Modulation of the activities of catalase-peroxidase HPI of Escherichia coli by site-directed mutagenesis

Catalase-peroxidases have a predominant catalatic activity but differ from monofunctional catalases in exhibiting a substantial peroxidatic reaction w hich has been implicated in the activation of the antitubercular drug isoni azid in Mycobacterium tuberculosis. Hydroperoxidase I of Escherichia coli e ncoded by katG is a catalase-peroxidase, and residues in its putative activ e site have been the target of a site directed-mutagenesis study. Variants of residues R102 and H106, on the distal side of the heme, and H267, the pr oximal side ligand, were constructed, all of which substantially reduced th e catalatic activity and, to a lesser extent, the peroxidatic activity. In addition, the heme content of the variants was reduced relative to the wild -type enzyme. The relative ease of heme loss from I-IPI and a mixture of te trameric enzymes with 2, 3, and 4 hemes was revealed by mass spectrometry a nalysis. Conversion of W105 to either an aromatic (F) or aliphatic (I) resi due caused a 4-5-fold increase in peroxidatic activity, coupled with a >99% inhibition of catalatic activity. The peroxidatic-to-catalatic ratio of th e W105F variant was increased 2800-fold such that compound I could be ident ified by both electronic and EPR spectroscopy as being similar to the porph yrin cation radical formed in other catalases and peroxidases, Compound I, when generated by a single addition of H2O2, decayed back to the native or resting stale within 1 min. When H2O2 was generated enzymatically in situ a t low levels, active compound I was evident for up to 2 h. However, such pr olonged treatment resulted in conversion of compound I to a reversibly inac tivated and, eventually, to an irreversibly inactivated species, both of wh ich were spectrally similar to compound I.