Kinetic analysis of the catalytic mechanism of serotonin N- acetyltransferase (EC 2.3.1.87)

Jacqueline De Angelis, Jonathan Gastel, David C. Klein, Philip A. Cole

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)3045-3050
Number of pages6
JournalJournal of Biological Chemistry
Volume273
Issue number5
DOIs
StatePublished - Jan 30 1998
Externally publishedYes

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Arylalkylamine N-Acetyltransferase
Acetyl Coenzyme A
Kinetics
Melatonin
Glutathione Transferase
Biosynthesis
Substrates
Enzymes
Reaction products
Vertebrates
Fusion reactions
tryptamine
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic analysis of the catalytic mechanism of serotonin N- acetyltransferase (EC 2.3.1.87). / De Angelis, Jacqueline; Gastel, Jonathan; Klein, David C.; Cole, Philip A.

In: Journal of Biological Chemistry, Vol. 273, No. 5, 30.01.1998, p. 3045-3050.

Research output: Contribution to journalArticle

De Angelis, Jacqueline ; Gastel, Jonathan ; Klein, David C. ; Cole, Philip A. / Kinetic analysis of the catalytic mechanism of serotonin N- acetyltransferase (EC 2.3.1.87). In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 5. pp. 3045-3050.
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