Key role of alkanoic acids on the spectral properties, activity, and active-site stability of iron-containing nitrile hydratase from Brevibacterium R312
Key role of alkanoic acids on the spectral properties, activity, and active-site stability of iron-containing nitrile hydratase from Brevibacterium R312
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DOI：
10.1111/j.1432-1033.1996.0239h.x
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发表时间：
1996-08-15
期刊：
影响因子：
--
通讯作者：
Mansuy, D
中科院分区：
文献类型：
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作者：
Kopf, MA;Bonnet, D;Mansuy, D
正丁酸与来自短杆菌R312的透析腈水合酶的相互作用，其特征在于在680 nm处的电荷转移带和具有Δ g = 0.22的低自旋Fe（III）的典型EPR信号，导致显示不同光谱性质的形式（λ = 710 nm，Δ g = 0.31）。丁酸也作为腈水合酶催化的丙烯腈水合的竞争性抑制剂，K-i值为0.9 mM。酶和丁酸之间的复合物的形成高度依赖于后者的浓度和pH值。当与高水平的丁酸一起储存时，腈水合酶是完全无活性的。活性未复合的酶在用于酶测定的高稀释条件下恢复，而复合形式在酸性pH下有利，并且在pH高于8时不形成。此外，丁酸的抑制效力随pH增加而降低（IC 50从pH 6.2时的0.8 mM增加至pH 8.2时的12 mM）。这些数据表明腈水合酶与丁酸的酸形式以高亲和力相互作用（在pH7.2时K1 ′约为4 μ M）。在pH
Interaction of n-butyric acid with dialyzed nitrile hydratase from Brevibacterium R312, which is characterized by a charge-transfer band at 680 nm and EPR signals typical of a low-spin Fe(III) with Delta g = 0.22, leads to a form displaying different spectral properties (lambda = 710 nm, Delta g = 0.31). Butyric acid also acts as a competitive inhibitor of nitrile-hydratase-catalyzed hydration of acrylonitrile with a K-i value of 0.9 mM. Formation of the complex between the enzyme and butyric acid is highly dependent on the concentration of the latter and on pH. When stored with high levels of butyric acid, nitrile hydratase is completely inactive. The active uncomplexed enzyme is restored under the high dilution conditions used for the enzymatic assays, while the complexed form is favored at acidic pH and is not formed at pH above 8. Furthermore, the inhibitory potency of butyric acid decreases upon increasing pH (IC50 increases from 0.8 mM at pH 6.2 to 12 mM at pH 8.2). These data show that nitrile hydratase interacts with the acid form of butyric acid with a high affinty (K-i' approximate to 4 mu M at pH 7.2). At pH