Abstract
Tryptophan 2,3-dioxygenase (EC 1.13.1.12) is a hemoprotein which catalyzes the first step in the oxidative degradation of tryptophan. The reaction is believed to proceed by addition of O2 across the 2,3-bond of the indole ring, followed by decomposition of the resultant dioxetane to give N-formylkynurenine. A primary D2O isotope effect of 4.4 on Vmax/Km was observed at the pH optimum, pH 7.0. This implies that abstraction of the indole proton is at least partially rate-determining. An inverse secondary isotope effect of 0.96 was observed for L-[2-3H]tryptophan at this pH. The secondary isotope effect signals the formation of the C-O bond at C-2. As the rate of proton abstraction increased with increasing pH, the D2O isotope effect decreased to 1.2 at pH 8.5 and the secondary isotope effect increased to 0.92. The rate-determining steps therefore change with increasing pH, and bond formation at C-2 becomes more rate-limiting. The secondary isotope effect did not change significantly with varying O2 concentration so that substrate binding is primarily ordered with O2 binding first. The specificity of the enzyme towards substituted tryptophans shows that substitution of the phenyl ring of the indole is sterically unfavorable. Steric hindrance is highest at the 4- and 7-positions, while the 5- and 6-positions are less sensitive. 6-Fluoro-L-tryptophan was more reactive than tryptophan, and the increased reactivity can be explained by an electronic effect that enhances of the rate of C-O bond formation at C-2.
Highlights
Tryptophan 2,3-dioxygenase (EC 1.13.1.12) is a for tryptophan at neutral pH (Schutz et al, 1972).a-Methylhemoprotein which catalyzes the first stepin the oxi- DL-tryptophan is not a substrate but is an allosteric effector
A Equilibrium binding studies with Pseudomonas tryptophan primary DzO isotope effect of 4.4 on V,aJKm was dioxygenase and indoleamine dioxygenase
It is assumed that oxidative cleavage of the indole ring procedes via formation and decomposition of a dioxetane intermediate
Summary
Contained 4.9 D M L-tryptophan, 20 mM sodium formate, 2 mM sodium ascorbate, 0.46 p~ hematin, and ~-[2-"C]tryptophan(typi-. A number of other tryptophan analogues were tested but perchloric acid was added and the samples were incubated an additional 30 min. The columns were eluted for 5 analogue relative to tryptophan when the two substrates were incubated together in the same reaction mixture (Abeles et al, 1960). The assay mixture contained 0.1 mM L-tryptophan, 3 mM a-methyl-DLtryptophan, 50 mM sodium formate, 4 mM sodium ascorbate, 0.92 g M range of reactivities. 6-Fluoro-~-tryptophanwas actryptophan per100 pl.For assays at pH7.0, a single reaction mixture tually a better substratethan tryptophan,. This may was prepared in 0.1 M potassium phosphate. Samples for all conditions were analyzed by HPLC, and 1-min fractions were collected and counted for 20 min
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