Spectroscopic studies of ligand and substrate binding to human indoleamine 2,3-dioxygenase.
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.
Lu
Changyuan
C
Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Lin
Yu
Y
Yeh
Syun-Ru
SR
D001665:Binding Sites; D002384:Catalysis; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006801:Humans; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000737:chemistry*; D007700:Kinetics; D008024:Ligands; D011190:Potassium Cyanide / Q000737:chemistry; D011485:Protein Binding; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry*
D005290:Ferric Compounds; D005296:Ferrous Compounds; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D008024:Ligands; D014364:Tryptophan; D011190:Potassium Cyanide