NAD‐Linked l(+)‐Lactate Dehydrogenase from the Strict Aerobe Alcaligenes eutrophus

An NAD‐linked l (+)‐lactate dehydrogenase (EC 1.1.1.27) was isolated from the strictly respiratory bacterium Alcaligenes eutrophus and purified about 700‐fold to homogeneity. Derepression of enzyme formation required prolonged incubation of the cell suspension under conditions of restricted oxygen supply. The purification procedure included precipitation by cetyltrimethylammonium bromide, and subsequent chromatography on Matrex TM green A gel, DEAE‐Sephacel and Cibacron blue F3G‐A Sepharose. Affinity chromatography on Matrex green A resulted in an about 140‐fold purification. The overall enzyme recovery was 41.7% of the initial activity, and the specific activity (pyruvate reduction) of the final preparation was 825 units · mg protein −1 . The enzyme is a dimer consisting of two equal subunits of a relative molecular mass of 37000 each as determined at pH 7.0 in potassium phosphate buffer. It is specific for the l (+)‐stereoisomer of lactate. It is able to reduce, besides pyruvate, a variety of 2‐oxocarbonic acids and 3‐fluoropyruvate. The optimum temperature is 42–45°C, and the activation energy amounts to E A = 19.2 kJ · mol −1 for both directions. The enzyme is highly sensitive to p ‐chloromercuribenzoate and heavy metal ions. It does not belong to the type of lactate dehydrogenase dependent on fructose 1,6‐bisphosphate for activity. Among various compounds tested for affecting enzyme activity only NAD, phospho enol pyruvate, 3‐fluoropyruvate, oxalate, oxamate and oxaloacetate exhibited significant effects, the latter inhibiting the enzyme almost completely at 1 μM concentration.