Biochemical characterization of recombinant serotonin N-acetyltransferase.
Pineal and retinal melatonin synthesis is controlled by the enzymatic activity of arylalkylamine N-acetyltransferase (AA-NAT, EC 2.3.1.87), which is regulated by light/dark signals and circadian factors. This enzyme converts serotonin to N-acetylserotonin by the transfer of an acetyl group from acetyl coenzyme A. Endogenous AA-NAT instability during routine purification has made enzyme characterization difficult, but now a stable recombinant protein for AA-NAT has been synthesized to investigate the intrinsic biochemical properties of AA-NAT from a rat pineal cDNA encoding a 205 amino acid, 23 kilodalton protein, by using a glutathione-S-transferase (GST) fusion protein system. Recombinant GST-AA-NAT showed substrate specificity for arylalkylamines and stability at 4 degrees C; however, the enzyme activity was reduced by 40% upon preincubation at 37 degrees C for 2 hr. GST-AA-NAT is preferentially phosphorylated by either cyclic AMP- or cyclic GMP-dependent kinases in vitro, but no detrimental effect was observed on AA-NAT enzymatic activity. Among the metal cations tested in this study, Ca2+, Mg2+, Mn2+, Fe2+, and Co2 showed little or no inhibitory potency, while either 1 mM Zn2+ or 0.1 mM Cu2+ nearly abolished the enzymatic activity. GST-AA-NAT enzyme activity is also inhibited by reagents that are known biochemically to modify thiol groups (N-ethylmaleimide, NEM) and histidine residues (p-chloromercuribenzoate, NBS and diethyl pyrocarbonate, DEPC), suggesting the presence of essential cysteine and histidine moieties. Moreover, preincubation of acetyl CoA completely protects the recombinant AA-NAT from inactivation by NEM and DEPC, indicating that specific cysteine and histidine residues may be at the acetylation site. The conclusion is that the biochemical properties of rat recombinant AA-NAT is similar to the endogenous pineal and retinal AA-NAT with respect to the sensitivity to temperature, metal cations, as well as the thiol modification reagents. These data also suggest that the phosphorylation status of the AA-NAT does not affect enzymatic activity directly, and histidine residues are potentially important residues required for high catalytic activity.
Zhan-Poe
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Doheny Eye Institute, Department of Cell and Neurobiology, University of Southern California School of Medicine, Los Angeles 90033, USA.
Craft
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D000107:Acetylation / Q000187:drug effects; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D005990:Glycerol / Q000494:pharmacology; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012709:Serum Albumin / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects
D000596:Amino Acids; D002413:Cations, Divalent; D002614:Chelating Agents; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D001191:Arylamine N-Acetyltransferase; D005982:Glutathione Transferase; D005990:Glycerol