Protein Radicals in Fungal Versatile Peroxidase
Protein Radicals in Fungal Versatile Peroxidase
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真菌多功能过氧化物酶中的蛋白质自由基
DOI：
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发表时间：
2009
影响因子：
4.8
通讯作者：
Angel T. Martı́nez
中科院分区：
文献类型：
--
作者：
F. J. Ruiz;R. Pogni;M. Morales;S. Giansanti;M. Maté;A. Romero;M. Martínez;R. Basosi;Angel T. Martı́nez
木质素降解过氧化物酶是一组生物技术上感兴趣的酶，通过暴露的蛋白质自由基氧化高氧化还原电位的芳香族化合物。低温EPR的Plepherynamic通用过氧化物酶（VP）揭示，第一次在真菌过氧化物酶，存在的双电子（VPI）和单电子（VPII）激活形式的酶的一个乙酰基自由基。使用定点诱变将该色氨酸（Trp-164）替换为酪氨酸和组氨酸残基。没有观察到晶体结构的变化，表明改变的行为完全是由于引入的突变。EPR揭示了在VPI和VPII的W164 Y变体的酪氨酰基自由基的形成。然而，在W164 H变体中没有检测到蛋白质自由基，其VPI光谱表明与无活性W164 S变体相同的卟啉自由基。停流分光光度法表明，W164 Y突变降低了10倍的表观二阶速率常数VPI还原（k2 app）藜芦醇（VA），相比之下，超过50倍的减少W164 S，揭示了一些催化活性的酪氨酸自由基。其一级速率常数（k2）的影响大于解离常数（KD 2）。此外，通过VA的VPII还原受到上述突变的损害，这表明Trp-164自由基参与了VPI和VPII的催化。低的一级速率常数（k3）的值是相似的W164 Y，W164 H，和W164 S的变体，表明VPII中的酪氨酰自由基不能氧化VA（与VPI观察到的相反）。VPII自还原也被抑制，揭示了Trp-164参与了这一自催化过程。
Lignin-degrading peroxidases, a group of biotechnologically interesting enzymes, oxidize high redox potential aromatics via an exposed protein radical. Low temperature EPR of Pleurotus eryngii versatile peroxidase (VP) revealed, for the first time in a fungal peroxidase, the presence of a tryptophanyl radical in both the two-electron (VPI) and the one-electron (VPII) activated forms of the enzyme. Site-directed mutagenesis was used to substitute this tryptophan (Trp-164) by tyrosine and histidine residues. No changes in the crystal structure were observed, indicating that the modified behavior was due exclusively to the mutations introduced. EPR revealed the formation of tyrosyl radicals in both VPI and VPII of the W164Y variant. However, no protein radical was detected in the W164H variant, whose VPI spectrum indicated a porphyrin radical identical to that of the inactive W164S variant. Stopped-flow spectrophotometry showed that the W164Y mutation reduced 10-fold the apparent second-order rate constant for VPI reduction (k2app) by veratryl alcohol (VA), when compared with over 50-fold reduction in W164S, revealing some catalytic activity of the tyrosine radical. Its first-order rate constant (k2) was more affected than the dissociation constant (KD2). Moreover, VPII reduction by VA was impaired by the above mutations, revealing that the Trp-164 radical was involved in catalysis by both VPI and VPII. The low first-order rate constant (k3) values were similar for the W164Y, W164H, and W164S variants, indicating that the tyrosyl radical in VPII was not able to oxidize VA (in contrast with that observed for VPI). VPII self-reduction was also suppressed, revealing that Trp-164 is involved in this autocatalytic process.
控制酵母细胞色素 c 过氧化物酶中血红素铁和自由基位点之间氧化当量的转移。
DOI：
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发表时间：
1983
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Ho,PS;Hoffman,BM;Kang,CH;Margoliash,E
通讯作者：
Margoliash,E
影响因子：
56.9
作者：
PELLETIER, H;KRAUT, J
通讯作者：
KRAUT, J