Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.12.7.2 (hydrogenase)
3,522 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Uptake hydrogenase (EC 1.12) from Azotobacter vinelandii has been purified 250-fold from membrane preparations. Purification involved selective solubilization of the enzyme from the membranes, followed by successive chromatography on DEAE-cellulose, Sephadex G-100, and hydroxylapatite. Freshly isolated hydrogenase showed a specific activity of 110 mumol of H2 uptake (min X mg of protein)-1. The purified hydrogenase still contained two minor contaminants that ran near the front on sodium dodecyl sulfate-polyacrylamide gels. The enzyme appears to be a monomer of molecular weight near 60,000 +/- 3,000. The pI of the protein is 5.8 +/- 0.2. With methylene blue or ferricyanide as the electron acceptor (dyes such as methyl or benzyl viologen with negative midpoint potentials did not function), the enzyme had pH optima at pH 9.0 or 6.0, respectively, It has a temperature optimum at 65 to 70 degrees C, and the measured half-life for irreversible inactivation at 22 degrees C by 20% O2 was 20 min. The enzyme oxidizes H2 in the presence of an electron acceptor and also catalyzes the evolution of H2 from reduced methyl viologen; at the optimal pH of 3.5, 3.4 mumol of H2 was evolved (min X mg of protein)-1. The uptake hydrogenase catalyzes a slow deuterium-water exchange in the absence of an electron acceptor, and the highest rate was observed at pH 6.0. The Km values varied widely for different electron acceptors, whereas the Km for H2 remained virtually constant near 1 to 2 microM, independent of the electron acceptors.
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PMID:Purification and properties of membrane-bound hydrogenase from Azotobacter vinelandii. 637 82

The hydrogen in methane produced by cultures of Methanobacterium thermoautotrophicum originates from water. In H2O/D2O mixtures, a methane product isotope effect is observed that increases rapidly as the water deuterium enrichment approaches 100%. This effect is due to the intracellular production of protons from H2, catalyzed by hydrogenase, which occurs at 12% the rate of water diffusion through the cell membrane. We estimate that water diffusion through the thick cell membrane of M. thermoautotrophicum is retarded by a factor of 10(6) over the free diffusion rate. The intracellular production of H+ suggests that either (1) hydrogenase is not directly involved in the production of a chemiosmotic proton gradient or (2) if it is involved, the proton gradient exists between the cytosol and the interior of vesicles observed in this bacterium. The intrinsic deutrium product isotope effect in methanogenesis is 1.20 +/- 0.1, comparable to anabolic deuterium product isotope effects in other autotrophs. An algebraic model incorporating the intracellular H2 to H+ flux accurately predicts the distribution of deuterated methane species at all levels of water deuterium enrichment.
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PMID:Product isotope effects on in vivo methanogenesis by Methanobacterium thermoautotrophicum. 699 9

The periplasmic hydrogenase of Desulfovibrio desulfuricans was isolated and purified. Cells were washed with Tris-EDTA and the enzyme precipitated from the wash with ammonium sulfate. Absorption chromatography on DEAE and hydroxyapatite yielded the enzyme at better than 95% purity as judged by gel electrophoresis. The hydrogenase catalyzed the production of more than 9000 mumol H2/min mg protein(-1) from reduced methyl viologen at 37 degrees C. It is very stable and resists inactivation by heat (50% activity remained after 5 min in air at 65 degrees C) and by enzyme inhibitors (except N-ethylmaleimide and potassium ferricyanide). After storage in air at 4 degrees C for 1 month no activity was lost. The enzyme activity is sensitive to ionic environmental changes. With methyl viologen the optimum pH was 5.5 but with p-xylene polymeric viologen the optimum was about pH 7 but less sharp. The molecular weight was 47 X 10(3)(+/- 2 X 10(3), 52 X 10(3)(+/- X 10(3), and 56 X 19(3)(+/- 2 X 10(3) by SDS-gel electrophoresis, gel chromatography, and sedimentation equilibrium, respectively, and the isoelectric point was at pH 6.0. They enzyme might be useful in the production of hydrogen from water and solar energy.
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PMID:Purification and properties of the periplasmic hydrogenase from Desulfovibrio desulfuricans. 700 65

