Reconstitution and characterization of the polynuclear iron-sulfur cluster in pyruvate formate-lyase-activating enzyme -: Molecular properties of the holoenzyme form
Reconstitution and characterization of the polynuclear iron-sulfur cluster in pyruvate formate-lyase-activating enzyme -: Molecular properties of the holoenzyme form
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DOI：
10.1074/jbc.273.9.4897
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发表时间：
1998-02-27
影响因子：
4.8
通讯作者：
Knappe, J
中科院分区：
文献类型：
--
作者：
Külzer, R;Pils, T;Knappe, J
大肠杆菌的丙酮酸甲酸裂解酶（PFL）的活性形式中所含的甘氨酰自由基（Gly-734）由S-腺苷甲硫氨酸依赖性丙酮酸甲酸裂解酶活化酶（PFL活化酶）产生。由活化酶产生的5 ′-脱氧腺苷自由基中间体已被认为是提取PFL中甘氨酸734残基的pro-S氢的物质（Frey，M.，Rothe，M.，瓦格纳，A. F.五、和Knappe，J.（1994）J.Biol.Chem.269，12432-12437）。为了能够对该系统进行机理研究，我们已经制定了一种方便的大规模制备功能活性PFL活化酶的方法。根据W-可见光谱和EPR光谱信息，先前推断的金属辅因子被鉴定为氧化还原可互变的多核铁硫簇，最可能是[4Fe-4S]型，通过定点诱变确定Cys-29的Cys-> Ser置换，Cys-33和Cys-36对于产生活性全酶至关重要，凝胶过滤层析显示脱辅基酶和全酶形式均为单体结构（28 kDa），铁硫簇互补被证明是有效结合的先决条件腺苷甲硫氨酸，其诱导还原酶形式（[4Fe-4S]（+））的EPR信号形状从轴对称到菱形对称的特征性移位。
The glycyl radical (Gly-734) contained in the active form of pyruvate formate-lyase (PFL) of Escherichia coli is generated by the S-adenosylmethionine-dependent pyruvate formate-lyase-activating enzyme (PFL activase), A 5'-deoxyadenosyl radical intermediate produced by the activase has been suggested as the species that abstracts the pro-S hydrogen of the glycine 734 residue in PFL (Frey, M., Rothe, M., Wagner, A. F. V., and Knappe, J. (1994) J. Biol. Chem. 269, 12432-12437). To enable mechanistic investigations of this system we have worked out a convenient large scale preparation of functionally competent PFL activase from its apoform, The previously inferred metallic cofactor was identified as redox-interconvertible polynuclear iron-sulfur cluster, most probably of the [4Fe-4S] type, according to W-visible and EPR spectroscopic information, Cys --> Ser replacements by site-directed mutagenesis determined Cys-29, Cys-33, and Cys-36 to be essential to yield active holoenzyme, Gel filtration chromatography showed a monomeric structure (28 kDa) for both the apoenzyme and holoenzyme form, The iron-sulfur cluster complement proved to be a prerequisite for effective binding of adenosylmethionine, which induces a characteristic shift of the EPR signal shape of the reduced enzyme form ([4Fe-4S](+)) from axial to rhombic symmetry.