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- W3042300475 abstract "The first step of the kynurenine pathway for l-tryptophan (l-Trp) degradation is catalyzed by heme-dependent dioxygenases, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase. In this work, we employed stopped-flow optical absorption spectroscopy to study the kinetic behavior of the Michaelis complex of Cupriavidus metallidurans TDO (cmTDO) to improve our understanding of oxygen activation and initial oxidation of l-Trp. On the basis of the stopped-flow results, rapid freeze-quench (RFQ) experiments were performed to capture and characterize this intermediate by Mossbauer spectroscopy. By incorporating the chlorite dismutase-chlorite system to produce high concentrations of solubilized O2, we were able to capture the Michaelis complex of cmTDO in a nearly quantitative yield. The RFQ-Mossbauer results confirmed the identity of the Michaelis complex as an O2-bound ferrous species. They revealed remarkable similarities between the electronic properties of the Michaelis complex and those of the O2 adduct of myoglobin. We also found that the decay of this reactive intermediate is the rate-limiting step of the catalytic reaction. An inverse α-secondary substrate kinetic isotope effect was observed with a kH/kD of 0.87 ± 0.03 when (indole-d5)-l-Trp was employed as the substrate. This work provides an important piece of spectroscopic evidence of the chemical identity of the Michaelis complex of bacterial TDO." @default.
- W3042300475 created "2020-07-23" @default.
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- W3042300475 creator A5069795151 @default.
- W3042300475 creator A5076148265 @default.
- W3042300475 date "2020-07-13" @default.
- W3042300475 modified "2023-10-12" @default.
- W3042300475 title "Kinetic and Spectroscopic Characterization of the Catalytic Ternary Complex of Tryptophan 2,3-Dioxygenase" @default.
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- W3042300475 doi "https://doi.org/10.1021/acs.biochem.0c00179" @default.
- W3042300475 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7470927" @default.
- W3042300475 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/32659080" @default.
- W3042300475 hasPublicationYear "2020" @default.
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