Purification and Allosteric Properties of a Nicotinamide Adenine Dinucleotide-linked d(-)-Specific Lactate Dehydrogenase from Butyribacterium rettgeri

A procedure is described for the purification of a nicotinamide adenine dinucleotide-linked, d (-)-specific lactate dehydrogenase from extracts of Butyribacterium rettgeri . This procedure yields a 300-fold purified enzyme with a 28% recovery of the activity present in crude, cell-free extracts. The purified enzyme is inhibited by sulfhydryl reagents but does not depend upon reduced sulfhydryl compounds for catalytic activity. It is relatively insensitive to inhibition by oxamate. The enzyme actively catalyzes the reduction of pyruvate to lactate with NADH as the coenzyme, but it is unable to catalyze the reverse reaction under conditions where lactate dehydrogenases from other sources can be shown to reduce NAD + with lactate. It does catalyze the reduction of the 3-acetylpyridine analogue of NAD + with d (-)-lactate, although several other NAD + analogues were found to be ineffective as cofactors for lactate oxidation. Kinetic studies on the purified enzyme suggest that it possesses at least two binding sites for pyruvate, which interact with one another in a cooperative manner. The availability of these sites or the interaction strength between them (or both) is markedly influenced by pH. At pH 5.8, the concentration of pyruvate required for one-half maximal velocity is substantially lower than it is at pH 7.5, although pH changes in this range have no effect on V max . The data presented are consistent with the hypothesis that the B. rettgeri lactate dehydrogenase is an allosteric protein which is potentially capable of being regulated by one or more physiological effector ligands.