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)

The enzyme hydrogenase, from the photosynthetic bacterium Chromatium, was purified to homogeneity after solubilization of the particulate enzyme with deoxycholate. The purification procedure included ammonium sulfate fractionation, treatment with manganous phosphate gel, heating at 63 degrees, DEAE-cellulose chromatography, and isoelectric focusing. The last step gave two active enzyme fractions with isoelectric points of 4.2 and 4.4. It was shown that the two fractions were different forms of the same protein. The enzyme was obtained in 23% yield and was purified 1700-fold. The enzyme had a molecular weight of 98,000, a sedimentation coefficient of 5.16 S and gave a single protein and activity band on disc gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis gave a single band of mol wt 50,000, suggesting that the active enzyme was composed of two subunits of the same molecular weight. The pure hydrogenase contained four atoms of iron and four atoms of acid-labile sulfide, and had a visible absorption peak at 410 nm. Electron paramagnetic resonance (EPR) spectroscopy at 10--15 K showed a free-radical signal at g' = 2.003 in the oxidized enzyme and signals at g' = 2.2 and 2.06 in the reduced enzyme. These findings suggest that Chromatium hydrogenase is an iron-sulfur protein. The pure hydrogenase catalyzed the exchange reaction between H2 and HDO or HTO, the reduction of Benzyl Viologen and Methylene Blue, and the evolution of hydrogen from reduced Methyl Viologen. The mechanism of hydrogen activation was shown to be heterolytic cleavage to an enzyme hydride and a proton. Hydrogenase could not catalyze reduction of pyridine nucleotides or ferredoxin with H2. The effect of pH and various inhibitors on the enzymatic activity has been studied.
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PMID:Structural and catalytic properties of hydrogenase from Chromatium. 23 60

The activation kinetics of the H2-oxidizing activity of the soluble hydrogenase from Alcaligenes eutrophus H16 were investigated. Activation with Na2S2O4 plus 101 kPa H2 resulted in a rapid increase in activity over 1 h and constant activity after 3 h incubation. Less-stable activations were achieved if enzyme was incubated with Na2S2O4 under 1 kPa H2 or 101 kPa N2. The enzyme could also be partly activated either with NADH alone or with H2 alone. The level of activity obtained with both 101 kPa H2 and NADH present was greater than that obtained with either 101 kPa H2 or NADH alone. Activation with H2 plus NADH was virtually independent of NADH concentration but highly dependent on H2 concentration. The effects of various concentrations of H2 and constant concentration of NADH on the level of activation were the same whether H2 oxidation was assayed by H2-dependent Methylene Blue or NAD+ reduction. Diaphorase activity did not require activation and was little affected by the treatments that activated H2-oxidizing activity. The results suggest that H2 plays an important role in regulating the level of H2-oxidizing activity in this soluble hydrogenase.
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PMID:Role of hydrogen in the activation and regulation of hydrogen oxidation by the soluble hydrogenase from Alcaligenes eutrophus H16. 305 35

Non-autotrophic ( Aut -) mutants of Rhodopseudomonas capsulata B10 were tested for their efficiency of nitrogenase-mediated H2 production. Three of these mutants ( IR3 , IR4 and IR5 ) showed an increase stoichiometry of H2 production, mediated by nitrogenase, from certain organic substrates. For example, in a medium containing 7 mM-L-glutamate as nitrogen source, strain IR4 produced 10-20% more H2 than did the wild type with DL-lactate or L-malate as major carbon source, 20-50% more H2 with DL-malate, and up to 70% more with D-malate. Strain IR4 was deficient in 'uptake' hydrogenase activity as measured by H2-dependent reduction of Methylene Blue or Benzyl Viologen. However, this observation did not explain the increased efficiency of H2 production, since H2 uptake (H2 recycling) was undetectable in cells of the wild type. Instead, increased H2 production by the mutant appeared to be due to an improved conversion of organic substrates to H2 and CO2, presumably due to an altered carbon metabolism. The metabolism of D-malate by different strains was studied. An NAD+-dependent D-malic enzyme was synthesized constitutively by the wild type, and showed a Km for D-malate of 3 mM. The activity of this enzyme was approx. 50% higher in strain IR4 than in the wild type, and the mutant also grew twice as fast as the wild type with D-malate as sole carbon source.
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PMID:Increased photoproduction of hydrogen by non-autotrophic mutants of Rhodopseudomonas capsulata. 614 10

The agarose-coupled triazine dye Procion Red HE-3B has been demonstrated to be applicable as an affinity gel for the purification of five diverse hydrogenases, namely the soluble, NAD-specific and the membrane-bound hydrogenase of Alcaligenes eutrophus, the membrane-bound hydrogenase of the N2-fixing Alcaligenes latus, the reversible H2-evolving and the unidirectional H2-oxidizing hydrogenase of Clostridium pasteurianum. In the case of the soluble hydrogenase of A. eutrophus, chromatography on Procion Red-agarose even permitted the separation of inactive from active enzyme, thus yielding a 2-3-fold increase in specific activity. For the homogeneous enzyme preparation obtained after two column steps (Procion Red-agarose, DEAE-Sephacel), a specific activity of 121 mumol of H2 oxidized/min per mg of protein was determined. Kinetic studies with free Procion Red provided evidence that the diverse hydrogenases are competitively inhibited by the dye, each with respect to the electron carrier (NAD, Methylene Blue, Methyl Viologen), indicating a specific interaction between Procion Red and the catalytic centres of the enzymes. For the highly purified preparations of the soluble and the membrane-bound hydrogenase of A. eutrophus, in 50 mM-potassium phosphate, pH 7.0, Ki values for Procion Red of 103 and 19 microM have been determined.
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PMID:Purification of hydrogenases by affinity chromatography on Procion Red-agarose. 635 40