Catalase-Peroxidase (Mycobacterium tuberculosis KatG) Catalysis and Isoniazid Activation

Abstract

Resonance Raman spectra of native, overexpressed M. tuberculosis catalase-peroxidase (KatG), the enzyme responsible for activation of the antituberculosis antibiotic isoniazid (isonicotinic acid hydrazide), have confirmed that the heme iron in the resting (ferric) enzyme is high-spin five-coordinate. Difference Raman spectra did not reveal a change in coordination number upon binding of isoniazid to KatG. Stopped-flow spectrophotometric studies of the reaction of KatG with stoichiometric equivalents or small excesses of hydrogen peroxide revealed only the optical spectrum of the ferric enzyme with no hypervalent iron intermediates detected. Large excesses of hydrogen peroxide generated oxyferrous KatG, which was unstable and rapidly decayed to the ferric enzyme. Formation of a pseudo-stable intermediate sharing optical characteristics with the porphyrin pi-cation radical-ferryl iron species (Compound I) of horseradish peroxidase was observed upon reaction of KatG with excess 3-chloroperoxybenzoic acid, peroxyacetic acid, or tert-butylhydroperoxide (apparent second-order rate constants of 3.1 x 10(4), 1.2 x 10(4), and 25 M(-1) s(-1), respectively). Identification of the intermediate as KatG Compound I was confirmed using low-temperature electron paramagnetic resonance spectroscopy. Isoniazid, as well as ascorbate and potassium ferrocyanide, reduced KatG Compound I to the ferric enzyme without detectable formation of Compound II in stopped-flow measurements. This result differed from the reaction of horseradish peroxidase Compound I with isoniazid, during which Compound II was stably generated. These results demonstrate important mechanistic differences between a bacterial catalase-peroxidase and the homologous plant peroxidases and yeast cytochrome c peroxidase, in its reactions with peroxides as well as substrates.

Enzyme-catalyzed Mechanism of Isoniazid Activation in Class I and Class III Peroxidases

“…Studies have shown that Compound II can be stably generated in the reaction of HRP Compound I with INH, further supporting the operation of a classic peroxidatic mechanism of INH oxidation for this enzyme (19). The same authors also identified Compound I in the reaction of M. tuberculosis CP with INH and speculated that a similar peroxidatic pathway may be utilized (19). Based upon these data and the amenability of HRP to characterization of enzymearomatic compound complexes using NMR methods (20 -28), we have, therefore, undertaken a high resolution NMR characterization of the interactions between horseradish peroxidase C (HRPC) and INH.…”

“…Later work from the same group demonstrated similar activity using partially purified M. tuberculosis CP (6). Studies have shown that Compound II can be stably generated in the reaction of HRP Compound I with INH, further supporting the operation of a classic peroxidatic mechanism of INH oxidation for this enzyme (19). The same authors also identified Compound I in the reaction of M. tuberculosis CP with INH and speculated that a similar peroxidatic pathway may be utilized (19).…”

Identification and Characterization of Tyrosyl Radical Formation in Mycobacterium tuberculosisCatalase-Peroxidase (KatG)

“…Frozen sample powders were packed into precision-bore quartz EPR tubes immersed in an isopentane bath and were examined using a Varian EPR spectrometer operating at X-band. A finger Dewar inserted into the EPR cavity was used for recording spectra at liquid nitrogen (77 K) temperature, while a liquid helium cryostat and Heli-Tran liquid helium transfer system (Advanced Research Systems, Inc., Allentown, PA) were used for EPR at 5.5 K. EPR data acquisition and manipulation was made using WinEPR software (14). Simulation of EPR spectra was performed using software provided by F. Neese (22,23).…”

“…Optical stopped-flow experiments have shown that Mycobacterium tuberculosis KatG, like other peroxidases, forms the oxyferryl iron porphyrin -cation radical known as Compound I (Cmpd I) when the resting (ferric) enzyme is treated with alkyl hydroperoxides (14). Catalase-peroxidases from other organisms behave similarly (15,16).…”

“…M. tuberculosis KatG Cmpd I could be reduced by various substrates including INH, though optical evidence for Compound II was not found in these reactions. In the case of horseradish peroxidase, the expected reduction of Cmpd I to Cmpd II by INH demonstrated that the antibiotic could serve as a single-electron reducing substrate in both steps of a peroxidase cycle (14).…”