Abstract
An essential protein component of acetyl CoA carboxylase, isolated and extensively purified from cell-free extracts of Escherichia coli, has been identified as malonyl CoA: d-biotin carboxyl transferase. This enzyme, which does not contain covalently-bound biotin, catalyzes carboxyl transfer from malonyl CoA to free d-biotin, a model reaction for the second step in the carboxylation of acetyl CoA. The transcarboxylation product, after stabilization by methylation, was identified as 1′-N-carboxy-d-biotin dimethyl ester. These results indicate the presence of a biotin site on the carboxyl transferase, distinct from that on the biotin carboxylase, which carries out the first step in the overall process. In addition, the carboxyl transferase catalyzes a slower abortive decarboxylation of malonyl CoA, thus indicating that carboxyl abstraction and protonation do not require the participation of biotin.
It is now evident that the half-reactions of acetyl CoA carboxylation are catalyzed by biotin carboxylase and carboxyl transferase. Both components are devoid of biotin and have specific binding sites for free d-biotin, as well as for their respective substrates; hence, the acetyl CoA carboxylation mechanism must involve intersubunit translocation of the carboxylated biotinyl group, which is bound covalently to carboxyl-carrier-protein, a noncatalytic polypeptide.
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Selected References
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