LOCUS OF ACTION OF ACETYL COA IN THE BIOTIN CARBOXYLATION REACTION OF PYRUVATE-CARBOXYLASE
LOCUS OF ACTION OF ACETYL COA IN THE BIOTIN CARBOXYLATION REACTION OF PYRUVATE-CARBOXYLASE
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DOI：
10.1021/bi00210a024
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发表时间：
1993-11-30
期刊：
影响因子：
2.9
通讯作者：
ATTWOOD, PV
中科院分区：
文献类型：
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作者：
ATTWOOD, PV
根据Phillips等人的描述，酶与(HCO3-)-C-14，镁-三磷酸腺苷和镁离子孵育形成[C-14]羧基磷酸酶复合体，并通过凝胶过滤分离。[(1992)生化31,9445-9450]。当研究[C-14]羧基从络合物转移到丙酮酸的时间过程时，发现在第一个时间点(15 S)，在有或没有乙酰辅酶A的情况下，[C-14]草酸酯的形成是相同的。然而，在没有乙酰辅酶A的情况下，固定在[C-14]草酸乙酯中的放射性在随后的15分钟内迅速下降，而在乙酰辅酶A存在的情况下，[C-14]草酸乙酸酯的形成一直持续到大约10分钟。在没有乙酰辅酶A的情况下，[C-14]草酸乙酯的降解率被发现是由于该酶对草酸乙酯的酶依赖脱羧化。将分离的[C-14]羧基磷酸酶复合体与MgADP和Mg~(2+)孵育，在添加乙酰辅酶A和丙酮酸的情况下，[C-14]草乙酸酯的生成没有明显减少。将分离的[P-32]羧基磷酸酶复合体与丙酮酸孵育，在添加镁ADP和镁离子的情况下，[γ-P-32]ATP的生成没有明显减少。这一新的证据使人怀疑乙酰辅酶A激活酶的位置是促进羧基从羧基磷酸转移到生物素的位置，以及分离的酶中间体的身份[Phillips等人]。(1992)生物化学31,9445-9450]。
The [C-14]carboxyphospho-enzyme complex formed by incubation of the enzyme with (HCO3-)-C-14, MgATP, and Mg2+ was prepared and isolated by gel filtration as described by Phillips et al. [(1992) Biochemistry 31, 9445-9450]. When time courses of transfer of the [C-14]carboxyl group from the complex to pyruvate were studied, it was found that at the first time point (15 s) the formation of [C-14]oxalacetate was the same in the presence or absence of acetyl CoA. However, in the absence of acetyl CoA, the radioactivity fixed in [C-14]oxalacetate declined rapidly over the subsequent 15 min, whereas in the presence of acetyl CoA the formation of [C-14]oxalacetate continued up to about 10 min. The decline in [C-14]oxalacetate in the absence of acetyl CoA was found to be due to enzyme-dependent decarboxylation of the oxalacetate by the enzyme. Incubation of the isolated [C-14]carboxyphospho-enzyme complex with MgADP and Mg2+ resulted in no significant reduction in the formation of [C-14]oxalacetate on addition of acetyl CoA and pyruvate. Incubation of the isolated [P-32]carboxyphospho-enzyme complex with pyruvate resulted in no significant reduction in the formation of [gamma-P-32]ATP on the addition of MgADP and Mg2+. This new evidence casts doubt on the suggested locus of activation of the enzyme by acetyl CoA being the facilitation of the transfer of the carboxyl group from carboxyphosphate to biotin and indeed on the identity of the isolated enzyme intermediate [Phillips et al. (1992) Biochemistry 31, 9445-9450].