Steady-State Kinetic Isotope Effects Support a Complex Role of Arg226 in the Proposed Desulfonation Mechanism of Alkanesulfonate Monooxygenase

The alkanesulfonate monooxygenase system catalyzes the desulfonation of alkanesulfonates through proposed acid–base mechanistic steps that involves the abstraction of a proton from the alkane peroxyflavin intermediate and protonation of the FMN-O– intermediate. Both solvent and kinetic isotope studies were performed to define the proton transfer steps involved in the SsuD reaction. Substitution of the protium at the C1 position of octanesulfonate with deuterium resulted in an observed primary isotope effect of 3.0 ± 0.2 on the kcat parameter, supporting abstraction of the α-proton from the alkane peroxyflavin as the rate-limiting step in catalysis. Previous studies implicated Arg226 as the acid involved in the reprotonation of the hydroxyflavin intermediate. Solvent isotope kinetic studies gave an inverse isotope effect on D2Okcat of 0.75 ± 0.04 with no observable effect on D2Okcat/Km. This resulted in equivalent solvent isotope effects on D2Okcat and D2O(kcat)D, suggesting a solvent equilibrium isotope e...