Indoleamine 2,3-dioxygenase. Kinetic studies on the binding of superoxide anion and molecular oxygen to enzyme.

Abstract

To elucidate the reaction mechanism of indoleamine 2,3-dioxygenase, a protohemoprotein, the binding of molecular oxygen (02) and the superoxide anion (02-) to the enzyme in its ferrous and ferric states, respectively, was investigated. The second order rate constant for the binding of 01to the ferric enzyme was determined to be 1.1 x 10” M-’ s-’ at 24°C and pH 8.0. The rate constants for the binding of Opto the ferric enzyme in the absence and presence of 0.2 mM L-tryptophan were also estimated to be 3.3 X lo6 and 2.3 X lo6 M-’ s-l, respectively, by a different procedure. The ferrous form of enzyme was generated in situ by flash photolysis of the carbon monoxide complex of the enzyme in an air-saturated buffer. It reacted with O2 to form the oxygenated enzyme which was identified on the basis of its spectrum and the isosbestic points with the ferrous enzyme. The rate constants for the binding of 0, to the ferrous enzyme in the absence and presence of 0.2 mM I.-tryptophan were determined to be 7.4 x lo6 and 6.3 x lo6 M-’ s-l at 24°C and pH 8.0, respectively. The oxygenated form thus prepared in the presence of L-tryptophan produced approximately 50 mol of Nformylkynurenine/mol of enzyme and was oxidized to the ferric form during this process. It appears that 02and the ferric enzyme were not involved in product formation because neither superoxide dismutase nor potassium cyanide inhibited product formation. These results indicated that in the presence of the organic substrate the ternary complex of enzyme l O2 l substrate can be formed either by the reaction of Oawith the ferric enzyme or that of O2 with the ferrous enzyme. Once the ternary complex is formed, the reaction proceeds using O2 until the enzyme molecule is completely oxidized to the ferric form, for which 02is indispensable to yield the ternary complex.