Enzyme kinetic and spectroscopic studies of inhibitor and effector interactions with indoleamine 2,3-dioxygenase. 1. Norharman and 4-phenylimidazole binding to the enzyme as inhibitors and heme ligands.
The effects of norharman, one of the few known inhibitors of the heme protein indoleamine 2,3-dioxygenase, and of 4-phenylimidazole (4-PheImid), a heme ligand, on the catalytic (Vmax, Km) and spectroscopic properties (optical absorption, CD, and magnetic CD) of the rabbit small intestinal dioxygenase were investigated. Assays were performed with the substrate L- or D-tryptophan (Trp) and an ascorbic acid-methylene blue cofactor system at 25 degrees C. This study has revealed that both norharman and 4-PheImid exhibit noncompetitive inhibition with respect to L-Trp and D-Trp. The binding of norharman to the enzyme results in the formation of a low-spin complex in both the ferric and ferrous enzyme with comparable dissociation constants (Kd = approximately 10 microM at pH 7.0) that are about 10 times smaller than the observed Ki value. L-Trp exerts no effect for the ferric enzyme and slight negative cooperative effects for the ferrous enzyme on norharman binding. Close spectral similarities are observed between the adducts of the enzyme with norharman and 4-PheImid in the respective oxidation states. This, together with competition experiments using cyanide, demonstrates that norharman binds directly to the heme iron of the enzyme as a nitrogen donor ligand. Thus, norharman competes with O2 for the heme iron of the ferrous (active) enzyme, resulting in the observed inhibition. L-Trp and 4-PheImid appear to compete for the heme binding site in the ferric enzyme and display slight negative cooperativity on binding to the ferrous enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Chemistry, University of South Carolina, Columbia 29208.
Cady
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D000470:Alkaloids / Q000378:metabolism*; D000818:Animals; D002243:Carbolines; D006247:Harmine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D006418:Heme / Q000378:metabolism; D007093:Imidazoles / Q000378:metabolism* / Q000494:pharmacology; D007421:Intestine, Small / Q000201:enzymology; D007700:Kinetics; D008024:Ligands; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet; D014366:Tryptophan Oxygenase
D000470:Alkaloids; D002243:Carbolines; D007093:Imidazoles; D008024:Ligands; D006418:Heme; D006247:Harmine; C033023:4(5)-phenylimidazole; C010262:norharman; D010105:Oxygenases; D014366:Tryptophan Oxygenase