TY - JOUR
T1 - Indoleamine analogs as probes of the substrate selectivity and catalytic mechanism of serotonin N-acetyltransferase
AU - Khalil, Ehab M.
AU - De Angelis, Jacqueline
AU - Cole, Philip A.
PY - 1998/11/13
Y1 - 1998/11/13
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.273.46.30321
DO - 10.1074/jbc.273.46.30321
M3 - Article
C2 - 9804794
AN - SCOPUS:0032514895
SN - 0021-9258
VL - 273
SP - 30321
EP - 30327
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 46
ER -