期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 278, 期 45, 页码 44121-44127出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M304757200
关键词
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资金
- NIAID NIH HHS [AI-43582] Funding Source: Medline
Catalase-peroxidases (KatG), which belong to Class I heme peroxidase enzymes, have high catalase activity and substantial peroxidase activity. The Y229F mutant of Mycobacterium tuberculosis KatG was prepared and characterized to investigate the functional role of this conserved residue unique to KatG enzymes. Purified, overexpressed KatG[ Y229F] exhibited severely reduced steady-state catalase activity while the peroxidase activity was enhanced. Optical stopped-flow experiments showed rapid formation of Compound (Cmpd) II ( oxyferryl heme intermediate) in the reaction of resting KatG[ Y229F] with peroxyacetic acid or chloroperoxybenzoic acid, without detectable accumulation of Cmpd I ( oxyferryl heme pi-cation radical intermediate), the latter being readily observed in the wild-type enzyme under similar conditions. Facile formation of Cmpd III ( oxyferrous enzyme) also occurred in the mutant in the presence of micromolar hydrogen peroxide. Thus, the lost catalase function may be explained in part because of formation of intermediates that do not participate in catalatic turnover. The source of the reducing equivalent required for generation of Cmpd II from Cmpd I was shown by rapid freeze-quench electron paramagnetic resonance spectroscopy to be a tyrosine residue, just as in wild-type KatG. The kinetic coupling of radical generation and Cmpd II formation was shown in KatG[ Y229F]. Residue Y229, which is a component of a newly defined three amino acid adduct in catalase-peroxidases, is critically important for protecting the catalase activity of KatG.
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