Evidence for distinct ligand-bound conformational states of the multifunctional Escherichia coli repressor of biotin biosynthesis.
Evidence for distinct ligand-bound conformational states of the multifunctional Escherichia coli repressor of biotin biosynthesis.
复制标题
生物素生物合成的多功能大肠杆菌阻遏物的不同配体结合构象状态的证据。
DOI：
10.1021/bi00051a010
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Beckett,D
中科院分区：
文献类型：
--
作者：
Xu,Y;Nenortas,E;Beckett,D
摘要：大肠杆菌生物素合成抑制因子（BirA）是一种独特的转录抑制因子，它可以催化其自身的辅抑制因子的合成，并催化辅因子与必需代谢酶的结合。BirA催化底物ATP和生物素合成生物素-5'-AMP，并将生物素部分从腺苷酸转移到乙酰辅酶a羧化酶亚基的赖氨酸残基上。此外，BirA-bio-5 '-AMP可以特异性地将序列结合到生物素操作符上，从而抑制生物素生物合成基因的转录。结合生物素和生物5′-AMP两种配体与BirA结合的动力学测量以及蛋白质水解消化实验，我们发现了BirA至少有三种离散构象状态的证据。两种配体与BirA结合的停流荧光测量结果表明，该过程涉及碰撞配合物的初始形成，随后是缓慢的构象变化。两种配体的构象变化动力学是不同的，这是两种蛋白质配体复合物热力学稳定性差异的基础。还观察到载脂蛋白BirA的蛋白水解消化率和BirA与两种配体的配合物的不同。综合分析结果表明，apoBirA、BirA-bio-5′-AMP和BirA-biotin复合物在构象上是不同的。BirA是一种35.3 kDa的大肠杆菌蛋白，具有酶和序列特异性DNA结合活性（Cronan, 1989; Barker & Campbell, 1981a, b）（图1）。它的酶功能包括催化合成生物素-5′-腺苷酸（bio-5′-AMP）和催化生物素从腺苷酸转移到乙酰辅酶a羧化酶的生物素羧基载体蛋白（BCCP）的赖氨酸残基。BirA还将site特异地结合到40碱基对生物素操作符上
Revised Manuscript Received October 23, 1995® abstract: The Escherichia coli repressor of biotin biosynthesis (BirA) is a unique transcriptional repressor which catalyzes synthesis of its own corepressor and catalyzes attachment of a cofactor to an essential metabolic enzyme. BirA both catalyzes synthesis of biotinyl-5'-AMP from the substrates ATP and biotin and transfer of the biotin moiety from the adenylate to a lysine residue of a subunit of the acetyl-CoA carboxylase. BirA—bio-5'-AMP, moreover, binds sequence specifically to the biotin operator to repress transcription of the biotin biosynthetic genes. Using a combination of kinetic measurements of binding of the two ligands, biotin and bio-5'-AMP, to BirA as well as proteolytic digestion experiments, we have found evidence for at least threediscrete conformational states of BirA. Results of stopped-flow fluorescence measurements of association of both ligands with BirA indicate that the process involves initial formation of a collision complex followed by a slow conformational change. The kinetics of the conformational change are distinct for the two ligands and are the basis for the differencein the thermodynamic stabilities of the two protein—ligand complexes. Different rates of proteolyticdigestion of apoBirA and complexes of BirA with the two ligands were also observed. Results of the combined approaches indicate that apoBirA, and the BirA—bio-5'-AMP and BirA—biotin complexes are conformationally distinct.BirA is a 35.3 kDa Escherichia coli protein that possesses both enzymatic and sequence-specific DNA binding activities (Cronan, 1989; Barker & Campbell, 1981a, b)(Figure 1). Its enzymatic functions include catalysis of synthesis of biotinyl-5'-adenylate (bio-5'-AMP) and catalysis of transfer of biotin from the adenylate to a lysine residue of the biotin carboxyl carrier protein (BCCP) of the acetyl CoA-carboxylase. BirA also binds site specifically to the 40 base pair biotin operator