Utilization of dihydroflavin mononucleotide and superoxide anion for the decyclization of L-tryptophan by murine epididymal indoleamine 2,3-dioxygenase.
Dihydroflavin mononucleotide (FMNH2) together with a regenerating enzyme system effectively supported L-tryptophan decyclization by indoleamine 2,3-dioxygenase isolated from murine epididymis. The native murine dioxygenase was a monomeric protein with Mr 40,000 +/- 1000, an apparent pI of 4.9 +/- 0.1, and an optimum pH within the range of 7 to 8. Using FMNH2 with FMN oxidoreductase, the enzyme attained significantly higher activity than the apparent maximal activity obtained by using the other electron donor systems examined (e.g., riboflavin, FAD, tetrahydrobiopterin, methylene blue). A kinetic study with the FMNH2 cofactor suggested the occurrence of a complex reaction (L-tryptophan-FMNH2 interdependency) and a theoretical K'm of 14 microM or a Km of 13 microM was estimated for the substrate. L-Tryptophan 2,3-dioxygenation was competitively inhibited by L-5-hydroxytryptophan with a Ki of 1 microM. The reaction rate was reduced to less than 50% of that of the control in the presence of superoxide dismutase and was decreased to 3% of the control in the absence of catalase. Thus, superoxide anion does not appear to be the only form of O2 participating in the reaction. However, these data indicate that the activation of molecular oxygen is an essential factor for an optimum catalysis and a mechanism of FMNH2-dependent oxygenation of L-tryptophan by murine indoleamine 2,3-dioxygenase.
Ozaki
Y
Y
Nichol
C A
CA
Duch
D S
DS
D006916:5-Hydroxytryptophan / Q000494:pharmacology; D000818:Animals; D002374:Catalase / Q000494:pharmacology; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004822:Epididymis / Q000201:enzymology*; D005486:Flavin Mononucleotide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007700:Kinetics; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D010105:Oxygenases / Q000494:pharmacology*; D013482:Superoxide Dismutase / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism*; D014366:Tryptophan Oxygenase
D013481:Superoxides; D005486:Flavin Mononucleotide; D014364:Tryptophan; D006916:5-Hydroxytryptophan; D002374:Catalase; D010105:Oxygenases; D014366:Tryptophan Oxygenase; D013482:Superoxide Dismutase