Guchhait R B, Moss J, Sokolski W, Lane M D
Proc Natl Acad Sci U S A. 1971 Mar;68(3):653-7. doi: 10.1073/pnas.68.3.653.
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.
从大肠杆菌无细胞提取物中分离并广泛纯化得到的乙酰辅酶A羧化酶的一种必需蛋白质成分，已被鉴定为丙二酸单酰辅酶A：d -生物素羧基转移酶。这种酶不含共价结合的生物素，催化羧基从丙二酸单酰辅酶A转移至游离的d -生物素，这是乙酰辅酶A羧化第二步的模拟反应。经甲基化稳定后的转羧化产物被鉴定为1'-N -羧基 - d -生物素二甲酯。这些结果表明羧基转移酶上存在一个生物素位点，与生物素羧化酶上的位点不同，生物素羧化酶负责整个过程的第一步。此外，羧基转移酶催化丙二酸单酰辅酶A进行较慢的无效脱羧反应，因此表明羧基夺取和质子化不需要生物素的参与。现在很明显，乙酰辅酶A羧化的半反应由生物素羧化酶和羧基转移酶催化。这两种成分都不含生物素，对游离的d -生物素以及它们各自的底物都有特异性结合位点；因此，乙酰辅酶A羧化机制必定涉及羧化生物素基团在亚基间的转运，该基团共价结合于羧基载体蛋白，一种非催化性多肽。
