title: Comparison of isoniazid oxidation catalyzed by bacterial catalase-peroxidases and horseradish peroxidase.
abstract: The physical properties and activities of the purified catalase-peroxidase hydroperoxidase I (HPI) of Escherichia coli (EcHPI) and HPI with a carboxyl-terminal extension of Mycobacterium tuberculosis (MtHPI-e) are compared to those of commercial preparations of horseradish peroxidase (HRP). The catalase-peroxidase proteins had similar absorption spectra and differed primarily in that MtHPI-e has a higher peroxidatic to catalatic activity ratio than EcHPI. Trypsin cleavage of MtHPI-e resulted in the formation of an active catalase-peroxidase lacking the carboxyl-terminal extension. The three enzymes, HRP, MtHPI-e, and EcHPI, mediated the isoniazid- and H2O2-dependent production of radical species, as detected by nitroblue tetrazolium reduction. A constant flux of H2O2, generated in situ from glucose oxidase and glucose was used. MtHPI-e was more effective at isoniazid-dependent radical production than EcHPI and HRP. Similar qualitative results were obtained by staining nondenaturing polyacrylamide gels for activity with nitroblue tetrazolium in the presence of isoniazid and H2O2. The absorbance spectrum of HRP exhibited changes during incubation with isoniazid and H2O2 consistent with the formation of several typical reaction intermediates, whereas the catalase-peroxidases exhibited no distinct spectral changes. The results suggest that the sensitivity of M. tuberculosis to isoniazid may be the result of isoniazid-dependent radical formation by the catalase-peroxidase in the absence of other catalase activities to remove substrate H2O2.
mesh_terms: D001426:Bacterial Proteins; D002374:Catalase / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005609:Free Radicals; D006735:Horseradish Peroxidase / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007538:Isoniazid / Q000378:metabolism*; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D010544:Peroxidases / Q000378:metabolism*; D013057:Spectrum Analysis
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005609:Free Radicals; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D010544:Peroxidases; D002374:Catalase; C076136:catalase HPI; C051253:katG protein, E coli; D007538:Isoniazid