Stereochemistry of the re‐Citrate‐Synthase Reaction

Abstract

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 ...

Enzymic Generation of Chiral Acetates. A Quantitative Evaluation of Their Configurational Assay

“…The same factual situation applies for the recitrate synthase reaction where the elucidated substrate stereochemistry, inversion of configuration during methyl-methylene interconversion, also depends on the operation of a normal isotopic effect [6]. Note that the experimental results are consistent only with the outlined ambiguities; other possibilities such as inversion of configuration combined with the operation of an inverse isotopic effect need not be considered.…”

“…It was, therefore, of additional interest to see whether evolution produced an enzyme with completely different stereochemistry, that is whether the condensation reaction on the re-synthase proceeded with retention of configuration. Chiral acetates were converted into citrates on the re-synthetase and the isolated citrates were degraded to the malates(c) by incubation with citrate lyase, NADH and malate dehydrogenase [6]. The expected malate specimens are qualitatively those shown in Scheme 1 for their formation from chiral acetyl-CoA and glyoxylate on malate synthase.…”

“…The isolated citrate was degraded enzymically to the correspondingly labelled malate (3.5 pmol) as described [6]. The results given here and below for R-acetate were obtained likewise with S-acetate.…”

“…The citrate specimens derived from chiral acetylCoA and oxaloacetate on the re-synthase are labelled stereospecifically at the pro-R side chain and can easily be degraded enzymically to the correspondingly C 3-labelled malates [6]. Since the stereochemical ambiguities are the same for both the re-synthase and malate synthase reactions, their solution is reducible to one: that is, how to differentiate between the pairs of malate specimens shown in Eqns 1 and 2.…”

“…The malate synthase ~H /~L H was determined from the data of this study as 3.9 k 0.2. 6. The average of the values given under paragraphs 1 and 5 for the isotopic discrimination on malate synthase corresponds to k~/ k 2~ = 3.8 & 0.1.…”