Partial Purification and Characterization of Tryptophan Hydroxylase from Rabbit Hindbrain

With the use of a rapid, sensitive fluorometric assay, tryptophan hydroxylase from rabbit hindbrain has been partially purified. The enzyme resembles phenylalanine and tyrosine hydroxylases in many respects. For example, the hydroxylation reaction shows an absolute requirement for a tetrahydropterin and is stimulated by reduced pyridine nucleotide, dihydropteridine reductase, and catalase. Fe2+ also stimulates the reaction but only in the absence of catalase; this finding suggests that catalase or Fe2+ protects a sensitive component of the tryptophan hydroxylation system from inactivation by H2O2. Just as with the other pterin-dependent aromatic amino acid hydroxylases, the apparent Km of tryptophan hydroxylase for its amino acid substrate and for oxygen are markedly lower in the presence of tetrahydrobiopterin than they are in the presence of the cofactor analogue, dimethyltetrahydropterin. In addition, high concentrations of tryptophan inhibit the reaction in the presence of tetrahydrobiopterin, but not in the presence of the dimethylpterin. The stoichiometry of the reaction catalyzed by tryptophan hydroxylase in the presence of tetrahydrobiopterin has been determined.