Spectroscopic Studies of Ligand and Substrate Binding to Human Indoleamine 2,3-Dioxygenase

摘要

Human indoleamine 2,3-dioxygenase (hIDO) is an intracellular heme-containing enzyme, whichncatalyzes the initial and rate-determining step of L-tryptophan (L-Trp) metabolism via the kynureninenpathway in nonhepatic tissues. Steady-state kinetic data showed that hIDO exhibits substrate inhibitionnbehavior, implying the existence of a second substrate binding site in the enzyme, although so far there is nondirect evidence supporting it. The kinetic data also revealed that the Km of L-Trp (15 μM) is ∼27-fold lowernthan the Kd of L-Trp (0.4 mM) for the ligand-free ferrous enzyme, suggesting that O2 binding proceeds L-Trpnbinding during the catalytic cycle. With cyanide as a structural probe, we have investigated the thermo-ndynamic and kinetic parameters associated with ligand and substrate binding to hIDO. Equilibrium titrationnstudies show that the cyanide adduct is capable of binding two L-Trp molecules, with Kd values of 18 μMandn26 mM. The data offer the first direct evidence of the second substrate binding site in hIDO. Kinetic studiesndemonstrate that prebinding of L-Trp to the enzyme retards cyanide binding by ∼13-fold, while prebinding ofncyanide to the enzyme facilitates L-Trp binding by ∼22-fold. The data support the view that during the activenturnover of the enzyme it is kinetically more favored to bind O2 prior to L-Trp.