Abstract
Dopamine is a neurotransmitter and associated with many physiological mechanisms, for example, cognition, circadian rhythm, aging, memory and learning. Dopamine N-acetyltransferase (Dat), an arylalkylamine N-acetyltransferase (AANAT), is identified in Drosophila melanogaster involves in the catabolism of monoamines and sclerotization. Dat transfers acetyl group from acetyl coenzyme A (AcCoA) to arylalkylamine and produces N-acetylarylalkylamine. Previous study in our laboratory revealed that Dat obeyed an ordered sequential mechanism: AcCoA binding first, substrates binding afterward, and then acetyl group transferring. In order to understand the detail of this catalysis process, isothermal titration calorimetry (ITC) was used to study the thermodynamics changes and binding affinities in reactions. To confirm the ordered sequential mechanism, sequential addition of cofactors and substrates into 15N labeled Dat protein was performed and monitored by nuclear magnetic resonance (NMR), and then the chemical shift changes of 15N heteronuclear single quantum coherence (HSQC) spectra were analyzed. ITC results were consistent with NMR results and proved the existence of ordered sequential mechanism. Recently, ternary structure which was co-crystalized by Dat, AcCoA, and substrate was resolved by our laboratory. However, this ternary structure was consisted of Dat, CoA, and acetyl-substrate. This indicated that final products did not release from Dat after catalysis. To further investigate how the products leave Dat and let the next reaction occur. Titration of AcCoA into Dat containing products was monitored by ITC, and the formation of new products was confirmed by DTNB assay. The results showed that AcCoA replaced CoA in Dat, and further let acetyl-substrate leave. Then a new Dat-AcCoA complex was ready to bind a new substrate and transfer acetyl group to it. To explore residues affecting cofactor or substrate binding, Ligplot+ was used to analyze the binary form of Dat structure (PDB code: 3TE4) and ternary form (unpublished). In Ligplot+ analysis report, R153 and K192 are related with AcCoA/CoA binding, while M121 may participate in substrate binding. In CAVER software analysis, M121 and D142 located in the narrowest of the substrate binding tunnel of Dat. Four residues were replaced to alanine, and ITC and enzyme activity assay was used to check their roles. Unexpectedly, M121A totally lost the AcCoA binding ability in ITC test, and remained only 13% of activity in functional assay. Considering the notable changes in secondary structure of M121A and far distance between M121 and cofactor, the loss of cofactor binding ability of M121A should be caused from the change in structure rather than the interaction with cofactor. R153 was important residue for AcCoA binding because no cofactor binding was detected in ITC test of R153A. K192A showed less binding affinity to AcCoA/CoA in ITC test, but remained its catalytic ability. D142A showed no significant effects on binding with cofactor or substrate, and on catalysis. In conclusion, Dat obeyed an ordered sequential mechanism: AcCoA binding first, substrates binding afterward, and then acetyl group transferring. To start next reaction, AcCoA replaced CoA in Dat, and further let acetyl-substrate leave. Then a new Dat-AcCoA complex was ready to bind a new substrate and transfer acetyl group to it. Besides, M121 affected on protein structure and R153 was important residue participating in cofactors binding to Dat.