Purification and Properties of Reduced Diphosphopyridine Nucleotide Kinase from Yeast Mitochondria

An enzyme that phosphorylates DPNH to form TPNH has been isolated from aerobic yeast mitochondria and purified 127-fold. This enzyme differs from other reported DPN kinases in that it is specific for DPNH as a substrate and requires high concentrations of a carboxylic acid such as acetate for maximal activity. In the presence of 0.2 m sodium acetate, the apparent Km values for DPNH, ATP, and Mg++ are 42 µm, 1.0 mm, and 1.0 mm, respectively. Purified DPNH kinase is highly unstable; however, it can be protected from denaturation by 0.2 m ammonium sulfate. Ammonium sulfate itself does not activate the enzyme. Mitochondrial DPNH kinase represents about 5% of the total DPN and DPNH kinase activities of the yeast cell and appears to be involved in maintaining the level of triphosphopyridine nucleotide in the mitochondrion.