Abstract
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.
Highlights
Serotonin N-acetyltransferase is the penultimate enzyme in melatonin biosynthesis
Lyzed by hydroxyindole-O-methyltransferase (EC 2.1.1.4), which is expressed at relatively constant levels; the rate of this step is regulated by N-acetyl-5-hydroxytryptamine availability
The organic phase was dried over Na2SO4, and the resultant was concentrated in vacuo to afford N-acetyltryptamine as off-white crystals (88% yield)
Summary
Serotonin N-acetyltransferase (arylalkylamine Nacetyltransferase, 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. 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. Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT, EC 2.3.1.87) is the penultimate enzyme in the synthesis of the pineal hormone melatonin (5-methoxyN-acetyltryptamine). This enzyme is of special interest because large increases in its activity are responsible for the large increase in circulating melatonin levels in vertebrates [1, 2]. AANAT activity is linked closely to enzyme protein amounts, which reflects regulation at transcriptional and post-transcriptional levels [10]
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