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Volume 17, Number 19, Issue of October 1, 1997 pp. 7245-7251
Copyright ©1997 Society for Neuroscience

Molecular Mechanism of the Inactivation of Tryptophan Hydroxylase by Nitric Oxide: Attack on Critical Sulfhydryls that Spare the Enzyme Iron Center

Received May 27, 1997; revised July 14, 1997; accepted July 16, 1997.

Donald M. Kuhn^{1, 2} and Robert Arthur Jr¹

¹ Cellular and Clinical Neurobiology Program, Department of Psychiatry and Behavioral Neurosciences, and ² Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48201

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-S-transferase 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 holo-form by the addition of ferrous iron. Apo-TPH exposed to NO cannot be reactivated by iron. Treatment of holo-TPH (iron-loaded) with the disulfide 5,5-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 amphetamines

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