Oxidizing substrate specificity of Mycobacterium tuberculosis alkyl hydroperoxide reductase E: kinetics and mechanisms of oxidation and overoxidation |
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Authors: | Reyes Aníbal M Hugo Martín Trostchansky Andrés Capece Luciana Radi Rafael Trujillo Madia |
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Institution: | a Departamento de Bioquímica, Universidad de la República, 11800 Montevideo, Uruguayb Center for Free Radical and Biomedical Research, Universidad de la República, 11800 Montevideo, Uruguayc Departamento de Química Inorgánica, Analítica y Química Física/INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina |
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Abstract: | Alkyl hydroperoxide reductase E (AhpE), a novel subgroup of the peroxiredoxin family, comprises Mycobacterium tuberculosis AhpE (MtAhpE) and AhpE-like proteins present in many bacteria and archaea, for which functional characterization is scarce. We previously reported that MtAhpE reacted ~ 103 times faster with peroxynitrite than with hydrogen peroxide, but the molecular reasons for that remained unknown. Herein, we investigated the oxidizing substrate specificity and the oxidative inactivation of the enzyme. In most cases, both peroxidatic thiol oxidation and sulfenic acid overoxidation followed a trend in which those peroxides with the lower leaving-group pKa reacted faster than others. These data are in agreement with the accepted mechanisms of thiol oxidation and support that overoxidation occurs through sulfenate anion reaction with the protonated peroxide. However, MtAhpE oxidation and overoxidation by fatty acid-derived hydroperoxides (~ 108 and 105 M− 1 s− 1, respectively, at pH 7.4 and 25 °C) were much faster than expected according to the Brønsted relationship with leaving-group pKa. A stoichiometric reduction of the arachidonic acid hydroperoxide 15-HpETE to its corresponding alcohol was confirmed. Interactions of fatty acid hydroperoxides with a hydrophobic groove present on the reduced MtAhpE surface could be the basis of their surprisingly fast reactivity. |
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Keywords: | AhpE alkyl hydroperoxide reductase E BHT butylated hydroxytoluene 3-CPBA 3-chloroperoxybenzoic acid cumeneOOH cumene hydroperoxide DTPA diethylenetriamine pentaacetic acid DTNB 5 5&prime -dithiobis-(2-nitrobenzoic acid) DTT 1 4-dithiothreitol arachidonic acid 5Z 8Z 11Z 14Z-eicosatetraenoic acid 15-HETE (± )15-hydroxy-5Z 8Z 11Z 13E-eicosatetraenoic acid 12-HETE-d8 12S-hydroxy-5Z 8Z 10E 14Z-eicosatetraenoic-5 6 8 9 11 12 14 15-d8 acid 15-HpETE 15S-hydroperoxy-5Z 8Z 11Z 13E-eicosatetraenoic acid α-linolenic acid 9Z 12Z 15Z-octadecatrienoic acid LOX lipoxidase CP peroxidatic cysteine Prx peroxiredoxin t-BuOOH tert-butylhydroperoxide |
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