Competitive oxidation of key pentose phosphate pathway enzymes modulates the fate of intermediates and NAPDH production.
Free Radic Biol Med
; 222: 505-518, 2024 Sep.
Article
em En
| MEDLINE
| ID: mdl-38848786
ABSTRACT
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 (ROOâ¢) mediated oxidative inactivation of E. coli G6PDH, 6PGL, and 6PGDH. However, these data were obtained from experiments where each enzyme was independently exposed to ROOâ¢, a condition not reflecting biological reality. In this work we investigated how NADPH production is modulated when these enzymes are jointly exposed to ROOâ¢. Enzyme mixtures (111 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 â¼175 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 (â¼100 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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Oxirredução
/
Via de Pentose Fosfato
/
Fosfogluconato Desidrogenase
/
Escherichia coli
/
Glucosefosfato Desidrogenase
/
NADP
Idioma:
En
Revista:
Free Radic Biol Med
Assunto da revista:
BIOQUIMICA
/
MEDICINA
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Chile
País de publicação:
Estados Unidos