Stereochemistry of si‐Citrate Synthase and ATP‐Citrate‐Lyase Reactions

Abstract

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.

Large D/H variations in bacterial lipids reflect central metabolic pathways

“…Although their magnitudes vary greatly (up to 3,500‰, see Fig. S5) (49)(50)(51)(52), it is not possible to confidently predict in vivo fractionations, and thus the isotopic composition of generated NADPH, from these data. In part this is because kinetic isotope effects will not be fully expressed as isotopic fractionations in committed pathways where the reactant is completely consumed (53).…”

“…These upstream reactions are catalyzed by succinate dehydrogenase and aconitase. Both have been shown to express significant normal isotope effects (50,52,55), and are present in C. necator and E. coli (26,43). For example, an average kinetic fractionation of 4,400‰ was measured for the removal of pro-R H from methylene groups in succinate by flavoprotein succinate dehydrogenase (55).…”

Evidence from Inhibitor Studies for Conformational Changes of Citrate Synthase

“…Kinetic data suggest that this inhibitor represents an intermediate analogue. 4. The results given above indicate conformational changes of the synthase during the catalytic cycle.…”

Large‐Scale Purification and Some Properties of Malate Synthase from Baker's Yeast

“…It is also well documented that the use of chemically prepared chiral acetyl-CoA in this assay, because of mercaptan impurities present in the original coenzyme A sample, can yield erroneous results, indicating an apparently higher optical purity for (R)-acetate and an apparently less optical purity for (S)-acetate [33]. Thus, ( R ) and (S)-acetates, analyzed in the sequence acetate --$ acetyl phosphate -+ acetyl-CoA + malate --f fumarate, yielded a d 50% value of k 26%, whereas the acetyl-CoA specimens prepared chemically from these acetates yielded A 50 % values of -I 32 7; for (R)-acetyl-CoA, and -7 % for (S)-acetyl-CoA; the values were about f 26 7; if these acetyl-CoA specimens were purified prior to the assay [33]. Now, the purification procedure can readily be checked if [14C]acetyl-CoA is converted to [3H, 14C]acetyl-CoA by a malate-synthase-dependent isotopic exchange in tritiated water, performed in the presence of pyruvate [2].…”