Browsing by Author "Arenas, Felipe"

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    Implications of differential peroxyl radical-induced inactivation of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase for the pentose phosphate pathway
    (Nature Research, 2022) Reyes Valenzuela, Juan Sebastián; Figueroa Alegría, Juan David; Martínez Rojas, Francisco Javier; López Alarcon, Camilo Ignacio; Fuentes Lemus, Eduardo Felipe; Hagglund, P.M.; Davies, Michael J.; Fierro Huerta, Angelica María; Arenas, Felipe
    © 2022, The Author(s).Escherichia coli glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) are key enzymes of the pentose phosphate pathway, responsible for the NADPH production in cells. We investigated modification of both enzymes mediated by peroxyl radicals (ROO·) to determine their respective susceptibilities to and mechanisms of oxidation. G6PDH and 6PGDH were incubated with AAPH (2,2?-azobis(2-methylpropionamidine)dihydrochloride), which was employed as ROO· source. The enzymatic activities of both enzymes were determined by NADPH release, with oxidative modifications examined by electrophoresis and liquid chromatography (LC) with fluorescence and mass (MS) detection. The activity of G6PDH decreased up to 62.0 ± 15.0% after 180 min incubation with 100 mM AAPH, whilst almost total inactivation of 6PGDH was determined under the same conditions. Although both proteins contain abundant Tyr (particularly 6PGDH), these residues were minimally affected by ROO·, with Trp and Met being major targets. LC–MS and in silico analysis showed that the modification sites of G6PDH are distant to the active site, consistent with a dispersed distribution of modifications, and inactivation resulting from oxidation of multiple Trp and Met residues. In contrast, the sites of oxidation detected on 6PGDH are located close to its catalytic site indicating a more localized oxidation, and a consequent high susceptibility to ROO·-mediated inactivation.
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    Inactivation of human glucose 6-phosphate dehydrogenase (G6PDH) by peroxyl radicals is strongly modulated by its substrate and cofactor
    (2025) Sebastián Reyes, Juan; Fuentes Lemus, Eduardo Felipe; Fierro, Angélica; Rivero-Rodríguez, Karina; Arenas, Felipe; Davies, Michael J.; López-Alarcón, Camilo
    Glucose 6-phosphate dehydrogenase (G6PDH) is the rate-limiting enzyme of the pentose phosphate pathway (PPP). This enzyme catalyzes the oxidation of glucose 6-phosphate (G6P) into 6-phosphogluconolactone with concomitant reduction of NADP+to NADPH. Despite the link between the PPP and oxidative stress, the oxidation and consequences on the activity of the human G6PDH (hG6PDH) has not been investigated. In the present work we report the oxidative inactivation of hG6PDH mediated by peroxyl radicals (ROO•) generated by AAPH (2,2′- azobis(2-methylpropionamidine) dihydrochloride) thermolysis. hG6PDH (46.4 μM, monomers) was incubated at 37 ◦C with 10 or 100 mM AAPH. At defined times, enzyme activity was determined (NADPH release followed at 340 nm), mapping of modifications studied by LC-MS, structural changes analyzed by circular dichroism, and results rationalized by in silico analysis of the three-dimensional structure of the enzyme. Analogous experiments were developed in the presence of NADP+or G6P at excess or 1:1 (hG6PDH:NADP+or G6P) molar ratios. High susceptibility to inactivation by ROO• was observed, 3.6 mol of ROO • inactivated 1 mol of hG6PDH. This behavior is rationalized, at least in part, by oxidation at Trp349 which is located close to the structural site of NADP+. The presence of G6P significantly increased the ROO •-mediated inactivation of hG6PDH, while an opposite effect was observed in the presence of NADP+where, despite oxidation at different sites, the enzyme activity was practically unaltered by ROO•. These results demonstrate that hG6PDH is highly susceptible to inactivation mediated by ROO• with these processes strongly modulated by G6P and NADP+.
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    Peroxyl radicals modify 6-phosphogluconolactonase from Escherichia coli via oxidation of specific amino acids and aggregation which inhibits enzyme activity
    (ELSEVIER SCIENCE INC, 2023) Reyes Valenzuela, Juan Sebastián; Fuentes Lemus, Eduardo Felipe; Romero, Jefferson; Arenas, Felipe; Fierro Huerta, Angelica María; Davies, Michael J.; Lopez Alarcon Camilo Ignacio
    6-phosphogluconolactonase (6PGL) catalyzes the second reaction of the pentose phosphate pathway (PPP) converting 6-phosphogluconolactone to 6-phosphogluconate. The PPP is critical to the generation of NADPH and metabolic intermediates, but some of its components are susceptible to oxidative inactivation. Previous studies have characterized damage to the first (glucose-6-phosphate dehydrogenase) and third (6-phosphogluconate dehydrogenase) enzymes of the pathway, but no data are available for 6PGL. This knowledge gap is addressed here. Oxidation of Escherichia coli 6PGL by peroxyl radicals (ROO center dot, from AAPH (2,2 '-azobis(2-methylpropionamidine) dihydrochloride) was examined using SDS-PAGE, amino acid consumption, liquid chromatography with mass detection (LC-MS), protein carbonyl formation and computational methods. NADPH generation was assessed using mixtures all three enzymes of the oxidative phase of the PPP. Incubation of 6PGL with 10 or 100 mM AAPH resulted in protein aggregation mostly due to reducible (disulfide) bonds. High fluxes of ROO center dot induced consumption of Cys, Met and Trp, with the Cys oxidation rationalizing the aggregate formation. Low levels of carbonyls were detected, while LC-MS analyses provided evidence for oxidation of selected Trp and Met residues (Met1, Trp18, Met41, Trp203, Met220 and Met221). ROO center dot elicited little loss of enzymatic activity of monomeric 6PGL, but the aggregates showed diminished NADPH generation. This is consistent with in silico analyses that indicate that the modified Trp and Met are far from the 6-phosphogluconolactone binding site and the catalytic dyad (His130 and Arg179). Together these data indicate that monomeric 6PGL is a robust enzyme towards oxidative inactivation by ROO center dot and when compared to other PPP enzymes.