Identification of two electron-transfer sites in ascorbate peroxidase using chemical modification, enzyme kinetics, and crystallography
Identification of two electron-transfer sites in ascorbate peroxidase using chemical modification, enzyme kinetics, and crystallography
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DOI：
10.1021/bi981958y
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发表时间：
1998-12-15
期刊：
影响因子：
2.9
通讯作者：
Poulos, TL
中科院分区：
文献类型：
--
作者：
Mandelman, D;Jamal, J;Poulos, TL
化学和诱变修饰结合X射线晶体学已被用来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以阻断APX氧化抗坏血酸的能力，但不能阻断其他小的芳香族酚底物。DTNB修饰的APX（APX-TNB）在抗坏血酸作为底物时仅表现出1.3%的野生型活性，但当使用芳香族底物愈创木酚或焦性没食子酸时表现出完全活性。停流研究表明，APX-TNB通常与过氧化物反应得到化合物I，但抗坏血酸盐还原化合物I和II的速率显著减慢。Cys 32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，不带电的芳香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至Cys 32的TNB基团的清晰电子密度，表明完全和特异性修饰。看来抗坏血酸盐位点被DTNB修饰阻断，DTNB修饰从暴露的δ-血红素边缘很好地去除，其中芳香族底物被认为是结合的。这是第一个实验证据表明，抗坏血酸氧化不发生在暴露的血红素边缘，但在一个替代的结合位点附近的Arg 172和血红素丙酸附近的Cys 32。
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.