| Summary: | 碩士 === 國立清華大學 === 分子醫學研究所 === 103 === Drosophila dopamine N-acetyltransferase (Dat, EC 2.3.1.87) is an arylalkylamine N-acetyltransferase (AANAT), which can catalyze acetyl transfer from acetyl-coenzyme A to arylalkylamine, yielding N-acetylarylalkylamine. According to our X-ray structure, a tunnel was inside the protein with entrances at top and bottom. It has also been shown that the substrate located at the middle of Dat, and CoA located at the bottom which seemed to completely block the entrance. Dat exhibits an ordered sequential mechanism, with acetyl-CoA binding first, followed by substrate. Therefore, substrate should access to its binding site through the other entrance, and it looked like a tunnel existed. M121 and D142 located in the narrowest site of the tunnel (tunnel bottleneck). The replacement of these two residues with tryptophan resulted in a decrease in enzyme activity which may imply the hindrance to the substrate entrance by M121W and D142W. In the present study, the size effect of the substrate tunnel was on enzyme activity. We replaced M121 and D142 to alanine individually, which cause the tunnel bottleneck broader. We also made a double mutation, M121WD142W, to make the tunnel bottleneck even narrower than single mutation. The enzyme kinetic studies demonstrated that D142A had almost the same enzyme activity as that of wild type, and M121WD142W showed a significant decrease in the enzyme activity compared to wild type, M121W and D142W. These results confirmed that the size of tunnel bottleneck may affect the substrate specificity again. Although the tunnel bottleneck is broader, M121A exhibits a decrease in enzyme activity. After analysis by LIGPLOT and PyMol, we found that M121 should participate in substrate binding via S/π interactions. Therefore, changing M121 to alanine not only change the size of substrate tunnel bottleneck, but also was expected to significantly decrease the substrate binding.
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