The kinetic mechanism of the unidirectional H2-oxidizing hydrogenase from soybean nodule bacteroids has been investigated with highly purified enzyme. Measurements of the Km for H2 vary from 0.97 to 2.6 microM, and the Km for methylene blue varies from 6 to 17 microM. With H2 and methylene blue as substrates, the initial velocity patterns are intersecting. High levels of methylene blue are inhibitory (KI =2.4 mM): the inhibition is competitive vs. H2. CO is a competitive inhibitor of H2 (KI = 157 microM) and noncompetitive vs. methylene blue. O2 inactivates the enzyme (t 1/2 approximately 1 h) but also is a reversible inhibitor of hydrogenase activity upon short exposure to low concentrations. Inhibition by O2 is uncompetitive vs. H2 and noncompetitive vs. methylene blue. Hydrogenase was not inhibited by C2H2; preincubation under C2H2 inactivates the enzyme. Reduced methyl and benzyl viologens support low rates of H2 evolution by the hydrogenase. The Km for reduced methyl viologen is 11 microM. H2 is a potent inhibitor of H2 evolution: the inhibition is noncompetitive vs. reduced methyl viologen. The hydrogenase will catalyze a low rate of exchange in the reaction between D2 and H2O to form both HD and H2. We propose a two-site ping-pong mechanism for the enzyme in which H2 is reversibly activated at one site and e- carriers interact at the second site.
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PMID:Kinetic mechanism of the hydrogen-oxidizing hydrogenase from soybean nodule bacteroids. 701 76

H2-forming N5,N10-methylenetetrahydromethanopterin dehydrogenase from methanogenic Archaea, which is a novel hydrogenase containing neither nickel nor iron-sulfur clusters, catalyzes the reversible reduction of N5,N10-methenyltetrahydomethanopterin (CH identical to H4MPT+) with H2 to N5,N10-methylenetetrahydromethanopterin (CH2 = H4MPT) and a proton (delta G degree' = -5.5 kJ/mol). The enzyme also catalyzes a CH identical to H4MPT(+)-dependent H2/H+ exchange. We report here on kinetic deuterium isotope effects in these reactions. When CH identical to H4MPT+ reduction was performed with D2 instead of H2, Vmax and the Km did not change. A primary isotope effect of 1 was found at all pH and temperatures tested and independent of whether H2O or D2O was the solvent. The findings indicate that a step other than the activation of H2 was rate-determining in CH identical to H4MPT+ reduction with H2. This was substantiated by the observation that also the CH identical to H4MPT(+)-dependent H2/H+ exchange reaction did not exhibit an appreciable deuterium isotope effect. Vmax for CH2 = H4MPT dehydrogenation to CH identical to H4MPT+ and H2 was only 2-3 times higher than for CD2 = H4MPT dehydrogenation to CD identical to H4MPT+ and HD. Such a small primary isotope effect indicates that the breakage of the C-H bond in the methylene group of CH2 = H4MPT was only rate-limiting when hydrogen was substituted by a deuterium.
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PMID:Hydrogen isotope effects in the reactions catalyzed by H2-forming N5,N10-methylenetetrahydromethanopterin dehydrogenase from methanogenic Archaea. 758 69

Enzymological studies on the multienzyme acetyl-CoA decarbonylase synthase (ACDS) complex from Methanosarcina barkeri have been conducted in order to identify and characterize physiologically relevant substrates and reactions in acetyl-CoA synthesis and decomposition in methanogens. Whereas previous investigations employed carbon monoxide as substrate and reducing agent for acetyl-CoA synthesis, we discovered that bicarbonate (or CO2) acts as a highly efficient carbonyl group precursor substrate in the presence of either hydrogen or Ti3+.EDTA as reducing agent. In reactions with Ti3+.EDTA, synthesis of acetyl-CoA was strongly dependent on ferredoxin, and in reactions with H2, dependence on ferredoxin was absolute. Two major hydrogenases were resolved from the enzyme complex preparation by HPLC gel filtration. One of these hydrogenases was shown to be active in reconstitution of acetyl-CoA synthesis in CO2-containing reactions with H2 as reducing agent. The hydrogenase active in reconstitution was capable of reducing ferredoxin, but was unreactive toward the 8-hydroxy-5-deazaflavin derivative coenzyme F420. In contrast, the hydrogenase that did not reconstitute acetyl-CoA synthesis was reactive with F420 but was unable to reduce ferredoxin. Further experiments were performed in which the value of the equilibrium constant (Keq) was determined for the reaction: H2 + CO2 + CH3-H4SPt + CoASH <--> acetyl-CoA + H4SPt + H2O, where CH3-H4SPt and H4SPt stand for N5-methyl-tetrahydrosarcinapterin and tetrahydrosarcinapterin, respectively. Keq for this reaction was found to be 2.09 x 10(6) M-1ATMH2-1 at 37 degrees C. Calculations of thermodynamic values for additional, related reactions were made and are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Substrate and accessory protein requirements and thermodynamics of acetyl-CoA synthesis and cleavage in Methanosarcina barkeri. 771 64

