Abstract
Abstract The kinetic properties of highly purified hepatic l-tryptophan oxygenase have been studied at different hydrogen ion concentrations in the absence and presence of dl-α-methyltryptophan and 3-hydroxyanthranilic acid. At pH 8.0 plots of velocity versus l-tryptophan concentrations indicate classical hyperbolic Michaelis-Menten kinetics, whereas at pH 6.2 saturation by tryptophan is nonhyperbolic with a marked increase in the [Trp]0.5. This effect of pH is reversible. The tryptophan metabolite, 3-hydroxyanthranilic acid, is a negative modulator which at pH 7.9 converts saturation of tryptophan oxygenase by tryptophan from hyperbolic to sigmoidal. This regulatory effect is even more marked upon the protonated enzyme. Progressive saturation by 3-hydroxyanthranilic acid is itself sigmoidal, which is expressed only at higher substrate levels. α-Methyltryptophan increases the apparent affinity of hepatic tryptophan oxygenase for l-tryptophan at pH 6.2, but not at pH 8.0. α-Methyltryptophan, in concentrations where it is neither a substrate nor a competitive inhibitor, antagonizes the inactivation of the enzyme by sodium dodecyl sulfate. These findings indicate that hepatic tryptophan oxygenase is subject to allosteric regulation. The allosteric modulation of the enzyme activity can be shown with its substrate, l-tryptophan, as well as with positive, α-methyltryptophan, and negative, 3-hydroxyanthranilic acid, effectors. These allosteric properties of tryptophan oxygenase are strongly affected by the hydrogen ion concentration in the assay medium.
Highlights
PH 6.2 saturation by tryptophan is nonhyperbolic with a marked increase in the [Trp]O.s
The kinetic properties of tryptophan oxygenase have been studied at different pH values
8.0, the substrate saturation curve is transformed to a hyperbola which is reflected as a linear plot in the Lineweaver-Burk representation, suggestive of classical Michaelis-Menten kinetics
Summary
Tryptophan oxygenase was purified to homogeneity as described [3]. In certain experiments the final gel electrophoresis step has been omitted; the enzyme used, always had a specific activity above 1.5 units per mg of protein. The enzyme was passed through a Sephadex G-25 column equilibrated with 0.1 M sodium phosphate, pH 7.0, to remove free Ltryptophan. The enzyme was assayed as previously described [3]. Phosphate buffers were used throughout these experiments.
Sign-in/Register to view highlights & summary 
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call