Spectroscopic studies of ligand and substrate binding to human indoleamine 2,3-dioxygenase

Abstract

Human indoleamine 2,3-dioxygenase (hIDO) is an intracellular heme-containing enzyme, which catalyzes the initial and rate-determining step of l-tryptophan (l-Trp) metabolism via the kynurenine pathway in nonhepatic tissues. Steady-state kinetic data showed that hIDO exhibits substrate inhibition behavior, implying the existence of a second substrate binding site in the enzyme, although so far there is no direct evidence supporting it. The kinetic data also revealed that the K(m) of l-Trp (15 microM) is approximately 27-fold lower than the K(d) of l-Trp (0.4 mM) for the ligand-free ferrous enzyme, suggesting that O(2) binding proceeds l-Trp binding during the catalytic cycle. With cyanide as a structural probe, we have investigated the thermodynamic and kinetic parameters associated with ligand and substrate binding to hIDO. Equilibrium titration studies show that the cyanide adduct is capable of binding two l-Trp molecules, with K(d) values of 18 microM and 26 mM. The data offer the first direct evidence of the second substrate binding site in hIDO. Kinetic studies demonstrate that prebinding of l-Trp to the enzyme retards cyanide binding by approximately 13-fold, while prebinding of cyanide to the enzyme facilitates l-Trp binding by approximately 22-fold. The data support the view that during the active turnover of the enzyme it is kinetically more favored to bind O(2) prior to l-Trp.

Figure 1

Absorption spectra of ferrous hIDO (a) and equilibrium titration curve of ferrous hIDO with L-Trp (b). The absorption spectra in panel a were recorded in the absence (black curve) or presence (red curve) of 20 mM L-Trp. The ΔA values in panel b were measured at 428 nm. All the data were obtained with 5.5 μM hIDO in 100 mM Tris buffer (pH 7.4) at room temperature.

Figure 2

Absorption spectra of ferric hIDO (a) and equilibrium titration curve of ferric hIDO with L-Trp (b). The absorption spectra in panel a were recorded in the absence (black curve) or presence (red curve) of 20 mM L-Trp. The ΔA values in panel b were measured at 404 nm. All the data were obtained with 5.5 μM hIDO in 100 mM Tris buffer (pH 7.4) at room temperature.

Figure 3

Absorption spectra of cyanide-bound ferric hIDO (a) and equilibrium titration curve of the substrate-free ferric hIDO with cyanide (b) or that of the cyanide-bound hIDO with L-Trp (c). The absorption spectra in panel a were recorded in the absence (black curve) or presence of 1 mM L-Trp (red curve) or 44 mM L-Trp (blue curve). The ΔA values in panels b and c were measured at 403 and 423 nm, respectively. The data were obtained with 4.7 μM hIDO (a and c) and 1.5 μM hIDO (b) in 100 mM Tris buffer (pH 7.4) at room temperature. The cyanide concentration used for panels a and c was 1 mM.

Figure 4

L-Trp binding kinetics of cyanide-free and cyanide-bound ferric hIDO (a) and plot of the observed rate constants as a function of L-Trp concentration (b). The kinetic traces of the cyanide-free hIDO (2.5 μM) and cyanide-bound hIDO (4.0 μM) shown in panel a were obtained with 750 μM L-Trp at 403 nm and 75 μM Trp at 421 nm, respectively. The solid lines in panel a are the best-fit curves with single-exponential functions. All the samples were prepared in 100 mM Tris buffer (pH 7.4) at 20 °C, in the presence or absence of 1 mM cyanide.

Figure 5

Cyanide binding kinetics of substrate-free and L-Trp-bound ferric hIDO (a) and plot of the observed rate constants as a function of cyanide concentration (b). The kinetic traces of the substrate-free hIDO (1.5 μM)and L-Trp-bound hIDO (3.1 μM)shown in panel a were obtained with 87 μM cyanide at 404 and 410 nm, respectively. The solid lines in panel a are the best-fit curves with single-exponential functions. All the samples were prepared in 100 mM Tris buffer (pH 7.4) at 20 °C, in the presence or absence of 25 mM L-Trp.

Figure 6

Thermodynamic cycle describing the cyanide and L-Trp binding reactions of hIDO. The parameters labeled in red were obtained from equilibrium measurements. The parameters listed in the shaded boxes were obtained from kinetic measurements. The free energy (ΔG) values associated with ligand or substrate binding were calculated from K_d, on the basis of the relationship ΔG = RT ln(K_d). The side reaction labeled in green is associated with the binding of L-Trp to the second substrate binding site in the cyanide-bound enzyme.

Scheme 1

Dioxygenation Reaction of Tryptophan Catalyzed by IDO and TDO

Scheme 2

Catalytic Cycle of Human Indoleamine 2,3-Dioxygenase