The Identification of Critical Active-Site Groups and Catalytic Steps Governing the Desulfonation Reaction by Alkanesulfonate Monooxygenase

Abstract

The alkanesulfonate monooxygenase enzyme (SsuD) catalyzes the oxygenolytic cleavage of a carbon-sulfur bond of sulfonated substrates. A mechanism involving acid-base catalysis is proposed for the desulfonation mechanism by SsuD. In the proposed mechanism, base catalysis is involved in abstracting a proton from an alkane peroxyflavin intermediate, while acid catalysis is needed for the protonation of the FMNO- intermediate. The catalytic mechanism for SsuD was evaluated using several kinetic approaches including steady-state pH dependence studies, solvent deuterium and substrate deuterium kinetic isotope effects, temperature dependent studies, and single turnover kinetics. The pH dependence of kcat indicated SsuD requires a group with a pKa of 6.6 ± 0.1 to be deprotonated and a second group with a pKa of 9.5 ± 0.1 to be protonated for catalysis, while the pH dependence of kcat/Km indicated SsuD requires a single group with a pKa of 6.9 ± 0.1 to be deprotonated in order for the reaction to commit through the first irreversible step. Each observed isotope effect on the kcat and kcat/Km kinetic parameter was determined by analyzing the pH dependence of the solvent deuterium and substrate deuterium isotope effects, the latter obtained in both H2O and D2O. Labeled and unlabeled substrate yielded a solvent isotope effect on kcat of 0.75 ± 0.04 and 2.4 ± 0.2, respectively. The observed substrate isotope effect on kcat in H2O was 3.0 ± 0.2. Each isotope effect on kcat/Km was within an experimental error of one indicating product release to be the rate-limiting step. This result was reinforced by proton inventories exhibiting dome-shaped curves indicating large commitments to catalysis. Results from single-turnover experiments show increased stability of the C4a-(hydro)peroxyflavin intermediate in D2O, but that the overall rate of flavin oxidation by SsuD monitored at 370 and 450 nm was not altered in the deuterated solvent. The deuterated substrate failed to stabilize the C4a-(hydro)peroxyflavin intermediate under single-turnover conditions. These combined results have identified key chemical steps of the proposed catalytic mechanism by implicating Arg226 as playing a critical role in catalysis as well as the quantification of catalytic commitment factors.