| Serotonin N-acetyltransferase (AANAT) is a critical enzyme in the light-mediated regulation of melatonin production. It is a member of the GNAT (GCN5-related N-acetyltransferase) superfamily of enzymes, which catalyze a diverse array of biologically important acetyl transfer reactions from antibiotic resistance to chromatin remodeling. AANAT functions via an ordered BiBi ternary complex mechanism that seems to be conserved among other GNAT family members whose mechanisms have been determined.; The objective of this thesis was to fill in mechanistic pieces of AANAT that have remained elusive. We determined three residues to be catalytically important in the active site of AANAT. Tyrosine-168, with a pKa of 8.5, plays the role of a proton or hydrogen bond donor, and therefore must be protonated for optimal function. Tyr-168 contributes 150-fold to the chemical step of acetyl transfer, and its hydroxyl moiety destabilizes the ground state of the enzyme leading to an enhancement of catalysis. Histidine-122 contributes 6-fold to the chemical step of AANAT-catalyzed acetyl transfer, and acts as a remote catalytic base or a hydrogen bond acceptor. Histidine-120, situated directly adjacent to His-122, was found to act redundantly, and take over the role of His-122 when His-122 is mutated.; In addition, we determined the oligomeric state of AANAT in solution by a new method, Fluorescence Resonance Energy Transfer (FRET). To do this, we explored a new technique, expressed protein ligation, with which to label proteins with fluorophores in a specific, quantitative way for use in FRET. By determining that AANAT exists as an NSI oligomer in solution, we have gained evidence towards the theory that AANAT has two distinct active sites, one for each catalytic activity.; Finally, we explored the effect on AANAT stability in vivo of mutating residues important in the PKA phosphorylation of the enzyme (Thr-29 and His-28), as well as residues of catalytic importance. It appears that mutations of catalytic residues have no effect on phosphorylation or stability of AANAT. However, mutation of Thr-29 to alanine renders AANAT unable to be phosphorylated by PKA. Changing His-28 to a tyrosine also seems to affect the phosphorylation of AANAT, though to a lesser extent. |
