Substrate Specificity and Inhibition Studies of Human Serotonin N-Acetyltransferase.
Ferry G, Loynel A, Kucharczyk N, Bertin S, Rodriguez M, Delagrange P, Galizzi
JP, Jacoby E, Volland JP, Lesieur D, Renard P, Canet E, Fauchere JL,
Division de Pharmacologie Moleculaire et Cellulaire, Institut de Recherches Servier, 125 Chemin de Ronde, 78290 Croissy sur Seine, France.
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Arylalkylamine N-acetyltransferase (AANAT) catalyzes the reaction of serotonin
with acetyl-CoA to form N-acetylserotonin and plays a major role in the
the melatonin circadian rhythm in vertebrates. In the present study, the human cloned enzyme has been expressed in bacteria, purified, cleaved, and characterized. The
specificity of the human enzyme toward substrates (natural as well as synthetic arylethylamines) and cosubstrates (essentially acyl homologs of acetyl-CoA) has been
investigated. Peptide combinatorial libraries of tri-, tetra-, and pentapeptides with various amino acid compositions were also screened as potential sources of inhibitors.
We report the findings of several peptides with low micromolar inhibitory potency. For activity measurement as well as for specificity studies, an original and rapid
method of analysis was developed. The assay was based on the separation and detection of N-[(3)H]acetylarylethylamine formed from various arylethylamines and
tritiated acetyl-CoA, by means of high performance liquid chromatography with radiochemical detection. The assay proved to be robust and flexible, could accommodate
the use of numerous synthetic substrates, and was successfully used throughout this study. We also screened a large number of pharmacological bioamines among
which only one, tranylcypromine, behaved as a substrate. The synthesis and survey of simple arylethylamines also showed that AANAT has a large recognition pattern,
including compounds as different as phenyl-, naphthyl-, benzothienyl-, or benzofuranyl-ethylamine derivatives. An extensive enzymatic study allowed us to pinpoint the
amino acid residue of the pentapeptide inhibitor, S 34461, which interacts with the cosubstrate-binding site area, in agreement with an in silico study based on the
available coordinates of the hAANAT crystal.