INDOLEAMINE ANALOGS AS PROBES OF THE SUBSTRATE SELECTIVITY AND CATALYTIC MECHANISM OF SEROTONIN N-ACETYLTRANSFERASE

Serotonin N-acetyltransferase (arylallrylamine N-acetyltransferase (AA NAT)) catalyzes the reaction of serotonin (or tryptamine) with acetyl- CoA to form N acetyl serotonin (or N-acetyltryptamine) and is responsi ble for the melatonin circadian rhythm in vertebrates. This study eval uates a series of indoleamine analogs as alternate substrates of AANAT , 3-Indolepropylamine and 3-indolebutylamine were chemically synthesiz ed and found to be processed by AANAT, although 20- and 60-fold less e fficiently compared with the natural substrate serotonin, respectively . Racemic alpha-methyltryptamine and N-omega-methyltryptamine were als o shown to be substrates for AANAT, again with reduced k(cat) and k(ca t)/K-m compared with serotonin, The enzyme did exhibit similar to 9:1 stereoselectivity for the R-enantiomer of a methyltryptamine versus th e S-enantiomer, By measuring the enzymatic rates versus increasing buf fer microviscosity, it was demonstrated that diffusional release of pr oduct is most likely the principal rate-determining step for the enzym atic 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 versu s pH for the poor substrate N-omega-methyltryptamine showed that an io nizable group on the enzyme with pK(a) similar to 7, required to be in its deprotonated form, may be important in catalysis, The alpha-methy ltryptamine analog alpha-trifluoromethyltryptamine was not processed b y the enzyme, but served as a modest competitive inhibitor. Taken toge ther with the ps-rate analysis, these results favor a model in which t he serotonin substrate binds to the enzyme as the positively charged a mmonium salt, and nucleophilicity of the amine is important in enzyme- catalyzed acetyl transfer.