The Mechanism of Substrate Inhibition in Human Indoleamine 2,3-Dioxygenase

Abstract

Indoleamine 2,3-dioxygenase catalyzes the O2-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 O2 and l-Trp. Analysis of the data shows that at low concentrations of l-Trp, O2 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 (Em0) has a regulatory role in controlling the overall rate of catalysis (and hence the extent of inhibition) because there is a quantifiable correlation between Em0 (that increases in the presence of l-Trp) and the rate constant for O2 binding. This means that the initial formation of ferric superoxide (Fe3+–O2•–) from Fe2+-O2 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 (kcat and Em0) 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.

Measurement of Internal Substrate Binding in Dehaloperoxidase–Hemoglobin by Competition with the Heme–Fluoride Binding Equilibrium

“…63 Moreover, substrate inhibition is a recurrent theme in these studies of dioxygenases, as is observed in DHP as well. 13,64 ■ CONCLUSION The present study permits a comparison of substrates and inhibitors that have different modes of binding. Both substrates and inhibitors bind in the distal pocket of the DHP protein in sites we call the α-and β-sites in Figure 1.…”

Role of indoleamine 2,3-dioxygenase in health and disease

“…On the basis of equilibrium binding studies, Sono et al [136] proposed that the ferrous enzyme binds first with L-Trp and then with O 2 [136]. However, a more recent study that combined kinetic and electrochemical measurements showed that, at low concentrations of L-Trp, O 2 binds first followed by L-Trp binding, but, at higher L-Trp concentrations, the binding order is reversed [137]. Resonance Raman spectroscopic data for the Fe II -O 2 complex of human recombinant IDO1 indicated that it is best described as an Fe III -O 2…”

Tryptophan oxygenation: mechanistic considerations

“…Notably the ternary complex resulted in ordering a loop adjacent to the active site [15]. Measurement of binding affinity suggested an obligate order of substrate binding, with CN binding first [15], which was also recently shown to be the case for TDO [16]. Unusually, when bound to PrnB, CN adopted a bent conformation (like that expected for oxygen), with the nitrogen pointing towards an oxyanion hole.…”