An intramolecular disulfide bridge as a catalytic switch for serotonin N-acetyltransferase.
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 [(14)C]NEM. Cys(61) and Cys(177) were specifically labeled in an acetyl-CoA-protected manner. The AA-NAT with the Cys(61) to Ala and Cys(177) 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 Cys(61) and Cys(177) formed upon oxidation and cleaved upon reduction was identified. Furthermore, C61A/C177A-AA-NAT expressed in COS7 cells was relatively insensitive to H(2)O(2)-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 Cys(61) and Cys(177) 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.
Tsuboi
Seiji
S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Kotani
Yoshifumi
Y
Ogawa
Ken'ichi
K
Hatanaka
Tadashi
T
Yatsushiro
Shouki
S
Otsuka
Masato
M
Moriyama
Yoshinori
Y
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000737:chemistry* / Q000378:metabolism; D019556:COS Cells; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D006861:Hydrogen Peroxide / Q000494:pharmacology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018384:Oxidative Stress; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D013997:Time Factors
D004220:Disulfides; D010455:Peptides; D006861:Hydrogen Peroxide; D001191:Arylamine N-Acetyltransferase; D003545:Cysteine; D000409:Alanine