Characterization of the W321F mutant of Mycobacterium tuberculosis catalase-peroxidase KatG.
A single amino acid mutation (W321F) in Mycobacterium tuberculosis catalase-peroxidase (KatG) was constructed by site-directed mutagenesis. The purified mutant enzyme was characterized using optical and electron paramagnetic resonance spectroscopy, and optical stopped-flow spectrophotometry. Reaction of KatG(W321F) with 3-chloroperoxybenzoic acid, peroxyacetic acid, or t-butylhydroperoxide showed formation of an unstable intermediate assigned as Compound I (oxyferryl iron:porphyrin pi-cation radical) by similarity to wild-type KatG, although second-order rate constants were significantly lower in the mutant for each peroxide tested. No evidence for Compound II was detected during the spontaneous or substrate-accelerated decay of Compound I. The binding of isoniazid, a first-line anti-tuberculosis pro-drug activated by catalase-peroxidase, was noncooperative and threefold weaker in KatG(W321F) compared with wild-type enzyme. An EPR signal assigned to a protein-based radical tentatively assigned as tyrosyl radical in wild-type KatG, was also observed in the mutant upon reaction of the resting enzyme with alkyl peroxide. These results show that mutation of residue W321 in KatG does not lead to a major alteration in the identity of intermediates formed in the catalytic cycle of the enzyme in the time regimes examined here, and show that this residue is not the site of stabilization of a radical as might be expected based on homology to yeast cytochrome c peroxidase. Furthermore, W321 is indicated to be important in KatG for substrate binding and subunit interactions within the dimer, providing insights into the origin of isoniazid resistance in clinically isolated KatG mutants.
Yu
Shengwei
S
Department of Chemistry, Brooklyn College, and the Graduate Center of the City University of New York, Brooklyn, New York 11210-2889, USA.
Chouchane
Salem
S
Magliozzo
Richard S
RS
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010544:Peroxidases / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D015398:Signal Transduction; D013053:Spectrophotometry; D013997:Time Factors
D001426:Bacterial Proteins; D010544:Peroxidases; C081610:catalase-peroxidase, bacteria; C076136:catalase HPI