Inactivation of human glucose 6-phosphate dehydrogenase (G6PDH) by peroxyl radicals is strongly modulated by its substrate and cofactor.
| Title: | Inactivation of human glucose 6-phosphate dehydrogenase (G6PDH) by peroxyl radicals is strongly modulated by its substrate and cofactor. |
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| Authors: | Reyes JS; Departamento de Química Física, Facultad de Química y de Farmacia, Pontificia Universidad Católica de Chile, Chile.; Fuentes-Lemus E; Departamento de Química Física, Facultad de Química y de Farmacia, Pontificia Universidad Católica de Chile, Chile; Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Denmark.; Fierro A; Departamento de Química Orgánica, Facultad de Química y de Farmacia, Pontificia Universidad Católica de Chile, Chile.; Rivero-Rodríguez K; Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, Chile.; Arenas F; Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, Chile.; Davies MJ; Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Denmark.; López-Alarcón C; Departamento de Química Física, Facultad de Química y de Farmacia, Pontificia Universidad Católica de Chile, Chile. Electronic address: clopezr@uc.cl. |
| Source: | Free radical biology & medicine [Free Radic Biol Med] 2025 Jun; Vol. 233, pp. 55-69. Date of Electronic Publication: 2025 Mar 20. |
| Publication Type: | Journal Article |
| Language: | English |
| Journal Info: | Publisher: Elsevier Science Country of Publication: United States NLM ID: 8709159 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1873-4596 (Electronic) Linking ISSN: 08915849 NLM ISO Abbreviation: Free Radic Biol Med Subsets: MEDLINE |
| Imprint Name(s): | Publication: Tarrytown, NY : Elsevier Science; Original Publication: New York : Pergamon, c1987- |
| MeSH Terms: | Glucosephosphate Dehydrogenase*/chemistry ; Glucosephosphate Dehydrogenase*/metabolism ; Glucosephosphate Dehydrogenase*/antagonists & inhibitors ; NADP*/metabolism ; NADP*/chemistry ; Peroxides*/chemistry; Glucose-6-Phosphate/metabolism ; Glucose-6-Phosphate/chemistry ; Amidines/chemistry ; Amidines/pharmacology ; Humans ; Oxidation-Reduction ; Pentose Phosphate Pathway ; Oxidative Stress |
| Abstract: | 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+.; (Copyright © 2025 Elsevier Inc. All rights reserved.) |
| Competing Interests: | Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: MJD declares consultancy contracts with Novo Nordisk A/S, and is a Director and major shareholder in the start-up company Seleno Therapeutics plc. These funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. |
| Contributed Indexing: | Keywords: Enzyme activity; Glucose 6-phosphate; Glucose 6-phosphate dehydrogenase; NADP(+); NADPH; Pentose phosphate pathway; Peroxyl radicals; Protein oxidation |
| Substance Nomenclature: | EC 1.1.1.49 (Glucosephosphate Dehydrogenase); 53-59-8 (NADP); 0 (Peroxides); 3170-83-0 (perhydroxyl radical); 56-73-5 (Glucose-6-Phosphate); 7381JDR72F (2,2'-azobis(2-amidinopropane)); 0 (Amidines); EC 1.1.1.49 (G6PD protein, human) |
| Entry Date(s): | Date Created: 20250322 Date Completed: 20250502 Latest Revision: 20250502 |
| Update Code: | 20260130 |
| DOI: | 10.1016/j.freeradbiomed.2025.03.030 |
| PMID: | 40120653 |
| Database: | MEDLINE |
Journal Article