Functional and structural analysis of catalase oxidized by singlet oxygen
Functional and structural analysis of catalase oxidized by singlet oxygen
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DOI：
10.1016/j.biochi.2004.10.014
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发表时间：
2005-02-01
期刊：
影响因子：
3.9
通讯作者：
Hansberg, W
中科院分区：
文献类型：
--
作者：
Díaz, A;Muñoz-Clares, RA;Hansberg, W
从粗糙脉孢菌无性孢子中纯化的过氧化氢酶-1（CAT-1）被单线态氧氧化，产生具有不同电泳迁移率的活性酶形式。这些酶形式在体内应激条件下和在无性形态发生转变开始时的发育过程中检测到。CAT-1血红素B被单线态氧氧化为血红素d。在这里，我们描述的功能和结构的非氧化的酶与完全氧化的比较。使用宽H2 O2浓度范围（0.01-3.0 M）。在两种酶中发现非双曲线饱和动力学，表明动力学复杂性不是由血红素氧化引起的。动力学与存在两种底物亲和力相差10倍以上的活性位点一致。饱和曲线中的一个或两个的正协同性是可能的。在22 ℃下获得的动力学常数略有不同，两种组分的反应的表观活化能没有显着差异。两种酶的蛋白质荧光和圆二色性几乎相同，表明氧化没有总的构象变化。氰化物抑制的敏感性增加表明在氧化过氧化氢酶的活性位点的局部变化。氧化过氧化氢酶对高温、高胍离子浓度和枯草杆菌蛋白酶消化的耐受性较低。在酸性pH下，它也比非氧化酶稳定性差。总体数据表明，氧化酶在结构上不同于非氧化酶。尽管它保留了非氧化酶的大部分显著的稳定性和催化效率。由于细胞中的酶可以在生理条件下被氧化，过氧化氢酶的功能和结构特性的保存可能已经通过进化被选择，以确保在氧化应激条件下的活性酶。(c)2004年，Elsevier SAS。All rights reserved.
Purified catalase-1 (CAT-1) from Neurospora crassa asexual spores is oxidized by singlet oxygen giving rise to active enzyme forms with different electrophoretic mobility. These enzyme forms are detected in vivo under stress conditions and during development at the start of the asexual morphogenetic transitions. CAT-1 heme b is oxidized to heme d by singlet oxygen. Here, we describe functional and structural comparisons of the non-oxidized enzyme with the fully oxidized one. Using a broad H2O2 concentration range (0.01-3.0 M). non-hyperbolic saturation kinetics was found in both enzymes, indicating that kinetic complexity does not arise from heme oxidation. The kinetics was consistent with the existence of two kinds of active sites differing more than 10-times in Substrate affinity. Positive cooperativity for one or both of the saturation curves is possible. Kinetic constants obtained at 22 degrees C varied slightly and apparent activation energies for the reaction of both components are not significantly different. Protein fluorescence and circular dicroism of the two enzymes were nearly identical, indicating, no gross conformational change with oxidation. Increased sensitivity to inhibition by cyanide indicated a local change at the active site in the oxidized catalase. Oxidized catalase was less resistant to hi-h temperatures, high guanidinium ion concentration, and digestion with subtilisin. It was also less stable than the non-oxidized enzyme at an acid pH. The overall data show that the oxidized enzyme is Structurally different from the non-oxidized one. although it conserves most of the remarkable stability and catalytic efficiency of the non-oxidized enzyme. Because the enzyme in the cell can be oxidized under physiological conditions, preservation of functional and structural properties of catalase Could have been selected through evolution to assure an active enzyme under oxidative stress conditions. (c) 2004 Elsevier SAS. All rights reserved.