Kinetic analysis of the catalytic mechanism of serotonin N-acetyltransferase (EC 2.3.1.87).
Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT, EC 2.3.1.87) is the penultimate enzyme in melatonin biosynthesis. This enzyme is of special biological interest because large changes in its activity drive the large night/day rhythm in circulating melatonin in vertebrates. In this study the kinetic mechanism of AANAT action was studied using bacterially expressed glutathione S-transferase (GST)-AANAT fusion protein. The enzymologic behavior of GST-AANAT and cleaved AANAT was essentially identical. Two-substrate kinetic analysis generated an intersecting line pattern characteristic of a ternary complex mechanism. The dead end inhibitor analog desulfo-CoA was competitive versus acetyl-CoA and noncompetitive versus tryptamine. Tryptophol was not an alternative substrate but was a dead end competitive inhibitor versus tryptamine and an uncompetitive inhibitor versus acetyl-CoA, indicative of an ordered binding mechanism requiring binding of acetyl-CoA first. N-Acetyltryptamine, a reaction product, was a noncompetitive inhibitor versus tryptamine and uncompetitive with respect to acetyl-CoA. Taken together these results support an ordered BiBi ternary complex (sequential) kinetic mechanism for AANAT and provide a framework for inhibitor design.
De Angelis
J
J
Laboratory of Bioorganic Chemistry, The Rockefeller University, New York, New York 10021, USA.
Gastel
J
J
Klein
D C
DC
Cole
P A
PA
D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000378:metabolism*; D001667:Binding, Competitive; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012756:Sheep; D014363:Tryptamines / Q000037:antagonists & inhibitors / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D014363:Tryptamines; C043464:N-acetyltryptamine; C030820:tryptamine; D001191:Arylamine N-Acetyltransferase; D005982:Glutathione Transferase