Tetrahydrofolate enzymes involved in acetate biosynthesis from clostridium formicoaceticum (corrinoid path, methenyl tetrahydrofolate cyclohydrolase, methylene tetrahydrofolate reductase)
Athens, Ga., Univ. of Georgia, Diss., 1983
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Tag hinzufügen | Zusammenfassung: | Athens, Ga., Univ. of Georgia, Diss., 1983 Methenyltetrahydrofolate cyclohydrolase from C. formicoaceticum has been purified to homogeneity to a specific activity of 469 (mu)mol/min/mg at 35(DEGREES)C, pH 7.2. This is the first monofunctional cyclohydrolase purified which lacks methylenetetrahydrofolate dehydrogenase and formyltetrahydrofolate synthetase. The enzyme, with a molecular weight of 41,000 is a dimer. For a 0.1% solution, the enzyme has an extinction coefficient of 0.47 at 280 nm. The apparent Michaelis constant for methenyltetrahydrofolate is 0.19 mM and the apparent activation energy is 7500 cal/mol. Studies with other acetogens revealed that the cyclohydrolase from the mesophiles, Acetobacterium woodii and C. cylindrosporum are monofunctional and that the enzyme from the thermophile, C. thermoautotrophicum is bifunctional. The methylenetetrahydrofolate reductase from C. formicoaceticum has been purified to homogeneity to a specific activity of 140 (mu)mol/min/mg at 37(DEGREES)C, pH 7.2. A spectrophotometric assay with benzyl viologen was developed. The reductase is oxygen sensitive and requires strictly anaerobic conditions. The molecular weight is 237,000, with subunit molecular weights of 26,000 and 35,000, which are present in an equal molar ratio. Per 237,000, the enzyme contains 2 FAD, 15 iron, 19 inorganic sulfur and 2 zinc. The UV/visible absorbance spectrum shows a peak at 385 nm and a shoulder at 430 nm. When reduced with excess substrate, iron-sulfur centers and a semiquinone are detectible by EPR spectroscopy and have g-values of 2.02, 1.93 and 2.00. The benzyl viologen reductase reaction was studied at 37(DEGREES)C, pH 7.3. The kinetic data fit a substituted mechanism, with Michaelis constants of 11.1 and 0.12 mM for benzyl viologen and methyltetrahydrofolate, respectively. The reductase transfers electrons from FADH(,2) or reduced ferredoxin and in the reverse direction, the enzyme reduces rubredoxin. Formate dehydrogenase from C. formicoaceticum was purified to 50% homogeneity. The enzyme is oxygen labile. Electron carrier experiments indicated that ferredoxin is the physiological carrier. C. thermoautotrophicum, grown under 3 different conditions, has high levels of corrinoids and tetrahydrofolate enzymes. Cell-free extracts catalyzed the pyruvate-dependent formation of acetate from methyltetrahydrofolate. The results indicate that this acetogen used the corrinoid pathway. |
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| Beschreibung: | 213 S. |