Much understanding of metabolism is based on monitoring chemical reactions in cells with isotope tracers. For this purpose, 13C is well suited due to its stable incorporation into biomolecules and minimal kinetic isotope effect. For redox reactions, deuterium tracing can provide additional information. To date, studies examining NADPH production with deuterated carbon sources have failed to account for roughly half of NADPH's redox active hydrogen. We show the missing hydrogen is the result of enzyme-catalyzed H-D exchange between water and NADPH. Though isolated NADPH does not undergo H-D exchange with water, such exchange is catalyzed by Flavin enzymes and occurs rapidly in cells. Correction for H-D exchange is required for accurate assessment of biological sources of NADPH's high energy electrons. Deuterated water (D2O) is frequently used to monitor fat synthesis in vivo, but the chemical pathway of the deuterons into fat remains unclear. We show D2O labels fatty acids primarily via NADPH. Knowledge of this route enables calculation, without any fitting parameters, of the mass isotopomer distributions of fatty acids from cells grown in D2O. Thus, knowledge of enzyme-catalyzed H-D exchange between water and NADPH enables accurate interpretation of deuterium tracing studies of redox cofactor and fatty acid metabolism.
All Science Journal Classification (ASJC) codes
- Colloid and Surface Chemistry