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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 by a pKa of 8.8. That the higher pH values do not slow the rate of hydroxylation precludes that the pKa measured pertains to the proton of the hydroperoxflavin. Together, these observations indicate that the C4a-hydroperoxyflavin has a pKa >> 8.5, that a non-aromatic species is the immediate product of hydroxylation and that at least two solvent derived protons are in-flight during oxygen insertion to the substrate aromatic ring. A unifying mechanistic proposal for these observations is proposed.
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Inverse Solvent Isotope Effects in Enzyme-Catalyzed Reactions
Solvent isotope effects have long been used as a mechanistic tool for determining enzyme mechanisms. Most commonly, macroscopic rate constants such as kcat and kcat/Km are found to decrease when the reaction is performed in D2O for a variety of reasons including the transfer of protons. Under certain circumstances, these constants are found to increase, in what is termed an inverse solvent kinetic isotope effect (SKIE), which can be a diagnostic mechanistic feature. Generally, these phenomena can be attributed to an inverse solvent equilibrium isotope effect on a rapid equilibrium preceding the rate-limiting step(s). This review surveys inverse SKIEs in enzyme-catalyzed reactions by assessing their underlying origins in common mechanistic themes. Case studies for each category are presented, and the mechanistic implications are put into context. It is hoped that readers may find the illustrative examples valuable in planning and interpreting solvent isotope effect experiments.
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- Award ID(s):
- 1808449
- PAR ID:
- 10145805
- Date Published:
- Journal Name:
- Molecules
- Volume:
- 25
- Issue:
- 8
- ISSN:
- 1420-3049
- Page Range / eLocation ID:
- 1933
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/molecules25081933
