The role of Ala134 in controlling substrate binding and reactivity in ascorbate peroxidase.
The role of Ala134 in controlling substrate binding and reactivity in ascorbate peroxidase.
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DOI：
10.1016/j.jinorgbio.2017.12.018
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发表时间：
2017-12
影响因子：
3.9
通讯作者：
D. Turner;L. Lad;H. Kwon;J. Basran;K. Carr;P. Moody;E. Raven
中科院分区：
文献类型：
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作者：
D. Turner;L. Lad;H. Kwon;J. Basran;K. Carr;P. Moody;E. Raven
抗坏血酸过氧化物酶（APX）是一类血红素过氧化物酶。它有两个结合底物的位点。一个靠近γ-血红素边缘，用于抗坏血酸的氧化；另一个位于δ-血红素边缘，用于结合芳香底物[Gumiero et al.,(2010)]。物化学。生物工程学报，2003,13 - 20。在这项工作中，我们通过用脯氨酸残基取代APX中的Ala134，研究了控制δ-血红素边缘结合的结构因素，脯氨酸残基在其他II类和III类过氧化物酶中更常见。动力学数据表明，脯氨酸替代Ala134对抗坏血酸或愈创木酚氧化的催化机制影响不大。苯基肼的化学修饰表明，靠近δ-血红素边缘的血红素可及性仅受取代的影响很小。我们得出结论，A134P突变本身不足以实质性地影响APX对δ-血红素边缘结合的芳香底物的反应性。这些数据与最近APX （APEX）在细胞成像中的应用有关。
Ascorbate peroxidase (APX) is a class I heme peroxidase. It has two sites for binding of substrates. One is close to the γ-heme edge and is used for oxidation of ascorbate; the other is at the δ-heme edge and is used for binding of aromatic substrates [Gumiero et al., (2010) Arch. Biochem. Biophys. 500, 13–20]. In this work, we have examined the structural factors that control binding at the δ-heme edge by replacement of Ala134 in APX with a proline residue that is more commonly found in other class II and III peroxidases. Kinetic data indicate that replacement of Ala134 by proline has only a small effect on the catalytic mechanism, or the oxidation of ascorbate or guaiacol. Chemical modification with phenylhydrazine indicates that heme accessibility close to the δ-heme edge is only minorly affected by the substitution. We conclude that the A134P mutation alone is not enough to substantially affect the reactivity of APX towards aromatic substrates bound at the δ-heme edge. The data are relevant to the recent application of APX (APEX) in cellular imaging.