Abstract
Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase (AANAT)) is a critical enzyme in the light-mediated regulation of melatonin production and circadian rhythm. It is a member of the GNAT (GCN-5-related N-acetyltransferase) superfamily of enzymes, which catalyze a diverse array of biologically important acetyl transfer reactions from antibiotic resistance to chromatin remodeling. In this study, we probed the functional properties of two histidines (His-120 and His-122) and a tyrosine (Tyr-168) postulated to be important in the mechanism of AANAT based on prior x-ray structural and biochemical studies. Using a combination of steady-state kinetic measurements of microviscosity effects and pH dependence on the H122Q, H120Q, and H120Q/H122Q AANAT mutants, we show that His-122 (with an apparent pK(a) of 7.3) contributes approximately 6-fold to the acetyltransferase chemical step as either a remote catalytic base or hydrogen bond donor. Furthermore, His-120 and His-122 appear to contribute redundantly to this function. By analysis of the Y168F AANAT mutant, it was demonstrated that Tyr-168 contributes approximately 150-fold to the acetyltransferase chemical step and is responsible for the basic limb of the pH-rate profile with an apparent (subnormal) pK(a) of 8.5. Paradoxically, Y168F AANAT showed 10-fold enhanced apparent affinity for acetyl-CoA despite the loss of a hydrogen bond between the Tyr phenol and the CoA sulfur atom. The X-ray crystal structure of Y168F AANAT bound to a bisubstrate analog inhibitor showed no significant structural perturbation of the enzyme compared with the wild-type complex, but revealed the loss of dual inhibitor conformations present in the wild-type complex. Taken together with kinetic measurements, these crystallographic studies allow us to propose the relevant structural conformations related to the distinct alkyltransferase and acetyltransferase reactions catalyzed by AANAT. These findings have significant implications for understanding GNAT catalysis and the design of potent and selective inhibitors.
Highlights
Melatonin (N-acetyl-5-methoxytryptamine) is a hormone produced in the brain by the pineal gland and controls behavioral and physiological circadian rhythms
Using a combination of steady-state kinetic measurements of microviscosity effects and pH dependence on the H122Q, H120Q, and H120Q/H122Q AANAT mutants, we show that His-122 contributes ϳ6-fold to the acetyltransferase chemical step as either a remote catalytic base or hydrogen bond donor
The Y168F AANAT mutant displayed catalytic activity that was linear with time and followed Michaelis-Menten kinetics, suggesting that it was sufficiently stable for kinetic studies
Summary
Melatonin (N-acetyl-5-methoxytryptamine) is a hormone produced in the brain by the pineal gland and controls behavioral and physiological circadian rhythms The production of this hormone is dependent on the enzyme serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase (AANAT)1), which fluctuates in response to light and dark signals and is the rate-limiting enzyme in the biosynthesis pathway (see Fig. 1A) [1]. AANAT is a member of the GNAT (GCN-5-related N-acetyltransferase) superfamily of enzymes, the other members of which include GCN-5, PCAF (p300/CBP-associated factor), and the aminoglycoside N-acetyltransferases This superfamily has members found in all kingdoms of life and is characterized by a common substrate, acetyl-CoA, and a structural fold where acetyl-CoA binds [3]. The bisubstrate analog inhibitor has been used in complex with AANAT to obtain high resolution crystal structures of the enzyme [11, 12] These crystal structures show possible residues that may play a role in catalysis (Fig. 2).
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