Purification and properties of malate dehydrogenase from Pseudomonas testosteroni

Abstract

Nicotinamide adenine dinucleotide-linked malate dehydrogenase has been purified from Pseudomonas testosteroni (ATCC 11996). The purification represents over 450-fold increase in specific activity. The amino acid composition of the enzyme was determined and found to be quite different from the composition of the malate dehydrogenases from animal sources as well as from Escherichia coli. Despite this difference, however, the data show that the enzymatic properties of the purified enzyme are remarkably similar to those of other malate dehydrogenases that have been previously studied. The Pseudomonas enzyme has a molecular weight of 74,000 and consists of two subunits of identical size. In addition to L-malate, the enzyme slowly oxidizes other four-carbon dicarboxylates having an a-hydroxyl group of S configuration such as meso-and (-)-tartrate. Rate-determining steps, which differ from that of the reaction involving L-malate, are discussed for the reaction involving these alternative substrates. Oxidation of hydroxymalonate, a process previously undetected with other malate dehydrogenases, is demonstrated fluorometrically. Hydroxymalonate and D-malate strongly enhance the fluorescence of the reduced nicotinamide adenine dinucleotide bound to the enzyme. The enzyme is A-stereospecific with respect to the coenzyme. Malate dehydrogenase is present in a single form in the Pseudomonas. The susceptibility of the enzyme to activation or inhibition by its substrates-particularly the favoring of the oxidation of malate at elevated concentrations-strongly resembles the properties of the mitochondrial enzymes. The present study reveals that whereas profound variations in chemical composition have occurred between the prokaryotic and eukaryotic enzymes, the physical and catalytic properties of malate dehydrogenase, unlike lactate dehydrogenase, are well conserved during the evolutionary process.

Purification and characterization of the malate dehydrogenase from Streptomyces aureofaciens

“…4). This pH optimum is somewhat lower than the pH optima determined for most bacterial enzymes which are higher than 9 in several cases [19,21,23,30,31], but similar to the pH optimum of yeast [32], or plant chloroplast MDH [20]. All the K m values were in the same range as those reported for eubacterial and both mitochondrial and cytoplasmic eukaryotic MDHs.…”

Purification and characterization of the malate dehydrogenase fromStreptomyces aureofaciens

“…4). This pH optimum is somewhat lower than the pH optima determined for most bacterial enzymes which are higher than 9 in several cases [19, 21, 23, 30, 31], but similar to the pH optimum of yeast [32], or plant chloroplast MDH [20]. All the K m values were in the same range as those reported for eubacterial and both mitochondrial and cytoplasmic eukaryotic MDHs.…”

“…The great majority of MDHs are homodimers [15–19], including the chloroplastic NADP‐dependent form [20]. In some organisms however a homotetrameric MDH was found [16, 21, 22] and a homooctameric MDH was reported for Nitzschia alba [23].…”

The rapid purification of 3-hydroxybutyrate dehydrogenase and malate dehydrogenase on triazine dye affinity matrices

“…Malate dehydrogenase (L-malate :NAD+ oxidoreductase, EC 1.1.1.37) has been isolated from a large number of micro-organisms by means of conventional (Murphey et al, 1967a,b;Kohn & Jakoby, 1968;You & Kaplan, 1975;Kristjansson & Ponnamperuma, 1980) and affinity- (Wright & Sundaram, 1979) chromatographic procedures. The affinity-chromatographic procedures used employed AMP-Sepharose 4B and NAD+-hexyl-agarose.…”