Function of Escherichia coli biotin carboxylase requires catalytic activity of both subunits of the homodimer
Function of Escherichia coli biotin carboxylase requires catalytic activity of both subunits of the homodimer
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DOI：
10.1074/jbc.m104102200
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发表时间：
2001-08-10
影响因子：
4.8
通讯作者：
Cronan, JE
中科院分区：
文献类型：
--
作者：
Janiyani, K;Bordelon, T;Cronan, JE
生物素羧化酶催化生物素的ATP依赖性羧化，是多酶的组成部分之一。复合乙酰辅酶A羧化酶，催化脂肪酸合成的第一个关键步骤。大肠杆菌生物素羧化酶易于与乙酰辅酶A羧化酶复合物的其它组分分离，从而保持酶活性。生物素羧化酶的三维结构，由X-射线晶体学测定，表明该酶是一个homodimer的两个活性位点，其中每个亚基包含一个完整的活性位点组成。为了了解每个亚基如何对生物素羧化酶的整体功能做出贡献，我们制作了杂交分子，其中一个亚基具有野生型活性位点，另一个亚基含有已知显著影响酶活性的活性位点突变。两个基因中的一个在其N末端编码多组氨酸标签，而另一个基因具有N末端FLAG表位标签。这两个基因被组装成一个小操纵子，诱导这两种酶的高水平表达。通过在固定化镍螯合物和抗FLAG亲和基质柱上的连续色谱步骤获得由具有野生型活性位点的一个亚基和具有突变体活性位点的第二亚基组成的“杂交”二聚体。生物素羧化酶二聚体，其中两个亚基是野生型的体外动力学研究表明，N-末端标签的存在下，不改变酶的活性。然而，动力学测定的混合二聚体生物素羧化酶分子，其中一个亚基有一个活性位点突变（R292 A，N290 A，K238 Q或E288 K）和其他亚基有一个野生型活性位点，导致在39-，28-，94-和285-倍的这些酶的活性分别下降。这些突变体亚基的显性负效应也在体内检测到的[C-14]乙酸标记的细胞脂质通过监测脂肪酸生物合成的速率。从诱导型阿拉伯糖启动子的突变生物素羧化酶基因的表达导致脂肪酸合成速率相对于表达野生型基因的相同菌株显著降低。因此，体外和体内数据都表明，生物素羧化酶的两个亚基都是活性所必需的，并且这两个亚基在酶功能期间必须进行通信。
Biotin carboxylase catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme. complex acetyl-CoA carboxylase that catalyzes the first committed step in fatty acid synthesis. The Escherichia coli biotin carboxylase is readily isolated from the other components of the acetyl-CoA carboxylase complex such that enzymatic activity is retained. The three-dimensional structure of biotin carboxylase, determined by x-ray crystallography, demonstrated that the enzyme is a homodimer consisting of two active sites in which each subunit contains a complete active site. To understand how each subunit contributes to the overall function of biotin carboxylase, we made hybrid molecules in which one subunit had a wild-type active site, and the other subunit contained an active site mutation known to significantly affect the activity of the enzyme. One of the two genes encoded a poly-histidine tag at its N terminus, whereas the other gene had an N-terminal FLAG epitope tag. The two genes were assembled into a mini-operon that was induced to give high level expression of both enzymes. "Hybrid" dimers composed of one subunit with a wild-type active site and a second subunit having a mutant, active site were obtained by sequential chromatographic steps on columns of immobilized nickel chelate and anti-FLAG affinity matrices. In vitro kinetic studies of biotin carboxylase dimers in which both subunits were wild type revealed that the presence of the N-terminal tags did not alter the activity of the enzyme. However, kinetic assays of hy brid dimer biotin carboxylase molecules in which one subunit had an active site mutation (R292A, N290A, K238Q or E288K) and the other subunit had a wild-type active site resulted in 39-, 28-, 94-, and 285-fold decreases in the activity of these enzymes, respectively. The dominant negative effects of these mutant subunits were also detected in vivo by monitoring the rate of fatty acid biosynthesis by [C-14]acetate labeling of cellular lipids. Expression of the mutant biotin carboxylase genes from an inducible arabinose promoter resulted in a significantly reduced rate of fatty acid synthesis relative to the same strain that expressed the wild type gene. Thus, both the in vitro and in vivo data indicate that both subunits of biotin carboxylase are required for activity and that the two subunits must be in communication during enzyme function.