Indoleamine analogs as probes of the substrate selectivity and catalytic mechanism of serotonin N-acetyltransferase.
Serotonin N-acetyltransferase (arylalkylamine N-ace-tyltransferase (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 alpha-methyltryptamine and Nomega-methyltryptamine were also shown to be substrates for AANAT, again with reduced kcat and kcat/Km compared with serotonin. The enzyme did exhibit approximately 9:1 stereoselectivity for the R-enantiomer of alpha-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 kcat and kcat/Km compared with serotonin). Analysis of kcat and kcat/Km versus pH for the poor substrate Nomega-methyltryptamine showed that an ionizable group on the enzyme with pKa approximately 7, required to be in its deprotonated form, may be important in catalysis. The alpha-methyltryptamine analog alpha-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.[1]References
- Indoleamine analogs as probes of the substrate selectivity and catalytic mechanism of serotonin N-acetyltransferase. Khalil, E.M., De Angelis, J., Cole, P.A. J. Biol. Chem. (1998) [Pubmed]
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