The Molecular Mechanism of the Catalase-like Activity in Horseradish Peroxidase

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• 作者：Pablo Campomanes ; Ursula Rothlisberger ; Mercedes Alfonso-Prieto ; Carme Rovira
• 刊名：Journal of the American Chemical Society
• 出版年：2015
• 出版时间：September 2, 2015
• 年：2015
• 卷：137
• 期：34
• 页码：11170-11178
• 全文大小：619K
• ISSN：1520-5126

文摘

Horseradish peroxidase (HRP) is one of the most relevant peroxidase enzymes, used extensively in immunochemistry and biocatalysis applications. Unlike the closely related catalase enzymes, it exhibits a low activity to disproportionate hydrogen peroxide (H₂O₂). The origin of this disparity remains unknown due to the lack of atomistic information on the catalase-like reaction in HRP. Using QM(DFT)/MM metadynamics simulations, we uncover the mechanism for reduction of the HRP Compound I intermediate by H₂O₂ at atomic detail. The reaction begins with a hydrogen atom transfer, forming a peroxyl radical and a Compound II-like species. Reorientation of the peroxyl radical in the active site, concomitant with the transfer of the second hydrogen atom, is the rate-limiting step, with a computed free energy barrier (18.7 kcal/mol, 鈭?6 kcal/mol higher than the one obtained for catalase) in good agreement with experiments. Our simulations reveal the crucial role played by the distal pocket residues in accommodating H₂O₂, enabling formation of a Compound II-like intermediate, similar to catalases. However, out of the two pathways for Compound II reduction found in catalases, only one is operative in HRP. Moreover, the hydrogen bond network in the distal side of HRP compensates less efficiently than in catalases for the energetic cost required to reorient the peroxyl radical at the rate-determining step. The distal Arg and a water molecule in the 鈥渨et鈥?active site of HRP have a substantial impact on the reaction barrier, compared to the 鈥渄ry鈥?active site in catalase. Therefore, the lower catalase-like efficiency of heme peroxidases compared to catalases can be directly attributed to the different distal pocket architecture, providing hints to engineer peroxidases with a higher rate of H₂O₂ disproportionation.