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

Archives of Biochemistry and Biophysics
Brett A BeaupreGraham R Moran

Abstract

Kynurenine 3-monoxygenase (KMO) catalyzes the conversion of l-kynurenine (L-Kyn) to 3-hydroxykynurenine (3-OHKyn) in the pathway for tryptophan catabolism. We have investigated the effects of pH and deuterium substitution on the oxidative half-reaction of KMO from P. fluorescens (PfKMO). The three phases observed during the oxidative half reaction are formation of the hydroperoxyflavin, hydroxylation and product release. The measured rate constants for these phases proved largely unchanging with pH, suggesting that the KMO active site is insulated from exchange with solvent during catalysis. A solvent inventory study indicated that a solvent isotope effect of 2-3 is observed for the hydroxylation phase and that two or more protons are in flight during this step. An inverse isotope effect of 0.84 ± 0.01 on the rate constant for the hydroxylation step with ring perdeutero-L-Kyn as a substrate indicates a shift from sp2 to sp3 hybridization in the transition state leading to the formation of a non-aromatic intermediate. The pH dependence of transient state data collected for the substrate analog meta-nitrobenzoylalanine indicate that groups proximal to the hydroperoxyflavin are titrated in the range pH 5-8.5 and can be described b...Continue Reading

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Citations

Jun 3, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Lizaveta GotinaAe Nim Pae
Oct 23, 2021·The Journal of Physical Chemistry. a·Yılmaz Özkılıç, Nurcan Ş Tüzün

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