ISOTOPE EFFECTS AND ALTERNATIVE SUBSTRATE REACTIVITIES FOR TRYPTOPHAN2,3-DIOXYGENASE

Tryptophan 2,3-dioxygenase (EC 1.13.1.12) is a hemoprotein which catal yzes the first step in the oxidative degradation of tryptophan. The re action 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 V( max)/K(m) was observed at the pH optimum, pH 7.0. This implies that ab straction of the indole proton is at least partially rate-determining. An inverse secondary isotope effect of 0.96 was observed for L-[2-H-3 ]tryptophan at this pH. The secondary isotope effect signals the forma tion of the C-O bond at C-2. As the rate of proton abstraction increas ed 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-determ ining 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 b inding 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 hindra nce 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 tryp tophan, and the increased reactivity can be explained by an electronic effect that enhances of the rate of C-O bond formation at C-2.