PMID:10801338
| Citation |
Hillar, A, Peters, B, Pauls, R, Loboda, A, Zhang, H, Mauk, AG and Loewen, PC (2000) Modulation of the activities of catalase-peroxidase HPI of Escherichia coli by site-directed mutagenesis. Biochemistry 39:5868-75 |
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| Abstract |
Catalase-peroxidases have a predominant catalatic activity but differ from monofunctional catalases in exhibiting a substantial peroxidatic reaction which has been implicated in the activation of the antitubercular drug isoniazid in Mycobacterium tuberculosis. Hydroperoxidase I of Escherichia coli encoded by katG is a catalase-peroxidase, and residues in its putative active 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 proximal side ligand, were constructed, all of which substantially reduced the 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 HPI and a mixture of tetrameric enzymes with 2, 3, and 4 hemes was revealed by mass spectrometry analysis. Conversion of W105 to either an aromatic (F) or aliphatic (I) residue caused a 4-5-fold increase in peroxidatic activity, coupled with a >99% inhibition of catalatic activity. The peroxidatic-to-catalatic ratio of the W105F variant was increased 2800-fold such that compound I could be identified by both electronic and EPR spectroscopy as being similar to the porphyrin cation radical formed in other catalases and peroxidases. Compound I, when generated by a single addition of H(2)O(2), decayed back to the native or resting state within 1 min. When H(2)O(2) was generated enzymatically in situ at low levels, active compound I was evident for up to 2 h. However, such prolonged treatment resulted in conversion of compound I to a reversibly inactivated and, eventually, to an irreversibly inactivated species, both of which were spectrally similar to compound I. |
| Links | |
| Keywords |
Amino Acid Substitution/genetics; Bacterial Proteins; Binding Sites/genetics; Catalase/antagonists & inhibitors; Catalase/chemistry; Catalase/genetics; Catalysis; Electron Spin Resonance Spectroscopy; Enzyme Activation/genetics; Enzyme Inhibitors/pharmacology; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Heme/chemistry; Leucine/genetics; Mass Spectrometry; Mutagenesis, Site-Directed; Peroxidases/antagonists & inhibitors; Peroxidases/chemistry; Peroxidases/genetics; Phenylalanine/genetics; Recombinant Proteins/chemistry; Substrate Specificity/genetics; Tryptophan/genetics |
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