Please use this identifier to cite or link to this item: http://hdl.handle.net/2381/12082
Title: The mechanism of substrate inhibition in human indoleamine 2,3-dioxygenase.
Authors: Efimov, Igor
Basran, Jaswir
Sun, Xiao
Chauhan, Nishma
Chapman, S. K.
Mowat, C. G.
Raven, Emma Lloyd
First Published: 2-Feb-2012
Publisher: American Chemical Society
Citation: Journal of the American Chemical Society, 2012, 134 (6), pp. 3034-3041
Abstract: Indoleamine 2,3-dioxygenase catalyzes the O(2)-dependent oxidation of L-tryptophan (L-Trp) to N-formylkynurenine (NFK) as part of the kynurenine pathway. Inhibition of enzyme activity at high L-Trp concentrations was first noted more than 30 years ago, but the mechanism of inhibition has not been established. Using a combination of kinetic and reduction potential measurements, we present evidence showing that inhibition of enzyme activity in human indoleamine 2,3-dioxygenase (hIDO) and a number of site-directed variants during turnover with L-tryptophan (L-Trp) can be accounted for by the sequential, ordered binding of O(2) and L-Trp. Analysis of the data shows that at low concentrations of L-Trp, O(2) binds first followed by the binding of L-Trp; at higher concentrations of L-Trp, the order of binding is reversed. In addition, we show that the heme reduction potential (E(m)(0)) has a regulatory role in controlling the overall rate of catalysis (and hence the extent of inhibition) because there is a quantifiable correlation between E(m)(0) (that increases in the presence of L-Trp) and the rate constant for O(2) binding. This means that the initial formation of ferric superoxide (Fe(3+)-O(2)(•-)) from Fe(2+)-O(2) becomes thermodynamically less favorable as substrate binds, and we propose that it is the slowing down of this oxidation step at higher concentrations of substrate that is the origin of the inhibition. In contrast, we show that regeneration of the ferrous enzyme (and formation of NFK) in the final step of the mechanism, which formally requires reduction of the heme, is facilitated by the higher reduction potential in the substrate-bound enzyme and the two constants (k(cat) and E(m)(0)) are shown also to be correlated. Thus, the overall catalytic activity is balanced between the equal and opposite dependencies of the initial and final steps of the mechanism on the heme reduction potential. This tuning of the reduction potential provides a simple mechanism for regulation of the reactivity, which may be used more widely across this family of enzymes.
DOI Link: 10.1021/ja208694g
ISSN: 0002-7863
eISSN: 1520-5126
Links: http://hdl.handle.net/2381/12082
http://pubs.acs.org/doi/abs/10.1021/ja208694g
Type: Journal Article
Rights: Archived with reference to SHERPA/RoMEO and publisher website.
Description: PMCID: PMC3280726 Supporting Information: The derivation of relevant equations, steady-state data for variants (Figure S1) and representative reduction potential (Figure S2) and O2 binding (Figure S3) data. Complete ref 11. This material is available free of charge via the Internet at http://pubs.acs.org.
Appears in Collections:Published Articles, Dept. of Chemistry

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