for 232-MB.

scholarly journals An ADH toolbox for raspberry ketone production from natural resources via a biocatalytic cascade

2021 ◽  
Author(s):  
Aileen Becker ◽  
Dominique Böttcher ◽  
Werner Katzer ◽  
Karsten Siems ◽  
Lutz Müller-Kuhrt ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Genetically Modified Organisms ◽  
Optical Purity ◽  
High Yield ◽  
Alcohol Dehydrogenases ◽  
Flavor Compound ◽  
Raspberry Ketone ◽  
Cofactor Recycling ◽  
Cosmetic Industry ◽  
Key Points

Abstract Raspberry ketone is a widely used flavor compound in food and cosmetic industry. Several processes for its biocatalytic production have already been described, but either with the use of genetically modified organisms (GMOs) or incomplete conversion of the variety of precursors that are available in nature. Such natural precursors are rhododendrol glycosides with different proportions of (R)- and (S)-rhododendrol depending on the origin. After hydrolysis of these rhododendrol glycosides, the formed rhododendrol enantiomers have to be oxidized to obtain the final product raspberry ketone. To be able to achieve a high conversion with different starting material, we assembled an alcohol dehydrogenase toolbox that can be accessed depending on the optical purity of the intermediate rhododendrol. This is demonstrated by converting racemic rhododendrol using a combination of (R)- and (S)-selective alcohol dehydrogenases together with a universal cofactor recycling system. Furthermore, we conducted a biocatalytic cascade reaction starting from naturally derived rhododendrol glycosides by the use of a glucosidase and an alcohol dehydrogenase to produce raspberry ketone in high yield. Key points • LB-ADH, LK-ADH and LS-ADH oxidize (R)-rhododendrol • RR-ADH and ADH1E oxidize (S)-rhododendrol • Raspberry ketone production via glucosidase and alcohol dehydrogenases from a toolbox Graphical abstract

Receptor kinetics pertaining to blockade of nerve-released transmitter at synapses

1988 ◽  
Vol 233 (1273) ◽  
pp. 461-475 ◽  
Keyword(s):  
Channel Blocker ◽  
Competitive Inhibitor ◽  
Tissue Response ◽  
Receptor Density ◽  
Receptor Kinetics ◽  
Tissue Responses ◽  
Competitive Inhibitors ◽  
Transmitter Substance ◽  
Uncompetitive Inhibitor ◽  
Temporal Inhomogeneity

The question is raised as to whether competitive inhibitors should block responses of tissue to nerve-released neurotransmitter to the same extent as they block equivalent responses to exogenous agonist. From a simple dynamic model of synaptic events, which takes into account non-constancy of transmitter concentration in space and time, it is deduced that equal blockade of responses to nerve-released and exogenous transmitter substance will occur if: (i) there are locally many more receptor molecules than transmitter molecules; (ii) the active agonist–receptor complex, A n R, has n = 1 ; and (iii) tissue response is insensitive to spatial or temporal inhomogeneity of AR. In such a case there will also be equal sensitivity of responses to other modes of inhibition: irreversible competitive, uncompetitive, and non-competitive. Equal blockade of responses to equi-effective endogenous and exogenous agonist will also occur if nerve stimulation gives rise to a steady uniform concentration of agonist, so that equilibrium kinetics are applicable. When n > 1 and/or when tissue responses reflect local peak A n R, response to nerve-released transmitter will be relatively insensitive to receptor blockade by a competitive inhibitor. The same is true for irreversible competitive blockade or for modulation of receptor density. However, an uncompetitive inhibitor (e. g. a ‘channel blocker’) may be more effective against nerve-released agonist than against exogenous agonist.

scholarly journals Rat liver alcohol dehydrogenase. Purification and properties

1971 ◽  
Vol 125 (4) ◽  
pp. 1039-1047 ◽  
Author(s):  
M J Arslanian ◽  
E Pascoe ◽  
J G Reinhold
Keyword(s):  
Molecular Weight ◽  
Alcohol Dehydrogenase ◽  
Rat Liver ◽  
Gel Filtration ◽  
Rabbit Antiserum ◽  
Minor Component ◽  
Disc Electrophoresis ◽  
Supernatant Fraction ◽  
Liver Alcohol Dehydrogenase ◽  
A Minor

Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD+ and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn2+ at concentrations above 0.1mm.

Enzymatic sulfation of steroids. V. Partial purification and some properties of sulfotransferase III, the major glucocorticoid sulfotransferase of liver cytosols from male rats

1978 ◽  
Vol 56 (11) ◽  
pp. 1028-1035 ◽  
Author(s):  
Sanford S. Singer ◽  
James Gebhart ◽  
Edward Hess
Keyword(s):  
Molecular Weight ◽  
Sulfhydryl Groups ◽  
Male Rats ◽  
Partial Purification ◽  
Ph Optimum ◽  
Fold Enrichment ◽  
Competitive Inhibitors ◽  
Sequential Mechanism ◽  
Inhibition Studies ◽  
Estradiol 17Β

