Substrate specificity and inhibition studies of human serotonin N-acetyltransferase

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the reaction of seroto nin with acetyl-CoA to form N-acetylserotonin and plays a major role in the regulation of the melatonin circadian rhythm in vertebrates. In the presen t study, the human cloned enzyme has been expressed in bacteria, purified, cleaved, and characterized. The specificity of the human enzyme toward subs trates (natural as well as synthetic arylethylamines) and cosubstrates (ess entially acyl homologs of acetyl-CoA) has been investigated. Peptide combin atorial libraries of tri-, tetra- and pentapeptides with various amino acid compositions were also screened as potential sources of inhibitors. We rep ort 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 sepa ration and detection of N-[H-3]acetylarylethylamine formed from various ary lethylamines 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, a nd was successfully used throughout this study. me also screened a large nu mber of pharmacological bioamines among which only one, tranylcypromine, be haved as a substrate. The synthesis and survey of simple arylethylamines al so showed that AANAT has a large recognition pattern, including compounds a s different as phenyl-, naphthyl-, benzothienyl-, or benzofuranyl-ethylamin e derivatives. An extensive enzymatic study allowed us to pinpoint the amin o acid residue of the pentapeptide inhibitor, S 34461, which interacts with the cosubstrate-binding site area, in agreement with an in silico study ba sed on the available coordinates of the hAANAT crystal.