title: Enzyme kinetic and spectroscopic studies of inhibitor and effector interactions with indoleamine 2,3-dioxygenase. 2. Evidence for the existence of another binding site in the enzyme for indole derivative effectors.
abstract: To probe the active site of the heme protein indoleamine 2,3-dioxygenase, the effects of 3-indoleethanol (IET) (or tryptophol), one of the known indole derivative effectors, and indole (IND) on the catalytic (Vmax, Km) and spectroscopic properties (optical absorption and CD) of the enzyme 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 shown that, at millimolar concentrations, both IET and IND lower considerably the Km value for D-Trp by approximately 25% and approximately 60%, respectively, at pH 7.0, while neither affects the Km value for L-Trp. Interestingly, however, these effectors exert opposite effects with respect to each other on the Vmax values for both D-Trp and L-Trp: IET enhances the Vmax values by 40-60% while IND lowers them by 12-24%. These effects of IET and IND on the Vmax values may be attributed to the shift in the ferric (inactive) enzyme----ferrous (active) enzyme equilibrium either to the right (IET) or to the left (IND) caused by the binding of these effectors to the enzyme in the steady state of the catalytic reaction. Both effectors induce clearly detectable spectral changes, especially notable in CD spectra, upon binding (in a 1:1 molar ratio, Kd = 10(-4) to 2.5 X 10(-3) M) to the ferrous enzyme and its complexes with O2, CO, and NO, both in the presence and in the absence of L-Trp.(ABSTRACT TRUNCATED AT 250 WORDS)
authors: Department of Chemistry, University of South Carolina, Columbia 29208.
authors: M
authors: M
authors: Sono
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D007211:Indoles / Q000378:metabolism / Q000494:pharmacology*; D007421:Intestine, Small / Q000201:enzymology; D007700:Kinetics; D008962:Models, Theoretical; D010105:Oxygenases / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D013053:Spectrophotometry; D014366:Tryptophan Oxygenase; D000438:Alcohols
chemical_list: D007211:Indoles; D010105:Oxygenases; D014366:Tryptophan Oxygenase; C005949:tryptophol; D000438:Alcohols; C030374:indole