biostudies-literatureSimelis KRCUK | Engineering and Physical Sciences Research Council (EPSRC)Cancer Research UKEC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)305https://www.ebi.ac.uk/biostudies/studies/S-EPMC11662004biostudies-literatureUnknown7(1)Fe(II)- and 2-oxoglutarate (2OG)-dependent dioxygenases use 2OG and O₂ cofactors to catalyse substrate oxidation and yield oxidised product, succinate, and CO₂. Simultaneous detection of substrate and cofactors is difficult, contributing to a poor understanding of the dynamics between substrate oxidation and 2OG decarboxylation activities. Here, we profile 5-methylcytosine (^5mC)-oxidising Ten-Eleven Translocation (TET) enzymes using MS and ¹H NMR spectroscopy methods and reveal a high degree of substrate oxidation-independent 2OG turnover under a range of conditions. 2OG decarboxylase activity is substantial (>20% 2OG turned over after 1 h) in the absence of substrate, while, under substrate-saturating conditions, half of total 2OG consumption isCommunications chemistryUnravelling 2-oxoglutarate turnover and substrate oxidation dynamics in 5-methylcytosine-oxidising TET enzymes.PMC11662004101003111C8717/A28285EP/L015838/1Kawamura ASimelis KBelle RfalseUnravelling 2-oxoglutarate turnover and substrate oxidation dynamics in 5-methylcytosine-oxidising TET enzymes.Fe(II)- and 2-oxoglutarate (2OG)-dependent dioxygenases use 2OG and O₂ cofactors to catalyse substrate oxidation and yield oxidised product, succinate, and CO₂. Simultaneous detection of substrate and cofactors is difficult, contributing to a poor understanding of the dynamics between substrate oxidation and 2OG decarboxylation activities. Here, we profile 5-methylcytosine (^5mC)-oxidising Ten-Eleven Translocation (TET) enzymes using MS and ¹H NMR spectroscopy methods and reveal a high degree of substrate oxidation-independent 2OG turnover under a range of conditions. 2OG decarboxylase activity is substantial (>20% 2OG turned over after 1 h) in the absence of substrate, while, under substrate-saturating conditions, half of total 2OG consumption is2024-01-01T00:00:00Z2024 Dec2025-04-25T21:27:12.31Z2025-04-06T08:48:38.193ZS-EPMC116620043970688410.1038/s42004-024-01382-1