Hydrogen movements in the oxidative half-reaction of kynurenine 3-monooxygenase from Pseudomonas fluorescens reveal the mechanism of hydroxylation

摘要: Abstract Kynurenine 3-monoxygenase (KMO) catalyzes the conversion of l -kynurenine (L-Kyn) to 3-hydroxykynurenine (3-OHKyn) in pathway for tryptophan catabolism. We have investigated effects pH and deuterium substitution on oxidative half-reaction KMO from P. fluorescens (PfKMO). The three phases observed during half reaction are formation hydroperoxyflavin, hydroxylation product release. measured rate constants these proved largely unchanging with pH, suggesting that active site is insulated exchange solvent catalysis. A inventory study indicated a isotope effect 2–3 phase two or more protons flight this step. An inverse 0.84 ± 0.01 constant step ring perdeutero-L-Kyn as substrate indicates shift sp2 sp3 hybridization transition state leading non-aromatic intermediate. dependence transient data collected analog meta-nitrobenzoylalanine indicate groups proximal hydroperoxyflavin titrated range 5–8.5 can be described by pKa 8.8. That higher values do not slow precludes pertains proton hydroperoxflavin. Together, observations C4a-hydroperoxyflavin has ≫ 8.5, species immediate at least derived in-flight oxygen insertion aromatic ring. unifying mechanistic proposal proposed.