1. Oxalacetase from Aspergillus 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. 2S-[U-'4C,3-2H2]Malate in the presence of oxalacetase, NAD and malate dehydrogenase was partially converted to acetate and oxalate. The 3H/'4C 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. 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 l' or formation of acetate and oxalate; the isolated mixture 2 was treated likewise in normal water.6. The mixtures of symmetrically labelled [3Ht]acetate and chiral acetates thus produced were isolated and the configuration of the [2H1 ,3Hl]acetate specimens was determined in the sequence acetate -+ malate + fumarate, as usual. The L2HI, 3Hl]acetate derived from 2S, 3S-[3-'Hl]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 [2H1,3Hl]acetate derived from 2S,3R-[2-2H1, 3-3Hl]malate produced a malate which retained 35 7" 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 These results are taken from the Thesis of H. Lenz, Universitiit Regensburg, 1973; a preliminary account of part of this work has appeared elsewhere [I].
1. A new synthesis of R-and 8-acetic acid (R-and S-[2H,, 3H,]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 a t 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. (Table 1).From the results of the configurational assay of the product acetate it should be possible to reduce the stereochemical ambiguities from four to two. Since, however, citrate lyase is known to produce inversion of configuration [7], the formation of R-acetate in the above sequence would establish inversion and that of S-acetate would establish retention of configuration on the si-synthase. The chirality of the product acetate from, for example, R-acetate on combination of the si-synthase [Eqn (l)] with the ATP-citrate lyase reaction [Eqn (2)], will then likewise reveal the corresponding stereochemistry of this enzyme.
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 --f malate +-oxaloacetate -+ citrate -+ acetate.2. Acetate of opposite chirality was produced in the same sequence by replacing fumarate and tritiated water by [2,3-3H,]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 a t the methylene group which becomes methyl. [a]. The three different enzymes may thus be classified as si-citrate lyases and their common stereospecificity is shown below.Four known enzymes catalyse the synthesis or cleavage of citrate from, or to oxaloacetate and acetate (or acetyl-coenzyme A). The two synthases have hitherto been known as R-and S-synthases, for the reason that when oxaloacetate is condensed on the appropriate enzyme with an isotopically labelled acetate, R-citrate or S-citrate is formed. However, the chirality of the citrate is due entirely to the position of the isotope, and it is equally possible to produce R-citrate on the "S-synthase" by having the isotopic label in oxaloacetate instead of acetate. 14 Eur. J. Biochem., Vol. 24 meaningless description of the enzymes as R-and S-synthases will be abandoned in the three papers of which this is the fbst. Instead, we shall adopt, and we propose for general adoption, a suggestion of Cornforth to call "R-citrate synthase" re-citrate synthase and "S-citrate synthase" si-citrate synthase. The reason is that in the re-enzyme it is the re-side [l] of oxaloacetate that is attacked by the acetyl moiety, and in the si-enzyme it is the si-side that is attacked. This stereochemical truth is uneffected by the location of any label, or indeed by absence of any label.A more complete description of the stereochemistry of the reactions that these three si-citrate lyases catalyse requires insight into the enzymic transformation of the methyl group of the acetate unit into the methylene group of citrate and its reversal, respectively. At least two broad mechanisms can be considered for these processes. I n mechanism (1) the enzymically generated carbanion of the acetate group in an early-transition state participates in the formation of the C-C bond and the stereochemical consequence is more likely t o be inversion of the methyl group configuration since for retention the entering and leaving groups would be occupying much the same space. I n mechanism (2) the enolate anion of a late-transition state participates in the formation ofthe C-C bond. Depending on which side ofthe double bond ofthe enolate anion is involved in these processes the stereochemical consequence would be either retention or inversion of con6guration (Fig. 1).Experimentally it was possible to gain insight into these questions by application of (a) the th...
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 a t 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. I n 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 h ydratase .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. re-Citrate synthase (for nomenclature, see [14]), the fourth-known enzyme involved in citrate metabolism, was discovered by Gottschalk and Barker in extracts of anaerobic microorganisms [I]. Contrary to the action of the si-citrate lyases described in the preceding papers, this enzyme attaches the acetate unit at the re-side [2] of oxaloacetate, producing a citrate which carries the acetate derived group in pro-R position [I]. This is attacked by aconitate hydratase to yield cis-aconitate by a reversible trans-elimination of water formed from the hydroxyl group of citrate and the pro-R-hydrogen of the pro-R-acetate chain [3]. The stereochemistry of C-C bond formation on synthesis of citrate from acetylCoA and oxaloacetate consequently can be elucidated by (a) application of chiral acetates for this synthesis, and (b) use of aconitate hydratase for analysis of the product citrate.If a normal isotopic effect is operative on the re-synthase the tritium-containing products formed from R-acetate with inversion of configuration will consist of (2S,3R)-[2-2H,,2-3Hl]citrate preponderating over (2R,3R)-[2-3H,]citrate in a ratio equal to the intramolecular JCHIJCD for the re-synthase. This mixture on incubation with aconitate hydratase will retain more than 50°/, of its radioactive label. Retention of configuration would yield a mixture of (2R,3R)-[2-aHl,2-3H,]citrate and (2S,3R)-[2-3Hl]-citrate where the former outweighs the latter. This mixture on incubation with aconitate hydratase will lose more than 50°/, of the tritium label into the medium. Hence aconitate hydratase for analysis of stereospecifically labelled R-citrates provides the same analytical tool as does fumarase in case of the malates [4,5].Another approach to reveal the stereochemistry of the re-synthase makes use of the enzymes citrate lyase and malate dehydrogenase. Here the acetatederived group of R-citrate in the presence of NADH is converted to malate via oxaloacetate and malate is subjected to the usual analysis with fumarase. Melate dehydrogenase transfers hydrogen from NADH to the re side of oxaloacetate and fumarase, like aconitate hydratase, catalyzes a trans-elimination of water [3]. The hydrogen eliminated in the formation of water in ...
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