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.
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