KINETIC-ANALYSIS OF THE CATALYTIC MECHANISM OF SEROTONIN N-ACETYLTRANSFERASE (EC-2.3.1.87)

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AAN AT, 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 melatoni n 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 clea ved AANAT was essentially identical, Two-substrate kinetic analysis ge nerated an intersecting line pattern characteristic of a ternary compl ex mechanism, The dead end inhibitor analog desulfo-CoA was competitiv e versus acetyl-CoA and noncompetitive versus tryptamine. Tryptophol w as not an alternative substrate but was a dead end competitive inhibit or versus tryptamine and an uncompetitive inhibitor versus acetyl-CoA, indicative of an ordered binding mechanism requiring binding of acety l-CoA first, N-Acetyl-tryptamine, a reaction product, was a noncompeti tive inhibitor versus tryptamine and uncompetitive with respect to ace tyl-CoA, Taken together these results support an ordered BiBi ternary complex (sequential) kinetic mechanism for AANAT and provide a framewo rk for inhibitor design.