Kinetics of biotinyl-5'-adenylate synthesis catalyzed by the Escherichia coli repressor of biotin biosynthesis and the stability of the enzyme-product complex.
Kinetics of biotinyl-5'-adenylate synthesis catalyzed by the Escherichia coli repressor of biotin biosynthesis and the stability of the enzyme-product complex.
复制标题
大肠杆菌生物素生物合成阻遏物催化的生物素-5-腺苷酸合成动力学以及酶-产物复合物的稳定性。
DOI：
10.1021/bi00189a041
复制
发表时间：
1994
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;Beckett,D
大肠杆菌生物素生物合成阻遏物既是生物素连接酶，又是生物素生物合成操纵子转录起始的阻遏物。小分子生物素基-5 '-腺苷酸（bio-5'-AMP）是生物素连接反应的中间体，也是BirA与序列特异性DNA结合的正变构效应物。由底物生物素和ATP合成腺苷酸由BirA催化。虽然BirA和其他生物素全酶合成酶已成为生物化学研究的主题，但没有直接测量生物-5 '-AMP合成反应的报道。关于腺苷酸合成的机制和动力学参数还没有任何资料。除了缺乏动力学信息外，BirA-bio-5 '-AMP复合物的热力学稳定性也是未知的。由于BirA-腺苷酸复合物在生物素调节系统中起着关键作用，因此动力学和生物学信息对于定量了解该系统至关重要。我们已经开发了一种用于测量生物-5 '-AMP合成的时间过程的方法。这些测量的结果表明，时间过程的特点是由一个缓慢的线性阶段的初始突发。爆发对应于每摩尔酶快速合成1摩尔产物，并且缓慢线性相的速率受到产物从酶释放的限制。已分析数据以获得BirA-bio-5 '-AMP复合物的解离速率常数3.9 X Kh 4s-1。已利用停流荧光测量获得用于络合物形成的4.9 X 106M_1s_1的双分子缔合速率常数。由这两个动力学参数确定了BirA-bio-5 ′-AMP相互作用的平衡解离常数为7.9 × 10~（-1）M，复合物形成的吉布斯自由能为-13.5kcal/mol。
Revised Manuscript Received March 31, 1994· abstract: The Escherichia coli repressor of biotin biosynthesis is both a biotin ligase and the repressor of transcriptional initiation at the biotin biosynthetic operon. The small molecule, biotinyl-5'-adenylate (bio-5'-AMP), is the intermediate in the biotin ligation reaction and the positive allosteric effector for sequence-specific DNA binding by BirA. Synthesis of the adenylate from the substrates biotin and ATP is catalyzed by BirA. Although BirA and otherbiotin holoenzyme synthetases havebeen the subject of biochemical studies, no direct measurements of the bio-5'-AMP synthesis reaction have been reported. No information relating to the mechanism and kinetic parameters governing adenylatesynthesis is available. In addition to this lack of kineticinformation, the thermodynamic stability of the BirA-bio-5'-AMP complex is not known. Since the BirA-adenylate complex plays a pivotal role in the biotin regulatory system, both the kinetic and thermodynamicinformation are essential to a quantitative understanding of the system. We have developed a method for measuring the time course of bio-5'-AMP synthesis. The results of these measurements indicate that the time course is characterized by an initial burst followed by a slow linear phase. The burst corresponds to the rapid synthesis of 1 mol of product per mole of enzyme, and the rate of the slow linear phase is limited by the release of product from the enzyme. The data have been analyzed to obtain the dissociation rate constant of 3.9 X Kh4 s-1 for the BirA-bio-5'-AMP complex. Stopped-flow fluorescence measurements have been utilized to obtain the bimolecular association rate constant of 4.9 X 106 M" 1 s_1 for complex formation. The equilibrium dissociationconstant for the BirA-bio-5'-AMP interaction of 7.9 X 10~ n M has been determined from the two kinetic parameters, and the resulting Gibbs free energy for complex formation is-13.5 kcal/mol.
登录
查看更多内容
生物素生物合成酶水平降低的大肠杆菌突变体
影响因子：
3.2
作者：
C. Pai
通讯作者：
C. Pai
从 D-生物素 5-腺苷酸和脱辅基羧化酶酶促合成丙酰辅酶 A 全羧化酶。
DOI：
10.1016/s0021-9258(18)97532-0
发表时间：
1965
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
L. Siegel;J. Foote;M. J. Coon
通讯作者：
M. J. Coon
DOI：
10.1073/pnas.76.11.5592
发表时间：
1979-01-01
影响因子：
11.1
作者：
PRAKASH, O;EISENBERG, MA
通讯作者：
EISENBERG, MA
大肠杆菌 K-12 中生物素的主动转运
影响因子：
3.2
作者：
O. Prakash;M. Eisenberg
通讯作者：
M. Eisenberg
在人体细胞中制造的融合嵌合蛋白
影响因子：
64.5
作者：
P. Borst;R. Benne;H. Tabak
通讯作者：
H. Tabak