Abstract
Serotonin N-acetyltransferase (EC. 2.3.1.87) (NAT) is a melatonin rhythm-generating enzyme in pineal glands.Here we show novel regulation of NAT activity, in which an intramolecular disulfide bond may function as a switch for the catalysis. Recombinant-NAT activity was irreversibly inhibited by N-ethylmaleimide (NEM) in an acetyl CoA-protectable manner. Oxidized glutathione or dissolved oxygen reversibly inhibited NAT in an acetyl CoA-protectable manner. To identify the cysteine residues responsible for the inhibition, NAT was labeled with [14C]-NEM. Cys61 and Cys177 were specifically labeled in an acetyl CoA-protectable manner. An intramolecular disulfide bond between Cys61 and Cys177 formed upon oxidation and cleaved upon reduction was identified. Furthermore, Cys61Ala/Cys177Ala-NAT expressed in COS7 cells was relatively insensitive to H2O2-evoked oxidative stress, while wild type 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 NAT.
