Abstract
Drosophila melanogaster dopamine N-acetyltransferase (Dat, EC 2.3.1.87) belongs to the arylalkylamine N-acetyltransferase (AANAT) family, which catalyzes the synthesis of the hormone precursor (melatonin). We have solved the structures of Dat in apo form, binary complex (Dat / acetyl coenzyme A) and ternary complex form (Dat / acetylarylalkylamine / CoA) and proposed the catalytic mechanism previously. According to the binding study by isothermal titration calorimetry (ITC), the cofactor (Acetyl-CoA) needed to bind to the Dat prior to substrate, which would hinder the substrate entry to its binding site. Therefore, we speculate that an entry tunnel for substrate may exist to facilitate the substrate binding to the active site. In this study, we replaced two residues with tryptophan, M121 and D142, located inside the tunnel to see the effects of tunnel hindrance. Our DTNB-based enzyme activity measurements and enzyme kinetic studies showed that mutant M121W decreased the enzyme activity and the substrate binding comparing to wild type Dat. Among the four substrates (Dopamine, serotonin, phenylethylamine, tryptamine) tested, only the efficiency of dopamine remains. This result confirms that M121W and D142W may hinder the substrate entry, resulting in decreased binding efficiency of the binary complex. Our studies not only confirm the existence of a substrate tunnel, but also show the tunnel size may contribute to the substrate specificity.