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

Abstract

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 (D2O)kcat of 0.75 ± 0.04 with no observable effect on (D2O)kcat/Km. This resulted in equivalent solvent isotope effects on (D2O)kcat and (D2O)(kcat)D, suggesting a solvent equilibrium isotope effect on a step occurring after the first irreversible step through product release. Data from proton inventory studies on kcat were best fit to a dome-shaped curve consistent with a conformational change to an open conformation during product release. The solvent isotope effect data coupled with the corresponding proton inventory results support and extend our previous observations that Arg226 donates a proton to the FMN-O(-) intermediate, triggering a conformational change that opens the enzyme to solvation and promotes product release.