Effects of tryptophan and pH on the kinetics of superoxide radical binding to indoleamine 2,3-dioxygenase studied by pulse radiolysis.
The reaction of superoxide radical (O2-) with the heme protein indoleamine 2,3-dioxygenase has been investigated by the use of pulse radiolysis. In the absence of the substrate tryptophan (Trp), the ferric enzyme reacted quantitatively with O2- to form the oxygenated enzyme. The rate constant for the reaction (8.0 x 10(6) M-1 s-1 at pH 7.0) increased with a decrease in pH. In the presence of low concentrations of L-Trp (approximately 50 microM), under which the catalytic site of the ferric enzyme is greater than 99% Trp-free at pH 7.0, the only spectral species observed upon O2- binding was L-Trp-bound oxygenated enzyme, the ternary complex. This suggests that under the conditions employed O2- binds first to the ferric enzyme to form the oxygenated enzyme and is followed by rapid binding of L-Trp. It was also found that absorbance changes (delta A) for the enzyme after the pulse were significantly decreased when an increased L-Trp concentration was employed. A 50% decrease in delta A was caused with approximately 50 microM L-Trp at pH 7.0. Similar results were also observed with other indole derivatives with decreasing delta A values in the order of indole, 3-indoleethanol, alpha-methyl-DL-Trp, and D-Trp. These results suggest that there exists a binding site for these compounds in the dioxygenase different from the catalytic site for Trp and, most significantly, that binding of Trp to the effector binding site of the ferric enzyme markedly inhibits its reaction with O2-.
Kobayashi
K
K
Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Japan.
Hayashi
K
K
Sono
M
M
D000818:Animals; D001665:Binding Sites; D007421:Intestine, Small / Q000201:enzymology; D007700:Kinetics; D009130:Muscle, Smooth / Q000201:enzymology; D010105:Oxygenases / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D013053:Spectrophotometry; D013481:Superoxides / Q000378:metabolism*; D013997:Time Factors; D014364:Tryptophan / Q000494:pharmacology*; D014366:Tryptophan Oxygenase
D013481:Superoxides; D014364:Tryptophan; D010105:Oxygenases; D014366:Tryptophan Oxygenase