RESUMO
Arylalkylamine N-acetyltransferase (AANAT) catalyzes the penultimate step in the biosynthesis of melatonin and other N-acetylarylalkylamides from the corresponding arylalkylamine and acetyl-CoA. The N-acetylation of arylalkylamines is a critical step in Drosophila melanogaster for the inactivation of the bioactive amines and the sclerotization of the cuticle. Two AANAT variants (AANATA and AANATB) have been identified in D. melanogaster, in which AANATA differs from AANATB by the truncation of 35 amino acids from the N-terminus. We have expressed and purified both D. melanogaster AANAT variants (AANATA and AANATB) in Escherichia coli and used the purified enzymes to demonstrate that this N-terminal truncation does not affect the activity of the enzyme. Subsequent characterization of the kinetic and chemical mechanism of AANATA identified an ordered sequential mechanism, with acetyl-CoA binding first, followed by tyramine. We used a combination of pH-activity profiling and site-directed mutagenesis to study prospective residues believed to function in AANATA catalysis. These data led to an assignment of Glu-47 as the general base in catalysis with an apparent pKa of 7.0. Using the data generated for the kinetic mechanism, structure-function relationships, pH-rate profiles, and site-directed mutagenesis, we propose a chemical mechanism for AANATA.
Assuntos
Arilalquilamina N-Acetiltransferase/metabolismo , Biocatálise , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Ácido Glutâmico/química , Modelos Moleculares , Acetilcoenzima A/metabolismo , Acetilação/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Arilalquilamina N-Acetiltransferase/antagonistas & inibidores , Arilalquilamina N-Acetiltransferase/química , Arilalquilamina N-Acetiltransferase/genética , Biocatálise/efeitos dos fármacos , Domínio Catalítico , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ligantes , Mutagênese Sítio-Dirigida , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Conformação Proteica , Especificidade por Substrato , Tiramina/análogos & derivados , Tiramina/metabolismoRESUMO
We employed agarose gel preparative electrophoresis to separate gold nanoparticles based on size, shape, and charge. The separating technique was first demonstrated by size separation of 5 nm, 15 nm, and 20 nm spherical gold nanoclusters; and further evidenced through the purification of crude 15 +/- 2.7 nm nanoclusters to nanoclusters that were 15 +/- 0.4 nm. The ability to separate gold nanoparticles by shape was also shown by the purification of a mixture of gold spheres, plates, and long rods.
