Interplay Between Ligand and Substrate Binding in Human Indoleamine 2,3-Dioxygenase

Abstract

Human indoleamine 2,3-dioxygenase (hIDO) is a heme-containing enzyme that catalyzes the initial and rate-determining step of L-tryptophan (L-Trp) metabolism via the kynurenine pathway. Earlier kinetic studies showed that the Km of L-Trp (0.015 mM) is ∼27-fold lower than the Kd (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. Kinetic studies demonstrate that pre-binding of L-Trp to the enzyme retards cyanide binding by ∼13-fold, while pre-binding 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 favorable to bind O2 prior to L-Trp. Equilibrium titration studies show that the cyanide adduct is capable of binding two L-Trp molecules, with Kd values of 0.018 and 26 mM. The data offer the first direct evidence of the second substrate binding site in hIDO, underlying its substrate-inhibition behavior.