Characterization of the Isolated Transferase Subunit of Citrate Lyase as a CoA‐Transferase. Evidence against a Covalent Enzyme‐Substrate Intermediate

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Abstract
1. The largest subunit of citrate lyase from Klebsiella aerogenes represents an acetyl‐thio‐acyl carrier protein: citrate acyl carrier protein transferase but is shown to be also an acetyl‐CoA: citrate CoA‐transferase.2. The enzyme catalyzes an exchange of acetyl residues between acetyl‐thio‐acyl carrier protein or acetyl‐CoA and acetate. The reaction occurs in the presence and, at a much lower rate, also in the absence of acetate. The citrate‐independent reaction was attributed to a substitution of citrate by acetate as a substrate analogue.3. Additionally, the isolated enzyme, in the presence of acetyl‐CoA and citrate, catalyzed the formation of (3S)‐citryl‐CoA and was thus unequivocally shown to be a CoA‐transferase.4. The catalytic efficiency of the enzyme in the CoA‐transfer reaction is similar to that of other CoA‐transferases. It was concluded that the transferase subunit of citrate lyase and the classical CoA‐transferases could belong to one family to enzymes.5. Attempts to demonstrate this relationship were based on a comparison of the mechanism of action of the transferase subunit of citrate lyase with that established for CoA‐transferases. These form enzyme‐CoA and ‘anhydride’ intermediates.6. Evidence is presented for the generation of ‘anhydride’ intermediates on the transferase subunit of citrate lyase. The ‘anhydrides’ are ‘citric acetic anhydride’ and ‘acetic anhydride’ in the acetyl exchange reactions with and without citrate, respectively.7. In contrast to other CoA‐transferases, however, the transferase‐catalyzed CoA‐transfer from acetyl‐CoA to citrate, apparently involves no enzyme‐CoA intermediate. Neither the results of several different chemical approaches nor the kinetics were compatible with the formation of this intermediate.8. An explanation is proposed and the consequences are discussed.