COMPARISON OF ISONIAZID OXIDATION CATALYZED BY BACTERIAL CATALASE-PEROXIDASES AND HORSERADISH-PEROXIDASE

The physical properties and activities of the purified catalase-peroxi dase hydroperoxidase I (HPI) of Escherichia coil (EcHPI) and HPI with a carboxyl-terminal extension of Mycobacterium tuberculosis (MtHPI-e) are compared to those of commercial preparations of horseradish peroxi dase (HRP). The catalase-peroxidase proteins had similar absorption sp ectra and differed primarily in that MtHPI-e has a higher peroxidatic to catalatic activity ratio than EcHPI. Trypsin cleavage of MtHPI-e re sulted in the formation of an active catalase-peroxidase lacking the c arboxyl-terminal extension, The three enzymes, HRP, MtHPI-e, and EcHPI , mediated the isoniazid- and H2O2-dependent production of radical spe cies, 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 t han EcHPI and HRP, Similar qualitative results were obtained by staini ng nondenaturing polyacrylamide gels for activity with nitroblue tetra zolium in the presence of isoniazid and H2O2. The absorbance spectrum of HRP exhibited changes during incubation with isoniazid and H2O2 con sistent with the formation of several typical reaction intermediates, whereas the catalase-peroxidases exhibited no distinct spectral change s, The results suggest that the sensitivity of M. tuberculosis to ison iazid may be the result of isoniazid-dependent radical formation by th e catalase-peroxidase in the absence of other catalase activities to r emove substrate H2O2. (C) 1995 Academic Press, Inc.