Indoleamine analogs as probes of the substrate selectivity and catalytic mechanism of serotonin N-acetyltransferase

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase (AANAT)) catalyzes the reaction of serotonin (or tryptamine) with acetyl-CoA to form N-acetylserotonin (or N-acetyltryptamine) and is responsible for the melatonin circadian rhythm in vertebrates. This study evaluates a series of indoleamine analogs as alternate substrates of AANAT. 3-Indolepropylamine and 3-indolebutylamine were chemically synthesized and found to be processed by AANAT, although 20- and 60-fold less efficiently compared with the natural substrate serotonin, respectively. Racemic α-methyltryptamine and N(ω)- methyltryptamine were also shown to be substrates for AANAT, again with reduced k(cat) and k(cat)/K(m) compared with serotonin. The enzyme did exhibit 9:1 stereoselectivity for the R-enantiomer of α-methyltryptamine versus the S-enantiomer. By measuring the enzymatic rates versus increasing buffer microviscosity, it was demonstrated that diffusional release of product is most likely the principal rate-determining step for the enzymatic transformation of tryptamine (which has similar k(cat) and k(cat)K(m) compared with serotonin). Analysis of k(cat) and k(cat)/K(m) versus pH for the poor substrate N(ω)-methyltryptamine showed that an ionizable group on the enzyme with pK(α) ~7, required to be in its deprotonated form, may be important in catalysis. The αmethyltryptamine analog α- trifluoromethyltryptamine was not processed by the enzyme, but served as a modest competitive inhibitor. Taken together with the pH-rate analysis, these results favor a model in which the serotonin substrate binds to the enzyme as the positively charged ammonium salt, and nucleophilicity of the amine is important in enzyme-catalyzed acetyl transfer.