Reactive sulfur species are inactivated and excreted as trimethylsulfonium ion by thiopurine S-methyltransferase

. Kinetic analyses revealed substrate-dependent differences in methylation efficiency. In silico analyses suggested that steric hindrance of the substrate is a key determinant of the methylation efficiency. Collectively, these findings uncover an energy-dependent, non-oxidative pathway for the elimination of RSS beyond classical oxidative detoxification. Given that TPMT polymorphisms are clinically important determinants of thiopurine drug metabolism, variation in TPMT activity may likewise influence biological responses to RSS. Together, these results provide a foundation for understanding how endogenous methylation shapes RSS homeostasis.