研究果蠅多巴胺乙醯基轉移酶受質的入口通道

果蠅的多巴胺乙醯基轉移酶(dopamine N-acetyltransferase, Dat)，是屬於苯烷基胺乙醯轉移酶家族的蛋白酶(EC 2.3.1.87, arylalkylamine N-acetyltransferase, AANAT)，可促使褪黑激素(melatonin)前驅物質的產生，而褪黑激素(melatonin)在生理上的研究，已經被應用在人類的睡眠週期、情緒以及免疫反應當中。在前期的研究，我們實驗室已成功將多巴胺乙醯基轉移酶(apo form)、多巴胺乙醯基轉移酶/乙醯輔酶A複合晶體(complex form)兩者的蛋白質結構解出，隨後也將多巴胺乙醯基轉移酶和基質間結合的動力學機制進行一連串的分析，更進而成功解出了高達1.20 Å解析度的多巴胺乙醯基轉移酶的三元複合體(多巴胺乙醯基轉移酶/乙醯輔酶A/基質)，我們根據多巴胺乙醯基轉移酶的複合晶體結構得知，乙醯輔酶A是結合於多巴胺乙醯基轉移酶底部的孔洞通道之中，經由等溫滴定微量熱法(ITC)實驗發現到，多巴胺乙醯基轉移酶要先結合輔助因子乙醯輔酶A，才會與基質作用，這個結果顯示輔助因子能夠優先和酵素結合，因此基質只能由酵素頂端的孔洞進入基質接合空腔中。經由序列比對以及通道運算軟體，發現了兩個位於通道瓶頸中的氨基酸M121以及D142，我們將其分別突變為色胺酸。再經由酵素動力學實驗證實，M121W以及D142W確實會阻礙基質進入疏水性的基質接合空腔，證實了乙醯輔酶A與基質是通過不同的孔洞與多巴胺乙醯基轉移酶結合。經由四種不同的基質的實驗結果發現，多巴胺比較不受到通道阻礙的影響。我們的研究證實了多巴胺乙醯基轉移酶基質通道的存在，也有助於了解果蠅AANAT的通道大小對基質選擇性的影響。

關鍵字

乙烯基輔酶A ；乙烯基轉移酶；果蠅蛋白；酵素動力學；多巴胺 N-乙烯基轉移酶；受質特異性；受質通道；通道瓶頸

並列摘要

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.

並列關鍵字

Acetyl Coenzyme A ； Acetyltransferases ； Drosophila Proteins ； Enzyme Kinetics ； Dopamine N-acetyltransferase ； Substrate Specificity ； Substrate tunnel ； Tunnel bottleneck

參考文獻

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3. Vetting, M. W., S. d. C. LP, et al. (2005). "Structure and functions of the GNAT superfamily of acetyltransferases." Arch Biochem Biophys 433(1): 212-226.

4. Wolf, E., J. De Angelis, et al. (2002). "X-ray crystallographic studies of serotonin N-acetyltransferase catalysis and inhibition." J Mol Biol 317(2): 215-224.

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研究果蠅多巴胺乙醯基轉移酶受質的入口通道

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