This manuscript describes purification of sulfotransferase III (STIII), the major hepatic glucocorticoid sulfotransferase of male rats, 77.8 ± 16 fold from cytosol. This represents a probable 250–345 fold enrichment, compared with homogenates. Purified STIII has a molecular weight of 61 500 ± 2500 from Sephadex G-100 chromatography. It is markedly activated by 5 mM divalent Ba, Ca, Co, Cr, Mg, Mn, and Ni salts; inhibited strongly by 5 mM divalent Zn and Cd; and unaffected by 8 mM ADP, ATP, and AMP. Comparison of the ability of purified STIII to sulfate equimolar Cortisol, estradiol-17β, testosterone, and dehydroepiandrosterone suggests that the enzyme may sulfate glucocorticoids preferentially. However, its Cortisol sulfotransferase activity is inhibited by a variety of steroids. Of these, dehydroepiandrosterone, dexamethasone, and progesterone were tested extensively. They were found to be competitive inhibitors. STIII has a sharp pH optimum at pH 6.0 ± 0.1. However, it is routinely assayed at pH 6.8, as explained in the text. It exhibits a sequential mechanism and Km values of 6.82 ± 1.2 and 6.28 ± 0.64 μM for Cortisol and 3′-phosphoadenosine-5′-phosphosulfate, respectively. It also possesses essential sulfhydryl groups, as shown by p-hydroxymercuribenzoate inhibition studies.

Purification and characterization of ferro- and cobalto-chelatases

1988 ◽  
Vol 66 (1) ◽  
pp. 32-39 ◽  
Author(s):  
Eduardo T. Cánepa ◽  
Elena B.C. Llambías
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Purification And Characterization ◽  
Degree Of Unsaturation ◽  
Apparent Homogeneity ◽  
Optimum Ph ◽  
Single Protein ◽  
Metal Insertion

Pig liver ferrochelatase was purified 465-fold with about 30% yield, to apparent homogeneity, by a procedure involving solubilization from mitochondria, ammonium sulfate fractionation, and Sephacryl S-300 chromatography. The fraction of each purification step had cobaltochelatase as well as ferrochelatase activity. A purified protein of molecular weight 40 000 was found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A molecular weight of approximately 240 000 was obtained by Sephacryl S-300 chromatography. Both activities of the purified fraction increased linearly with time until 2 h. but nonlinear plots were obtained with increasing concentrations of protein. Their optimum pH values were similar. Km values were, for ferrochelatase activity, 23.3 μM for the metal and 30.3 μM for mesoporphyrin. and for cobaltochelatase activity. 27 and 45.5 μM, respectively. Fe2+ and Co2+ each protected against inactivation by heat. Pb2+, Zn2+, Cu2+, or Hg2+ inhibited both activities, while Mn2+ slightly activated; Mg2+ had no effect, at the concentrations tested. There appeared to be an involvement of sulfhydryl groups in metal insertion. Lipids, in correlation with their degree of unsaturation, activated both purified activities; phospholipids also had activation effects. We conclude that a single protein catalyzes the insertion of Fe2+ or Co2+ into mesoporphyrin.

scholarly journals Mechanism of d-fructose isomerization by Arthrobacterd-xylose isomerase

1992 ◽  
Vol 283 (1) ◽  
pp. 223-233 ◽  
Author(s):  
M Rangarajan ◽  
B S Hartley
Keyword(s):  
Ring Opening ◽  
Ph Dependence ◽  
Xylose Isomerase ◽  
Competitive Inhibitor ◽  
Apparent Dissociation Constant ◽  
Ph Values ◽  
Rate Limiting Step ◽  
Competitive Inhibitors ◽  
Rate Limiting ◽  
Very High

The mechanism of D-fructose isomerization by Arthrobacter D-xylose isomerase suggested from X-ray-crystallographic studies was tested by detailed kinetic analysis of the enzyme with various metal ions at different pH values and temperatures. At D-fructose concentrations used in commercial processes Mg2+ is the best activator with an apparent dissociation constant of 63 microM; Co2+ and Mn2+ bind more strongly (apparent Kd 20 microM and 10 microM respectively) but give less activity (45% and 8% respectively). Ca2+ is a strict competitive inhibitor versus Mg2+ (Ki 3 microM) or Co2+ (Ki 105 microM). The kinetics show a compulsory order of binding; Co2+ binds first to Site 2 and then to Site 1; then D-fructose binds at Site 1. At normal concentrations Mg2+ binds at Site 1, then D-fructose and then Mg2+ at Site 2. At very high Mg2+ concentrations (greater than 10 mM) the order is Mg2+ at Site 1, Mg2+ at Site 2, then D-fructose. The turnover rate (kcat.) is controlled by ionization of a residue with apparent pKa at 30 degrees C of 6.0 +/- 0.07 (Mg2+) or 5.3 +/- 0.08 (Co2+) and delta H = 23.5 kJ/mol. This appears to be His-219, which is co-ordinated to M[2]; protonation destroys isomerization by displacing M[2]; Co2+ binds more strongly at Site 2 than Mg2+, so competes more strongly against H+. The inhibition constant (Ki) for the two competitive inhibitors 5-thio-alpha-D-glucopyranose and D-sorbitol is invariant with pH, but Km(app.) in the Mg[1]-enzyme is controlled by ionization of a group with pKa 6.8 +/- 0.07 and delta H = 27 kJ/mol, which appears to be His-53. This shows that Km(app.) is a complex constant that includes the rate of the ring-opening step catalysed by His-53, which explains the pH-dependence. In the Mg[1]Mg[2]-enzyme or Co[1]Co[2]-enzyme, the pKa is lower (6.2 +/- 0.1 or 5.6 +/- 0.08) because of the extra adjacent cation. Hence the results fit the previously proposed pathway, but show that the mechanisms differ for Mg2+ and Co2+ and that the rate-limiting step is isomerization and not ring-opening as previously postulated.

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