Competitive oxidation of key pentose phosphate pathway enzymes modulates the fate of intermediates and NAPDH production
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Competitive oxidation of key pentose phosphate pathway enzymes modulates the fate of intermediates and NAPDH production. / Reyes, Juan Sebastián; Cortés-Ríos, Javiera; Fuentes-Lemus, Eduardo; Rodriguez-Fernandez, Maria; Davies, Michael J.; López-Alarcón, Camilo.
I: Free Radical Biology and Medicine, Bind 222, 2024, s. 505-518.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - Competitive oxidation of key pentose phosphate pathway enzymes modulates the fate of intermediates and NAPDH production
AU - Reyes, Juan Sebastián
AU - Cortés-Ríos, Javiera
AU - Fuentes-Lemus, Eduardo
AU - Rodriguez-Fernandez, Maria
AU - Davies, Michael J.
AU - López-Alarcón, Camilo
N1 - Publisher Copyright: © 2024 Elsevier Inc.
PY - 2024
Y1 - 2024
N2 - The oxidative phase of the pentose phosphate pathway (PPP) involving the enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL), and 6-phosphogluconate dehydrogenase (6PGDH), is critical to NADPH generation within cells, with these enzymes catalyzing the conversion of glucose-6-phosphate (G6P) into ribulose-5-phosphate (Ribu5-P). We have previously studied peroxyl radical (ROO•) mediated oxidative inactivation of E. coli G6PDH, 6PGL, and 6PGDH. However, these data were obtained from experiments where each enzyme was independently exposed to ROO•, a condition not reflecting biological reality. In this work we investigated how NADPH production is modulated when these enzymes are jointly exposed to ROO•. Enzyme mixtures (1:1:1 ratio) were exposed to ROO• produced from thermolysis of 100 mM 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH). NADPH was quantified at 340 nm, and protein oxidation analyzed by liquid chromatography with mass spectrometric detection (LC-MS). The data obtained were rationalized using a mathematical model. The mixture of non-oxidized enzymes, G6P and NADP+ generated ∼175 μM NADPH. Computational simulations showed a constant decrease of G6P associated with NADPH formation, consistent with experimental data. When the enzyme mixture was exposed to AAPH (3 h, 37 °C), lower levels of NADPH were detected (∼100 μM) which also fitted with computational simulations. LC-MS analyses indicated modifications at Tyr, Trp, and Met residues but at lower concentrations than detected for the isolated enzymes. Quantification of NADPH generation showed that the pathway activity was not altered during the initial stages of the oxidations, consistent with a buffering role of G6PDH towards inactivation of the oxidative phase of the pathway.
AB - The oxidative phase of the pentose phosphate pathway (PPP) involving the enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL), and 6-phosphogluconate dehydrogenase (6PGDH), is critical to NADPH generation within cells, with these enzymes catalyzing the conversion of glucose-6-phosphate (G6P) into ribulose-5-phosphate (Ribu5-P). We have previously studied peroxyl radical (ROO•) mediated oxidative inactivation of E. coli G6PDH, 6PGL, and 6PGDH. However, these data were obtained from experiments where each enzyme was independently exposed to ROO•, a condition not reflecting biological reality. In this work we investigated how NADPH production is modulated when these enzymes are jointly exposed to ROO•. Enzyme mixtures (1:1:1 ratio) were exposed to ROO• produced from thermolysis of 100 mM 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH). NADPH was quantified at 340 nm, and protein oxidation analyzed by liquid chromatography with mass spectrometric detection (LC-MS). The data obtained were rationalized using a mathematical model. The mixture of non-oxidized enzymes, G6P and NADP+ generated ∼175 μM NADPH. Computational simulations showed a constant decrease of G6P associated with NADPH formation, consistent with experimental data. When the enzyme mixture was exposed to AAPH (3 h, 37 °C), lower levels of NADPH were detected (∼100 μM) which also fitted with computational simulations. LC-MS analyses indicated modifications at Tyr, Trp, and Met residues but at lower concentrations than detected for the isolated enzymes. Quantification of NADPH generation showed that the pathway activity was not altered during the initial stages of the oxidations, consistent with a buffering role of G6PDH towards inactivation of the oxidative phase of the pathway.
KW - 6-phosphogluconate dehydrogenase
KW - 6-phosphogluconolactonase
KW - AAPH
KW - Glucose-6-phosphate dehydrogenase
KW - Mathematical simulations
KW - NADPH
KW - Peroxyl radicals
KW - Protein oxidation
U2 - 10.1016/j.freeradbiomed.2024.05.050
DO - 10.1016/j.freeradbiomed.2024.05.050
M3 - Journal article
C2 - 38848786
AN - SCOPUS:85197793234
VL - 222
SP - 505
EP - 518
JO - Free Radical Biology & Medicine
JF - Free Radical Biology & Medicine
SN - 0891-5849
ER -
ID: 399110325