Partial Purification and Some Properties of Oxalacetase from Aspergillus niger

Abstract

1. Oxalacetase from Asperigillus niger was found to be an inducible enzyme, the induction being dependent not only on neutralisation of the acidic growth medium but also on the presence of carbonate. An explanation is proposed. 2. Three methods were established for the quantitative determination of oxalacetase activity. These are based on the determination of the product acetate, on the absorbance of oxaloacetate and on coupling the hydrolysis of oxaloacetate to the oxidation of malate by NAD in the presence of malate dehydrogenase. 3. Oxalacetase was purified about 50-fold from cell-free extracts of A. niger and used to determine some of its properties such as kinetic constants. 4. 2S-[U-14C, 3-2H2] Malate in the presence of oxalacetase, NAD and malate dehydrogenase was partially converted to acetate and oxalate. The 3H/14C ratio of the isolated acetate was nearly twice as high as that of the malate used initially. The result demonstrates that the keto form of oxaloacetate, not the enol, is the substrate of the enzyme. 5. Equimolecular mixtures of 2S, 3S-[3-2H1] malate + 2S-[2-2H1] malate (mixture 1) and 2S, 3R-[3-2H1, 3H1] malate + 2S, 3R-[2-2H1, 3-3H1] malate (mixture 2) were prepared from 2S-[3-3H2] malate by incubation with fumarase in normal and tritiated water, respectively. The isolated mixture 1, in the presence of oxalacetase, NAD and malate dehydrogenase was incubated in tritiated water for formation of acetate and oxalate; the isolated mixture 2 was treated likewise in normal water. 6. The mixtures of symmetrically labelled [3H1] acetate and chiral acetates thus produced were isolated and the configuration of the [3H1, 3H1] acetate specimens was determined in the sequence acetate leads to malate leads to fumarate, as usual. The [2H1, 3H1] acetate derived from 2S, 3S-[3-2H1] malate (present in mixture 1( yielded a malate which on incubation with fumarase retained 65.0% of its total tritium content. This chiral acetate, therefore, had the R configuration. The [2H1, 3H1] acetate derived from 2S, 3R-[2-2H1, 3-3H1] malate produced a malate which retained 35% of its total tritium content, and therefore had the S configuration. 7. It was concluded that the detachment of the oxaloyl residue from oxaloacetate and its replacement by a proton proceed with inversion of configuration at the methylene group which becomes methyl during the hydrolysis.