The mechanism of substrate inhibition in human indoleamine 2,3-dioxygenase

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.

Scheme 1. Reaction Catalyzed by IDO

Scheme 2. Mechanistic Scheme Used for Analysis of Substrate Inhibition

Figure 1

Active-site structure in hIDO, showing the locations of the residues targeted by mutagenesis in this work.

Figure 2

Plots of rate (ΔAbs min^–1) versus substrate concentration for hIDO (■) and the S167A variant of hIDO (△). Lines show fits of the data to eq 1; steady-state parameters extracted from the fit are listed in Table 1. Conditions: 0.1 M Tris-HCl, pH 8.0, [enzyme] = 100 nM, 30 μg/mL catalase, [ascorbate] = 20 mM, [methylene blue] = 10 μM, [O₂] = 258 μM, 20.0 °C.

Figure 3

Logarithmic dependence of k₁ (see values given in Table 1), normalized to k_1 (hIDO), on the difference of the reduction potentials between the corresponding variant E_m⁰ (Fe³⁺/Fe²⁺) and hIDO (E_m⁰ (Fe³⁺/Fe²⁺ (hIDO)). The F164A variant is not included in this plot because the stability of its ferrous oxy was too low to allow a meaningful determination of k₁ (see also data in Table 1).

Figure 4

Logarithmic dependence of k_cat, normalized to k_cat (hIDO), on the difference of the reduction potentials between the corresponding variant E_m⁰ (Fe³⁺/Fe²⁺) and hIDO E_m⁰ (Fe³⁺/Fe²⁺ (hIDO)). See also data in Table 1 (and footnote k).

Figure 5

Logarithmic plot of the dependence of K_i^eff (Table 1), normalized to K_i^eff_(hIDO), on the difference of the reduction potentials between the corresponding variant E_m⁰ (Fe³⁺/Fe²⁺) and hIDO E_m⁰ (Fe³⁺/Fe²⁺ (hIDO)). See also data in Table 1 (and footnote d).

Figure 6

Plots of previously published data (taken from Table 1 of ref (25)) showing (A) ln V_max and (B) ln K_50(O2) for heme-substituted TDO as a function of reduction potential of the corresponding heme. V_max is the maximal turnover rate, and K_50(O2) is the O₂ affinity.