Characterization of a Large Subunit Catalase Truncated by Proteolytic Cleavage

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• 作者：Prashen Chelikani ; Xavier Carpena ; Rosa Perez-Luque ; Lynda J. Donald ; Harry W. Duckworth ; Jacek Switala ; Ignacio Fita ; and Peter C. Loewen
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：April 19, 2005
• 年：2005
• 卷：44
• 期：15
• 页码：5597 - 5605
• 全文大小：629K
• 年卷期：v.44,no.15(April 19, 2005)
• ISSN：1520-4995

文摘

The large subunit catalase HPII from Escherichia coli can be truncated by proteolysis to astructure similar to small subunit catalases. Mass spectrometry analysis indicates that there is someheterogeneity in the precise cleavage sites, but approximately 74 N-terminal residues, 189 C-terminalresidues, and a 9-11-residue internal fragment, including residues 298-308, are removed. Crystal structurerefinement at 2.8 Å reveals that the tertiary and quaternary structure of the native enzyme is retained withonly very subtle changes despite the loss of 36% of the sequence. The truncated variant exhibits a 1.8times faster turnover rate and enhanced sensitivity to high concentrations of H₂O₂, consistent with easieraccess of the substrate to the active site. In addition, the truncated variant is more sensitive to inhibition,particularly by reagents such as aminotriazole and azide which are larger than substrate H₂O₂. The mainchannel leading to the heme cavity is largely unaffected by the truncation, but the lateral channel is shortenedand its entrance widened by removal of the C-terminal domain, providing an explanation for easier accessto the active site. Opening of the entrance to the lateral channel also opens the putative NADPH bindingsite, but NADPH binding could not be demonstrated. Despite the lack of bound NADPH, the compoundI species of both native and truncated HPII are reduced back to the resting state with compound II beingevident in the absorbance spectrum only of the heme b-containing H392A variant.