Human heme dioxygenases

The L-kynurenine pathway, which leads to the formation of NAD, is the major catabolic route of L-tryptophan metabolism in biology. The initial step in this pathway is oxidation of L-tryptophan to N-formyl-kynurenine. In all biological systems examined to date, this is catalysed by one of two heme enzymes, indoleamine 2,3-dioxygenase (IDO) or tryptophan 2,3-dioxygenase (TDO). In this thesis the reaction mechanism, the reactive catalytic intermediates involved in this reaction and the nature of substrate (L-tryptophan and dioxygen)-protein interactions, if any, present within the active site of rhIDO have been examined.;In Chapter 2, we addressed the role of S I67 in rhIDO (S167A and S167H), which is replaced with a histidine residue in TDO enzymes. Kinetic and spectroscopic data for S I67A indicate that this residue is not essential for O2 or substrate binding. The data for S167H show that the ferrous-oxy complex is dramatically destabilised, which is similar to the behaviour observed in rhTDO. The implications of these results are discussed in terms of our current understanding of IDO and TDO catalysis.;In Chapter 3, it was shown that 1-methyL-tryptophan is a substrate for rhIDO and S167A. However, no activity was observed for rhTDO. Substitution of an active site histidine residue in rhTDO (H76S) allows accommodation of the additional methyl group and 1-methyL-tryptophan turnover to occur. These observations suggest that deprotonation of the indole N 1 is not essential for catalysis, and an alternative reaction mechanism is presented. Additional experiments using EPR and 1H ENDOR spectroscopy were used to examine the surrounding environment of the heme iron. The results reveal important information on the surrounding environment of the heme-bound dioxygen and the interactions present in the ternary complex. The mechanistic implications of such interactions are discussed in this work.;In Chapter 5, we undertook site-directed mutagenesis of several active site residues and the role of each residue on dioxygen, substrate binding and in catalysis was examined. We found the conserved residue R231 plays a key role in substrate binding and is likely to do so in all heme dioxygenase enzymes. The F227A variant was found to be catalytically competent for L-tryptophan turnover and suggests that this residue is not involved in substrate recognition like previously proposed.;In Chapter 6, we have shown that rhTDO and rhIDO can utilise hydrogen peroxide as an alternative oxygen source to dioxygen. For rhTDO, approximately two equivalents of H2O2 were consumed in the production of one molecule of N-formyl-kynurenine, suggesting that an alternative mechanistic pathway is used with hydrogen peroxide.