title: Investigation of the roles of catalytic residues in serotonin N-acetyltransferase.
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.
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Kara A
authors.initials: KA
authors.lastname: Scheibner
authors.forename: Jacqueline
authors.initials: J
authors.lastname: De Angelis
authors.forename: Stephen K
authors.initials: SK
authors.lastname: Burley
authors.forename: Philip A
authors.initials: PA
authors.lastname: Cole
mesh_terms: D001191:Arylamine N-Acetyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D001191:Arylamine N-Acetyltransferase