Proton NMR investigation of the heme active site structure of an engineered cytochrome c peroxidase that mimics manganese peroxidase.
Proton NMR investigation of the heme active site structure of an engineered cytochrome c peroxidase that mimics manganese peroxidase.
复制标题
对模拟锰过氧化物酶的工程细胞色素 c 过氧化物酶的血红素活性位点结构进行质子核磁共振研究。
DOI：
10.1021/bi990235r
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发表时间：
1999
期刊：
影响因子：
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通讯作者：
Lu,Y
中科院分区：
文献类型：
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作者：
Wang,X;Lu,Y
工程化的细胞色素过氧化物酶[MnCcP;参见Yeung，B. K.，等人（1997）Chem.Biol.4，215 - 221]，其紧密模拟锰过氧化物酶（MnP），已经通过一维和二维NMR光谱表征。所有超精细移位共振血红素口袋以及共振催化相关的氨基酸残基在拥挤的抗磁性信封已被分配。从NMR光谱分配和特定质子的NOESY光谱的MnCcP的线增宽模式，工程Mn（II）中心的位置是坚定地确定。此外，我们发现，在CcP的Mn（II）结合位点的创建导致在蛋白质的远端血红素口袋上没有可检测的结构变化。然而，在血红素腔的近端侧观察到显著的结构变化。近端组氨酸的CεH位移和结合的C15 N-的15 N位移表明，与WtCcP相比，MnCcP中的血红素Fe（III）−N（His）键较弱。我们的研究结果表明，CcP中的工程Mn（II）结合位点不仅导致相似的Mn（II）结合亲和力和提高的MNP活性，而且还削弱了模板蛋白CcP的Fe（III）−N（His）键强度，使其键强度与靶蛋白MNP的键强度相似。这里提出的结果有助于阐明设计金属结合位点对酶的局部和全局结构的影响，并为更紧密地模拟MnP的下一代MnCcP的工程设计提供了结构基础。
The heme active site structure of an engineered cytochromecperoxidase [MnCcP; see Yeung, B. K., et al. (1997)Chem. Biol. 4, 215−221] that closely mimics manganese peroxidase (MnP) has been characterized by both one- and two-dimensional NMR spectroscopy. All hyperfine-shifted resonances from the heme pocket as well as resonances from catalytically relevant amino acid residues in the congested diamagnetic envelope have been assigned. From the NMR spectral assignment and the line broadening pattern of specific protons in NOESY spectra of MnCcP, the location of the engineered Mn(II) center is firmly identified. Furthermore, we found that the creation of the Mn(II)-binding site in CcP resulted in no detectable structural changes on the distal heme pocket of the protein. However, notable structural changes are observed at the proximal side of the heme cavity. Both CεH shift of the proximal histidine and15N shift of the bound C15N-suggest a weaker heme Fe(III)−N(His) bond in MnCcP compared to WtCcP. Our results indicate that the engineered Mn(II)-binding site in CcP resulted in not only a similar Mn(II)-binding affinity and improved MnP activity, but also weakened the Fe(III)−N(His) bond strength of the template protein CcP so that its bond strength is similar to that of the target protein MnP. The results presented here help elucidate the impact of designing a metal-binding site on both the local and global structure of the enzyme, and provide a structural basis for engineering the next generation of MnCcP that mimics MnP more closely.
蛋白质和肽的测序。G.
DOI：
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发表时间：
1982
期刊：
影响因子：
--
作者：
M. N. Margolies
通讯作者：
M. N. Margolies