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Cross‐linking of M. tuberculosis Catalase‐Peroxidase (KatG)
Author(s) -
Suarez Javier,
Ranguelova Kalina,
Magliozzo Richard S
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.625.1
Subject(s) - chemistry , peroxidase , catalase , hydrogen peroxide , peracetic acid , heme , radical , peroxide , biochemistry , mutant , isoniazid , stereochemistry , enzyme , organic chemistry , tuberculosis , medicine , pathology , gene
The formation of inter and intra‐molecular cross‐links has been reported for many heme and metallo‐proteins that form endogenous radicals. M. tuberculosis catalase‐peroxidase (KatG) is a bifunctional enzyme in which tyrosyl and tryptophanyl radicals are known to be formed during turnover with peroxides. The main goal of the present study was to gain insights into the loci of radical formation through the analysis of cross‐linking during turnover of KatG in the presence and absence of reducing substrate. SDS and Native‐PAGE of KatG treated with peracetic acid or hydrogen peroxide under a variety of conditions demonstrate oligomers of molecular weight greater than that of the native dimmer. Isoniazid, the antibiotic activated by Mtb KatG, completely inhibit crosslink formation. The KatG concentration dependence of the distribution and yield of oligomers strongly suggest that the sites of crosslink formation are solvent exposed though tyrosyl radicals are known to be first formed at buried tyrosines during peroxide turnover. KatG mutants at positions Y197, Y353, Y304, W204 demonstrate that these residues are not involved in crosslinking. The results are consistent with the hypothesis that cross‐linking of KatG can occur in the absence of peroxidase substrates and that under physiological conditions, the activation of INH by KatG may be altered by this process. Experiments for the identification of the residues involved in the crosslinks and to study the properties of the higher molecular weight products are ongoing.