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Title: Tryptophan Oxidation by the Heme-Containing Dioxygenases
Authors: Booth, Elizabeth Suzanne
Supervisors: Raven, Emma
Handa, Sandeep
Award date: 1-Feb-2016
Presented at: University of Leicester
Abstract: In biology, the kynurenine pathway is the major degradation pathway of tryptophan (L-Trp). The first and rate-limiting step is the oxidation of L-Trp to N-formylkynurenine (NFK). The mechanism of this oxygen-dependent reaction has not been established, but is catalysed by two heme-containing dioxygenase enzymes: indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO). Early proposals suggested a base-catalysed abstraction mechanism, but most of the recent studies argue against that. Instead, formation of a ferryl (Compound II intermediate) seems more likely. In this thesis, aspects of the reaction mechanism have been investigated. A Compound II intermediate was detected during the oxidation of L-Trp by hIDO using stopped flow photodiode array spectroscopy. A Compound II intermediate was also detected during the oxidation of a number of different tryptophan analogues. The results suggest a common mechanism of oxidation between L-Trp and other substrates of hIDO. The difference in reactivity between the tryptophan analogues 5-hydroxy-tryptophan and 5-methoxy-tryptophan with hIDO have been interpreted to indicate that initial oxygen atom insertion occurs by radical rather than electrophilic addition. An intermediate was detected during the oxidation of L-Trp by hTDO and XcTDO. The spectrum of this intermediate did not appear to be characteristic of a Compound II based on comparison with the spectrum of Compound II from hIDO. Weaker binding of L-Trp to both hTDO and XcTDO has been used to interpret these results. It is suggested that Compound II in TDO has a different spectrum or that the rate-limiting step is altered and an alternative catalytic intermediate accumulates. Crystal trials have been conducted for hIDO and hTDO, with some conditions producing micro-crystals. The structure of XcTDO in complex with potassium cyanide and L-Trp was solved. The binding mode of L-Trp within the distal pocket does not correlate with the L-Trp binding mode in the published ferrous XcTDO- L-Trp binary complex structure. Oxidation of L-Trp by ferric TDO without the addition of reducing agent was investigated. The results suggest the recruitment of hydrogen peroxide from solution to activate ferric heme. A summary of the mechanistic information gathered from all of the above experiments is presented.
Type: Thesis
Level: Doctoral
Qualification: PhD
Rights: Copyright © the author. All rights reserved.
Appears in Collections:Leicester Theses
Theses, Dept. of Chemistry

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