The catalytic mechanism of Mycobacterium tuberculosis catalase-peroxidase (KatG) and isoniazid activation.
Item
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Title
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The catalytic mechanism of Mycobacterium tuberculosis catalase-peroxidase (KatG) and isoniazid activation.
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Identifier
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AAI3187421
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identifier
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3187421
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Creator
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Zhao, Xiangbo.
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Contributor
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Adviser: Richard S. Magliozzo
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Date
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2005
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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Mycobacterium tuberculosis catalase-peroxidase (KatG) is a bi-functional heme enzyme and is involved in the in vivo activation of the antituberculosis drug isoniazid. The resting enzyme (ferric) is usually activated by peroxide and gives rise to a highly oxidative intermediate. Like other heme peroxidases, the catalytic cycle of this enzyme involves heme-based and amino acid based radical intermediates.;In previous studies, a protein based tyrosyl radical was detected in the reaction of this enzyme with alkyl peroxide. However, the location of the radical forming residue(s) required special efforts to elucidate. In this study, nitric oxide (NO) was used as a radical scavenging reagent to label the tyrosyl radical forming residue(s). Quenching of the tyrosyl radical generated in the presence of NO was shown using electron paramagnetic resonance spectroscopy, and formation of nitrotyrosine was confirmed by proteolytic digestion followed by HPLC analysis of the NO-treated enzyme. Edman sequencing of nitrated peptides showed that only Tyr353 was labeled. In the mutant enzyme KatG[Y353F], which was constructed using site-directed mutagenesis, a tyrosyl radical was also formed upon turnover with peroxide, but in poor yield compared to wild-type KatG. Thus, we propose that Tyr353 is one of the tyrosyl radical forming site(s) in wild type KatG.;Previous studies did not clarify the oxidants that participate in the KatG-mediated activation of isoniazid. In this study, we mimic the in vivo oxidative environment by the generation of a continuous flow of small amounts of hydrogen peroxide enzymatically. For the first time, we demonstrated that hydrogen peroxide is a very effective species that accelerates the activation of isoniazid. We also verified that NAD, rather than NADH, is involved in formation of an important inhibitor molecule specific for the action of the antibiotic. A model of an INH activation mechanism is proposed, and the behavior of the isoniazid resistant KatG[S315T] mutant fits this model very well.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
