Catalytic activities and structural properties of horseradish peroxidase distal His42->Glu or Gln mutant
Catalytic activities and structural properties of horseradish peroxidase distal His42->Glu or Gln mutant
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DOI：
10.1021/bi970906q
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发表时间：
1997-08-12
期刊：
影响因子：
2.9
通讯作者：
Morishima, I
中科院分区：
文献类型：
--
作者：
Tanaka, M;Ishimori, K;Morishima, I
末端组氨酸（His）在过氧化物酶中高度保守，被认为是过氧化物酶反应循环的主要酸碱催化剂。然而，最近，来自海洋真菌烟状卡尔达酵母（Caldariomyces fumago）的氯过氧化物酶的X-射线结构已经揭示谷氨酸位于大多数过氧化物酶具有组氨酸残基的位置，这表明谷氨酸（Glu）中的羧基也可以帮助过氧化物中的O-O键的裂解[Sundaramoorthy，M.，Terner，J.，& Poulos，T. L.（1995）Structure 3，1367-1377];为了研究谷氨酸在远端腔的催化作用，制备了两种辣根过氧化物酶突变体，其中远端His 42已被Glu（H42 E）或Gln（H42 Q）取代。化合物I在H42 E突变体中的形成速率显著大于H42 Q突变体，表明远端Glu可以起到一般酸碱催化剂的作用。然而，与天然酶相比，H42 E突变体的过氧化物酶活性仍然较低。CD，共振拉曼和EPR光谱的基础上，有人建议，远端Glu的碱性低于远端组氨酸的碱性，远端Glu的位置不是固定在最佳位置作为催化氨基酸残基，虽然没有显着的结构变化周围血红素环境被检测到。较低的碱性和远端Glu的不适当定位将使过氧化物酶反应的血红素-H2 O2-远端Glu三元中间体不稳定。突变体的另一个特征是过氧合酶活性的增强。由于在H42 E突变体中过氧合酶活性显著增强，因此远端Glu对于促进过氧合酶活性以及由氨基酸取代引起的远端腔扩大也是至关重要的。这些观察结果表明，远端的氨基酸残基是必不可少的过氧化物酶的功能和周围的远端腔的细微构象变化将控制过氧化物酶的催化反应。
The distal histidine (His) is highly conserved in peroxidases and has been considered to play a major role as a general acid-base catalyst for peroxidase reaction cycle. Recently, however, the X-ray structure of chloroperoxidase from the marine fungus Caldariomyces fumago has revealed that a glutamic acid is located at the position where most of the peroxidase has a histidine residue, suggesting that the carboxyl group in the glutamic acid (Glu) can also assist cleavage of an O-O bond in peroxides [Sundaramoorthy, M., Terner, J., & Poulos, T. L. (1995) Structure 3, 1367-1377]; In order to investigate catalytic roles of the glutamic acid at the distal cavity, two horseradish peroxidase mutants were prepared, in which the distal His42 has been replaced by Glu (H42E) or Gln (H42Q). The formation rate of compound I in the H42E mutant was significantly greater than that for the H42Q mutant, indicating that the distal Glu can play a role as a general acid-base catalyst. However, the peroxidase activity of the H42E mutant was still lower, compared to that for native enzyme. On the basis of the CD, resonance Raman, and EPR spectra, it was suggested that the basicity of the distal Glu is lower than that of the distal His and the position of the distal Glu is not fixed at the optimal position as a catalytic amino acid residue, although no prominent structural changes around heme environment were detected. The less basicity and improper positioning of the distal Glu would destabilize the heme-H2O2-distal Glu ternary intermediate for the peroxidase reaction. Another characteristic feature in the mutants was the enhancement of the peroxygenase activity. Since the peroxygenase activity was remarkably enhanced in the H42E mutant, the distal Glu is also crucial to facilitate the peroxygenase activity as well as the enlarged distal cavity caused by the amino acid substitution. These observations indicate that the distal amino acid residue is essential for function of peroxidases and subtle conformational changes around the distal cavity would control the catalytic reactions in peroxidase.