Spectral Studies on the Catalytic Mechanism and Activation of Pseudomonas Tryptophan Oxygenase (Tryptophan Pyrrolase)

The substrate, tryptophan, forms spectrophotometrically detectable enzyme-substrate complexes with tryptophan oxygenase (EC 1.13.1.12)—commonly known as tryptophan pyrrolase—when the iron atom of the heme prosthetic group is either divalent, trivalent, or combined with carbon monoxide. 2. One-half of the enzyme-bound ferriprotoporphyrin IX undergoes a rapid cyclic valence change during catalysis owing to its sequential reduction by tryptophan and reoxidation by oxygen. During steady state catalysis rapid reoxidation maintains the enzyme predominantly as ferriprotoporphyrin, but addition of CO to the steady state catalytic system leads to the detection and trapping of the reduced form as the CO-ferroprotoporphyrin complex. 3. Activation of latent enzyme may be accomplished with sulfhydryl compounds or photochemically, as well as with ascorbate. The nature of the action spectrum and the inhibition of activation by catalase implicates photochemically generated hydrogen peroxide as the chemical mediator of the photoactivation. 4. Spectral studies indicate that reductive or photochemical activation of the enzyme is not due to net reduction of the ferriprotoporphyrin, but rather to the reduction of an unidentified enzyme component which then permits the ferriprotoporphyrin prosthetic group to undergo reduction by the substrate, tryptophan. 5. p-Chloromercuribenzoate competitively inhibits tryptophan oxygenase, indicating probable involvement of one or more sulfhydryl groups as binding sites for tryptophan.