Properties of tryptophan hydroxylase from human carcinoid tumor

Abstract

Tryptophan 5-hydroxylase ( l-tryptophan, tetra-hydropteridine: oxygen oxidoreductase (5-hydroxylating), EC 1.14.16.4) was purified 6-fold from a human carcinoid tumor containing a large amount of 5-hydroxytryptophan as well as 5-hydroxytryptamine. The enzyme was activated following anaerobic dialysis in the presence of 2-mercaptoethanol, and did not require 2-mercaptoethanol and Fe 2+ for full activity, though these reducing agents shifted the pH optimum for maximum enzyme activity on the more acidic side. The K m values were 13 μM for tryptophan in the presence of 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetra-hydropteridine, 1.2% and 7.1% for oxygen in the presence of either 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetra-hydropteridine or 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetra-hydropteridine, respectively, and 50 μM for 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetra-hydropteridine and 105 μM for 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetra-hydropteridine. The substrate inhibition by tryptophan was observed with 2-amino-4-hydroxy-6-methyl5,6,7,8-tetra-hydropteridine, but not with 2-amino-4-hydroxy-6,7-dimethyl5,6,7,8-tetra-hydropteridine, and high oxygen levels did not inhibit the hydroxylation in the presence of these reduced pterins. It was found that o-phenanthroline was also a potent inhibitor of tryptophan hydroxylase from this tumor similar to the mast-cell enzyme and the other pterin-dependent aromatic amino acid hydroxylases. The inhibition by this metal chelating agent was competitive with respect to both tryptophan and 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetra-hydropteridine, but not to molecular oxygen under the assay conditions employed. The results obtained support the idea that tryptophan hydroxylase found in some human carcinoids appears to function in the same manner as do most other mammalian hydroxylases.