La D-altronate: NAD-oxydoréductase d'Escherichia coli K12.

D-Altronate: NAD-oxidoreductase of Escherichia coli K12 has been purified. The purification consists of four steps: nucleic acids precipitation with protamine sulfate; ammonium sulfate precipitation; ion exchange chromatography on DEAE-cellulose and Sephadex G-150 gel filtration. The overall procedure results in a 53-fold purification with a yield of 60%. Altronate oxidoreductase is an inducible enzyme: galacturonate, tagaturonate, glucuronate and fructuronate are good inducers. The activity remains stable at 4 °C or at — 20 °C for several months. Optimal activity is obtained at pH 6.3 for tagaturonate reduction, and at pH 8.9 for altronate oxidation. Altronate oxidoreductase does not require any cofactor, except NADH. The apparent Michaelis constants have been determined: for tagaturonate, the Km is 350 μM; for NADH, 60 μM; for altronate, 90 μM, and for NAD+, 110 μM. Moreover, altronate oxidoreductase is active with NADPH as coenzyme and the apparent Km value for NADPH is 400 μM. Other carbohydrates tested are unsuitable as substrates. Many other carbohydrates tested do not prove to be inhibitors. The value for the energy of activation is 38 kJ/mol (9 kcal/mol); the Q10 is 1.5. The results of heat inactivation of altronate oxidoreductase (58.5 °C) indicate homogeneous enzyme activity in crude extract. Thermal inactivations also indicate that the same enzyme, altronate oxidoreductase, is induced by galacturonate and glucuronate, and catalyzes the direct reactions with NADH or NADPH. Finally, it has been confirmed by heat inactivation that there is no interference between specific assays of altronate and mannonate oxidoreduetases. Compounds such as NADH, NADPH, altronate and mannonate protect enzymatic activity from degradation by heat, whereas NAD+, tagaturonate and fructuronate do not. NADH, in particular, is effective as a stabilizer; this ability is proportional to its concentration. Enzyme inhibition with para-chloromercuribenzoate is non-competitive and the apparent Ki is 1 mM. para-Chloromercuribenzoate also inactivates altronate oxidoreductase indicating homogeneous activity and specificity of the assay method in crude extracts. Chromatographic properties of the enzyme are identical whether the inducer is galacturonate or glucuronate. Using paper chromatography, the reaction product of enzyme action on D-tagaturonate is identified as D-altronate. [ABSTRACT FROM AUTHOR]