In water-in-oil microemulsion the membrane-associated F420-hydrogenase of Methanobacterium thermoautotrophicum (strain Marburg) and the membrane-bound hydrogenase of Alcaligenes eutrophus H 16 (MBH) showed prolonged activity at elevated temperatures (measured as hydrogen production) as compared to aqueous buffer solution. The temperature optimum of the reactions was about 15 degrees C higher than in aqueous buffer solution. Activity of the almost completely inactivated F420-hydrogenase could be partially recovered by transfer into microemulsion.
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PMID:Stability and activity of hydrogenases of Methanobacterium thermoautotrophicum and Alcaligenes eutrophus in reversed micellar systems. 802 Jul 49

The thermodynamics of the nitrogenase reactions are discussed in terms of chemical equations and biochemical equations. Chemical equations balance all elements and electric charge. Biochemical equations represent changes at specified pH and specified free concentrations of metal ions that are bound by reactants, but they do not balance hydrogen or metal ions that have specified free concentrations. At a specified pH, it takes three separate biochemical equations to represent the changes catalyzed by nitrogenase. [formula; see text] The first two equations are required because the nitrogenase and hydrogenase activities of the enzyme have not been separated. The hydrolysis of ATP is necessary, but it is not coupled stoichiometrically to the first two equations. The function of the hydrolysis of ATP by nitrogenase may be to provide the 10 H+ required per mol of N2 consumed. However, reactions cannot generally be coupled stoichiometrically through H+ because H+ is potentially available by dissociation of protein, buffer, and H2O. The standard Gibbs energies of formation of the reactant species are calculated for 25 degrees C, 1 bar, and ionic strengths of 0 and 0.25 M. The standard transformed Gibbs energies of formation of the reactants are calculated at 25 degrees C, 1 bar, pH 7, and ionic strengths of 0 and 0.25 M.
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PMID:Thermodynamics of the nitrogenase reactions. 812 17

5,6,7,8-Tetrahydromethanopterin is a coenzyme playing a key role in the energy metabolism of methanogenic archaea. In Methanobacterium thermoautotrophicum, the reduction of N5, N10-methenyl-5,6,7,8-tetrahydromethanopterin at C(14a) with H2 to N5, N10-methylene-5,6,7,8-tetrahydromethanopterin can be catalyzed by H2-forming methylenetetrahydromethanopterin dehydrogenase, a new hydrogenase present in most methanogenic archaea, which is unique because it does not contain nickel or iron/sulfur clusters. In this work, the stereochemistry of this enzymatic hydride-transfer reaction is elucidated by means of a series of heteronuclear two-dimensional NMR experiments. It is found that the hydride from H2 is transferred by the enzyme into the rel-(pro-R) position of the C(14a) methylene group of the reaction product N5, N10-methylene-5,6,7,8-tetrahydromethanopterin. NMR experiments are described that show that the hydrogen nucleus of the hydride transferred to the oxidized coenzyme partially originates from water. The stereochemical course of this reaction is the same as that for direct hydride transfer. It is demonstrated that the diastereotopic atoms at C(14a) of the reaction product epimerize in an uncatalyzed reaction under the conditions of operation of the enzyme (k = 0.01 s-1 at 58 degree C and pH 6.5).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:H2-forming N5, N10-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum catalyzes a stereoselective hydride transfer as determined by two-dimensional NMR spectroscopy. 814 3

Oxidation of glycolate to 2 CO2 and 3 H2 (delta G degrees' = +36 kJ/mol glycolate) by the proton-reducing, glycolate-fermenting partner bacterium of a syntrophic coculture (strain FlGlyM) depends on a low hydrogen partial pressure (pH2). The first reaction, glycolate oxidation to glyoxylate (E zero' = -92 mV) with protons as electron acceptors (E zero' = -414 mV), is in equilibrium only at a pH2 of 1 microPa which cannot be maintained by the syntrophic partner bacterium Methanospirillum hungatei; energy therefore needs to be spent to drive this reaction. Glycolate dehydrogenase activity (0.3-0.96 U.mg protein-1) was detected which reduced various artificial electron acceptors such as benzyl viologen, methylene blue, dichloroindophenol, K3[Fe(CN)6], and water-soluble quinones. Fractionation of crude cell extract of the glycolate-fermenting bacterium revealed that glycolate dehydrogenase, hydrogenase, and proton-translocating ATPase were membrane-bound. Menaquinones were found as potential electron carriers. Everted membrane vesicles of the glycolate-fermenting bacterium catalyzed ATP-dependent H2 formation from glycolate (30-307 nmol H2.min-1 x mg protein-1). Protonophores, inhibitors of proton-translocating ATPase, and the quinone analog antimycin A inhibited H2 formation from glycolate, indicating the involvement of proton-motive force to drive the endergonic oxidation of glycolate to glyoxylate with concomitant H2 release. This is the first demonstration of a reversed electron transport in syntrophic interspecies hydrogen transfer.
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PMID:Hydrogen formation from glycolate driven by reversed electron transport in membrane vesicles of a syntrophic glycolate-oxidizing bacterium. 822 60


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