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首页> 外文期刊>Biochemistry >Identification of two electron-transfer sites in ascorbate peroxidase using chemical modification, enzyme kinetics, and crystallography
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Identification of two electron-transfer sites in ascorbate peroxidase using chemical modification, enzyme kinetics, and crystallography

机译：使用化学修饰，酶动力学和晶体学鉴定抗坏血酸过氧化物酶中的两个电子转移位点

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Chemical and mutagenic modification combined with X-ray crystallography has been used to probe the ascorbate binding site in ascorbate peroxidase (APX). Chemical modification of the single Cys residue in APX with Ellman's reagent (DTNB) blocks the ability of APX to oxidize ascorbate but not other small aromatic phenolic substrates. DTNB-modified APX (APX-TNB) exhibits only 1.3% wildtype activity when ascorbate is used as the substrate but full activity when aromatic substrate, guaiacol or pyrogallol, are used. Stopped-flow studies show that APX-TNB reacts normally with peroxide to give compound I but that the rates of reduction of both compounds I and II by ascorbate are dramatically slowed. Conversion of Cys32 to Ser leads to approximate to 70% drop in ascorbate peroxidase activity with no effect on guaiacol peroxidase activity. These results indicate that uncharged aromatic substrates and the anionic ascorbate molecule interact with different sites on APX. The 2.0 Angstrom X-ray crystal structure of APX-TNB shows clear electron density for the TNB group covalently attached to Cys32 in all four molecules of the asymmetric unit, indicating complete and specific modification. It appears that the ascorbate site is blocked by DTNB modification which is well removed from the exposed delta-heme edge where aromatic substrates are thought to bind. This is the first experimental evidence indicating that ascorbate oxidation does not occur at the exposed heme edge but at an alternate binding site in the vicinity of Cys32 near Arg172 and the heme propionates. [References: 25]
机译：化学和诱变修饰结合X射线晶体学已用于探测抗坏血酸过氧化物酶（APX）中的抗坏血酸结合位点。用Ellman试剂（DTNB）对APX中的单个Cys残基进行化学修饰可阻止APX氧化抗坏血酸的能力，但不能氧化其他小的芳族酚醛底物。当使用抗坏血酸盐作为底物时，DTNB修饰的APX（APX-TNB）仅表现出1.3％的野生型活性，而当使用芳香族底物，愈创木酚或邻苯三酚时，则具有完整的活性。停止流研究表明，APX-TNB与过氧化物正常反应生成化合物I，但抗坏血酸对化合物I和II的还原速度大大降低。 Cys32转化为Ser导致抗坏血酸过氧化物酶活性下降约70％，而对愈创木酚过氧化物酶活性没有影响。这些结果表明，不带电荷的芳族底物和阴离子抗坏血酸分子与APX上的不同位点相互作用。 APX-TNB的2.0埃X射线晶体结构显示，在不对称单元的所有四个分子中共价连接到Cys32的TNB基团具有清晰的电子密度。似乎抗坏血酸位点被DTNB修饰所阻断，而DTNB修饰已从暴露的δ-血红素边缘（认为芳香族底物会结合）上很好地去除了。这是第一个实验证据，表明抗坏血酸的氧化不会在暴露的血红素边缘发生，而是在靠近Arg172的Cys32和血红素丙酸酯附近的一个替代结合位点发生。 [参考：25]

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