An intramolecular disulfide bridge as a catalytic switch for serotonin N-acetyltransferase

Seiji Tsuboi, Yoshifumi Kotani, Ken'ichi Ogawa, Tadashi Hatanaka, Shouki Yatsushiro, Masato Otsuka, Yoshinori Moriyama

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9 Citations (Scopus)

Abstract

Serotonin N-acetyltransferase (EC. 2.3.1.87) (AA-NAT) is a melatonin rhythm-generating enzyme in pineal glands. To establish a melatonin rhythm, AA-NAT activity is precisely regulated through several signaling pathways. Here we show novel regulation of AA-NAT activity, in which an intramolecular disulfide bond may function as a switch for the catalysis. Recombinant AA-NAT activity was irreversibly inhibited by N-ethylmaleimide (NEM) in an acetyl-CoA-protected manner. Oxidized glutathione or dissolved oxygen reversibly inhibited AA-NAT in an acetyl-CoA-protected manner. To identify the cysteine residues responsible for the inhibition, AA-NAT was first oxidized with dissolved oxygen, treated with NEM, reduced with dithiothreitol, and then labeled with [14C]NEM. Cys61 and Cys177 were specifically labeled in an acetyl-CoA-protected manner. The AA-NAT with the Cys61 to Ala and Cys177 to Ala double substitutions (C61A/C177A-AA-NAT) was fully active but did not exhibit sensitivity to either oxidation or NEM, whereas the AA-NATs with only the single substitutions retained about 40% of these sensitivities. An intramolecular disulfide bond between Cys61 and Cys177 formed upon oxidation and cleaved upon reduction was identified. Furthermore, C61A/C177A-AA-NAT expressed in COS7 cells was relatively insensitive to H2O2-evoked oxidative stress, whereas wild-type AA-NAT was strongly inhibited under the same conditions. These results indicate that the formation and cleavage of the disulfide bond between Cys61 and Cys177 produce the active and inactive states of AA-NAT. It is possible that intracellular redox conditions regulate AA-NAT activity through switching via an intramolecular disulfide bridge.

Original languageEnglish
Pages (from-to)44229-44235
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number46
DOIs
Publication statusPublished - Nov 15 2002

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Tsuboi, S., Kotani, Y., Ogawa, K., Hatanaka, T., Yatsushiro, S., Otsuka, M., & Moriyama, Y. (2002). An intramolecular disulfide bridge as a catalytic switch for serotonin N-acetyltransferase. Journal of Biological Chemistry, 277(46), 44229-44235. https://doi.org/10.1074/jbc.M203305200