The mechanism of substrate regulation in L-lysine monooxygenase Mycobacterium smegmatis G (MbsG) has been investigated using steady-state and rapid reaction kinetic experiments. L-lysine regulates flavin reduction and has no effect on the oxidation, which occurs in a one-step process, without the observation of a C4a-hydroperoxyflavin intermediate. Kinetic isotope studies indicate that MbsG is preferential for transfer of the pro-R-hydride of NADH with a Dkcat and Dkred value of 2.0 and 1.8, respectively. A D2Okcat value of 1.3 was also determined. These results show that bond breaking and forming are only partially rate limiting in the reaction. At low pH, reduction is accelerated by a single ionizable group with a pKa of ~6. These results suggest a form of regulation by pH and substrate in the reductive half-reaction which is uncharacteristic of other enzymes in this class of flavin-containing monooxygenases. This work was supported by a grant from the NSF MCB-1021384. 

pH, primary, and solvent kinetic isotope effects on the reaction catalyzed by lysine N6-monooxygenase from M. smegmatis

245th National Meeting (2013)

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