Tryptophan hydroxylase (TPH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin (5-HT), is irreversibly inactivated by nitric oxide (NO). We have expressed brain TPH as a recombinant glutathione-Stransferase fusion protein and delineated the catalytic domain of the enzyme as the region spanning amino acids 99-444. Highly purified TPH catalytic core, like the native enzyme from brain, is inactivated by NO in a concentration-dependent manner. Removal of iron from TPH produces an apoenzyme with low activity that can be reconverted to its highly active holoform by the addition of ferrous iron. Apo-TPH exposed to NO cannot be reactivated by iron. Treatment of holo-TPH (ironloaded) with the disulfide 5,5Ј-dithio-bis (2-nitrobenzoic acid) (DTNB) causes an inactivation of TPH that is readily reversed by dithiothreitol (DTT). DTNB-treated TPH [sulfhydryl (SH)-protected] exposed to NO is returned to full activity by thiol reduction with DTT. The inactivation of native TPH by NO cannot be reversed by either iron or DTT. These data indicate that NO inactivates TPH by selective action on critical SH groups (i.e., cysteine residues) while sparing catalytic iron sites within the enzyme. The results are interpreted with reference to the substituted amphetamines, which are neurotoxic to 5-HT neurons, that inactivate TPH in vivo and are now known to produce NO and other reactive oxygen species in vivo.
Key words: tryptophan hydroxylase; nitric oxide; sulfhydryls; catalytic iron site; serotonin; neurotoxic amphetaminesTryptophan hydroxylase [EC 1.14.16.4; L-tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating)] (TPH) is the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin (5-HT) (Jequier et al., 1967). The synthesis of 5-HT can proceed only through this enzymecatalyzed step. The activity of TPH controls the amount of 5-HT produced and released from neurons (Gartside et al., 1992;Oluyomi et al., 1994), indicating that this enzyme regulates not just 5-HT synthesis but its f unction as well. As a neurotransmitter, 5-HT mediates pain, sleep, thermoregulation, and food intake. From a clinical perspective, altered 5-HT f unction has been implicated in depression, obsessive-compulsive disorder, autism, and impulsive self-destructive behaviors such as aggression, suicide, and drug taking (Schatzberg and Nemeroff, 1995).Selected amphetamines have profound effects on the 5-HT neuronal system. Methylenedioxymethamphetamine (ecstasy) (MDM A) and p-chloroamphetamine cause extensive destruction of 5-HT neurons. An early manifestation of their effects is a significant inactivation of TPH (for reviews, see Gibb et al., 1993;Steele et al., 1994;Seiden and Sabol, 1996). The mechanisms underlying these effects on TPH are not known, but emerging data implicate drug-induced production of reactive oxygen species (ROS) and nitric oxide (NO). The cellular effects of ROS or NO cannot be predicted a priori: NO can be toxic to some cells (Lipton et al., 1993;Da...