Multiple cycles of acetyl‐CoA enolization catalyzed by citrate synthase (CS) and ATP‐citrate lyase (ACL)

Abstract

Enolization of acetyl‐CoA on CS had been identified in Eggerer¡s experiments with [3H]acetyl‐CoA (Biochem. Z. 343: 111, 1965). To test whether CS catalyzes sequential cycles of acetyl‐CoA enolization, we incubated [2H3, 13C2]acetyl‐CoA with pig heart CS + oxaloacetate (OAA) (added as either a bolus, a bolus of OAA/malate, or malate + NAD + malate dehydrogenase). We measured the mass isotopomer distribution (MID) of acetyl‐CoA and citrate. The productions of M4 + M3 + M2 acetyl‐CoA, and of M4 + M3 + M2 citrate revealed up to 3 cycles of acetyl‐CoA enolization. This was confirmed when condensation of unlabeled acetyl‐CoA + OAA was conducted in 50% D2O. Acetyl‐CoA was up to M3 labeled and citrate was M1 labeled. Thus, up to 3 cycles of acetyl‐CoA enolization were catalyzed by CS.We hypothesized that ACL could also catalyze enolization of acetyl‐CoA. We incubated [2,2,4,4‐2H4]citrate + Mg++, CoA, ATP and rat liver ACL. The production of M2 + M1 + M acetyl‐ CoA, and the progressive de‐labeling of acetyl‐CoA demonstrated multiple cycles of enolization of acetyl‐CoA on ACL. This was confirmed when cleavage of unlabeled citrate was conducted in 50% D2O, by the accumulation of M1 + M2 + M3 acetyl‐CoA. Thus, multiple cycles of acetyl‐CoA enolization were catalyzed by ACL.(Supported by the NIDDK Metabolomic Initiative grant R33DK070291).