La d‐altronate: NAD‐oxydoréductase d' Escherichia coli K12

D-Mannonate : NAD oxidoreductase of Escherichia coli K12 has been purified. The purification consists of four steps: precipitation of nucleic acids with protamine sulfate; ammonium sulfate precipitation; ion-exchange chromatography on DEAE-cellulose and Sephadex G-200 gel filtration. The overall procedure results in a 53-fold purification with a yield of 70%. Mannonate oxidoreductase is an inducible enzyme: glucuronate and fructuronate are good inducers, galacturonate is not. Optimal activity is obtained at pH 6.3 for fructuronate reduction, and at pH 8.4 for mannonate oxidation. The apparent Michaelis constants for the purified enzyme have been determined: for fructuronate, the Km is 0.5 mM; for NADH 0.03 mM; for mannonate, 1.8 mM and for NAD+, 0.2 mM. Mannonate oxidoreductase and altronate oxidoreductase have been separated on DEAE-Sephadex in order to study mannonate-oxidoreductase specificity. After separation, mannonate oxidoreductase is found to be active with both fructuronate and tagaturonate as substrates. Kinetic studies, differential inhibition of activities by p-chloromercuribenzoate and thermal inactivation studies confirm this double specificity. Other carbohydrates tested are unsuitable as substrates. Mannonate oxidoreductase is active with NADPH as coenzyme and the apparent Km value for NADPH is 0.7 mM. Kinetic inhibition studies of the enzyme by direct and reverse reactional products suggest a random mechanism for mannonate oxidoreductase. Enzyme inhibition specificity appears to be restricted. The value of the energy of activation is 34 kJ/mol (8 kcal/mol); the Q10 is 1.5. The results of thermal inactivation of mannonate oxidoreductase (53°C) indicate homogeneousenzyme activity with fructuronate in the crude extract. In the absence of inducer, basal activity is dependent on mannonate oxidoreductase only. Thermal inactivations also indicate that the same enzyme, mannonate oxidoreductase, catalyzes the direct reactions with NADH or NADPH, and the reverse reaction. Enzyme substrates, such as NADH, NADPH, fructuronate, tagaturonate, NAD+, mannonate and altronate protect enzymatic activity from degradation by heat. NADH in particular, is effective as a stabilizer; this ability is proportional to its concentration. p-Chloromercuribenzoate is an active inhibitor: at 5 μM, activity is 100% inhibited. Inhibition is non-competitive, with an apparent Ki of 0.1 μM. The end product of the reaction of mannonate oxidoreductase with fructuronate has been identified by means of paper chromatography as D-mannonate.