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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.

1 Using a medium of tritiated water for the conversion of fumarate to malate and using a medium of deuterium oxide for the cleavage of citrate, chiral acetate was generated enzymically in the sequence fumarate malate oxaloacetate citrate acetate. 2 Acetate of opposite chirality was produced in the same sequence by replacing fumarate and tritiated water by [2,3-3H2]fumarate and ordinary water, respectively. 3 The configuration of acetate generated in (1) was shown to be R, and the configuration of acetate generated in (2) was shown in to be S. 4 It was concluded that citrate lyase cleaves citrate with inversion of configuration at the methylene group which becomes methyl.

By enzymic reactions using and generating asymmetrically labelled acetic acids, it is shown that R and S citrate synthases, citrate lyase and ATP citrate lyase all produce inversion of configuration at the methyl group when citrate is synthesized, or at the methylene group where citrate is cleaved.

1 R-Acetate and S-acetate, as the coenzyme-A esters, were converted into citrates on recitrate synthase. 2 The citrate were assayed for chirality at the 2-position (a) by incubation with aconitate hydratase (aconitase) and (b) by cleavage to malate by citrate lyase combined with malate dehydrogenase, followed by incubation of the isolated malate with fumarate hydratase (fumarase). 3 In each case, the citrate (or malate) derived from R-acetate retained most of its tritium, and the citrate (or malate) derived from S-acetate lost most of its tritium, on incubation with the hydratase. 4 It was concluded that if a normal intramolecular deuterium-isotope effect is operative, the condensation of oxaloacetate with acetyl-coenzyme A on re-citrate synthase proceeds with inversion of configuration at the methyl group.

1 A new synthesis of R- and S-acetic acid (R- and S-[2H1, 3H1]acetate) is described. 2 R-Acetic acid after conversion into citrate on si-citrate synthase and cleavage of the citrate with citrate lyase regenerates an acetate which is still predominantly R. 3 S-Acetic acid when put through the same sequence yields acetic acid which is predominantly S. 4 Essentially the same results as in (2) and (3) are obtained when ATP-citrate lyase is substituted for citrate lyase. 5 The conclusion is drawn that on si-citrate synthase, the replacement of hydrogen by oxaloacetic acid proceeds with inversion of configuration at the methyl group. 6 It also follows that ATP-citrate lyase, like citrate lyase, cleaves citrate with inversion of configuration. 7 It is further deduced that there is a normal intramolecular deuterium-isotope effect, with respect to the chiral-methyl group, in the condensation of chiral acetate with oxaloacetate on si-citrate synthase.