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 Km of L-Trp (15 μM) is ~27-fold lower than the Kd of L-Trp (0.4 mM) for the ligand-free ferrous enzyme, suggesting that O2 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 Kd values of 18 μM 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 ~13-fold, while prebinding of cyanide to the enzyme facilitates L-Trp binding by ~22-fold. The data support the view that during the active turnover of the enzyme it is kinetically more favored to bind O2 prior to L-Trp.

Discovery and preliminary SARs of keto-indoles as novel indoleamine 2,3-dioxygenase (IDO) inhibitors

“…The optical absorption spectrum of the substrate-free ferric IDO exhibits a Soret maximum at 404 nm and the binding of L-Trp causes, as reported previously, a red-shift of the Soret maximum to 410 nm together with a hypochromic effect (Fig. 5a) [37]. In the case of 1a addition (Fig.…”

Ferryl Derivatives of Human Indoleamine 2,3-Dioxygenase

“…The inhibitory site has been identified in the CObound ferrous complex at low temperature (44) and the CNbound ferric adduct at room temperature (31) but not in ligandfree ferric and ferrous states (31,39). The current data demonstrate that the ferryl species also possesses two substrate-binding sites, with …”

“…Steady-state kinetic studies of the dioxygenase reaction of hIDO (31,39) show that the K m (L-Trp) is 15 M, which is considerably lower than the K d (Trp) for the ligand-free ferric (900 M) or ferrous enzyme (400 M). Accordingly, we have proposed that O 2 binding occurs prior to L-Trp binding during the multiple turnover of the dioxygenase reaction under physiological conditions (31,39).…”

“…Although the dioxygenase reaction of IDO does not consume any electrons, a small portion of the ternary complex can leak out the active cycle by releasing O 2 as superoxide (1,31). Consequently, a significant amount of ferric enzyme accumulates during multiple turnovers, in particular under electron-deficient conditions.…”

“…The L-Trp binding curve was obtained by plotting ⌬A (defined by ⌬A 418 nm Ϫ ⌬A 450 nm ) as a function of L-Trp. A binding model with two independent binding sites, each described by the following Equation 1, was used to fit the data by using the Origin 6.1 program (Microcal, Inc., Northampton, MA) (31). Here, K d is the substrate dissociation constant; ⌬A max is the maximum ⌬A.…”