Carbon assimilation by Pseudomonas oxalaticus (OX1). 5. Purification and properties of glyoxylic dehydrogenase

Abstract

A purification of glyoxylic dehydrogenase, obtained from oxalate-grown Pseudomonas oxalaticus, is described. The purification involves ammonium sulphate precipitation, gel absorption and ion-exchange chromatography. The pH optimum for the coenzyme A-and triphosphopyridine nucleotide-linked oxidation of glyoxylate to oxalate is 8.6. The glyoxylic dehydrogenase is free of the following enzymic activities reduced pyridine nucleotide oxidase, pyridine nucleotide-linked glyoxylic acid reductase or glycollic acid dehydrogenase, and glyoxylic acid carboligase. The enzyme is specific for both triphosphopyridine nucleotide and coenzyme A. Diphosphopyridine nucleotide will not replace the former nor will cysteine, glutathione, thioethanolamine or pantetheine replace the latter. In the presence of triphosphopyridine nucleotide and coenzyme A, the enzyme will not oxidize formaldehyde, acetaldehyde, glycolaldehyde, pyruvate, oxaloacetate, a-oxoglutarate or hydroxypyruvate. The presence of any of these compounds at 1 m[image] concentration does not inhibit the oxidation of glyoxylic acid catalysed by the enzyme. No metal-ion requirement for the action of glyoxylic dehydrogenase has been found. Km values of 5.7 x 10-4[image] and 3.4 x 10-5[image], at pH 8.6 and 25[degree], have been found for glyoxylate and triphosphopyridine nucleotide respectively. Coenzyme A is required for the oxidation in catalytic quantities. The free-energy changes involved in the oxidation of glyoxylate are discussed. Glyoxylic dehydrogenase has also been found in extracts of oxalate-grown Pseudomonas ODI. This activity is decreased to one-sixth when the same organism is grown on malate.