研究果蠅多巴胺乙醯基轉移酶的次序連續性機制及結合位點上的關鍵殘基

Study of the ordered sequential mechanism and critical residues in binding site of Drosophila melanogaster dopamine N-acetyltransferase

丁韋丞(Ding, Wei Cheng)

指導教授 ： 呂平江

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2016年)

摘要

多巴胺是一種神經傳導物質，與許多生理功能相關，例如：認知、生理時鐘、老化、記憶和學習。從果蠅發現的多巴胺乙醯基轉移酶 (Dopamine N-Acetyltransferase, Dat) 是一種芳烴基烷基胺乙醯基轉移酶 (arylalkylamine N-acetyltransferase, AANAT)，參與單胺 (monoamines) 的代謝和昆蟲外骨骼硬化。多巴胺乙醯基轉移酶催化乙醯化作用 (N-acetylation)，是將乙醯輔酶A (acetyl-coenzyme A) 上的乙醯基轉移到芳烴基烷基胺，並且製造出乙醯芳烴基烷基胺 (N-acetylarylalkylamine)。本實驗室先前研究顯示，多巴胺乙醯基轉移酶遵守次序連續性機制 (the ordered sequential mechanism)，先與乙醯輔酶結合，再結合基質，之後進行乙醯基轉移。為了了解這個催化過程的細節，等溫滴定微量熱儀 (isothermal titration calorimetry，ITC) 被用來研究反應中的熱力學變化和結合親和力。為了確認次序連續性機制，利用核磁共振儀 (nuclear magnetic resonance，NMR) 監測氮15異核單量子相干性 (HSQC) 光譜的化學位移，以了解輔助因子 (cofactor) 和基質依次加入氮15標定蛋白質後產生之變化。等溫滴定微量熱儀的實驗結果與核磁共振儀的實驗結果一致，都證明了次序連續性機制的存在。
最近，本實驗解出了多巴胺乙醯基轉移酶的三元結構 (ternary structure)，此三元結構的晶體是由多巴胺乙醯基轉移酶、乙醯輔酶A和基質共同結晶而得。然而，解出的三元結構卻顯示其組成為多巴胺乙醯基轉移酶、乙醯輔酶A和基質。這個發現暗示了最終產物生成後並未從多巴胺乙醯基轉移酶離開。為了進一步地研究產物如何離開多巴胺乙醯基轉移酶並讓下一個反應發生。利用等溫滴定微量熱儀監測滴定乙醯輔酶A到含有產物的多巴胺乙醯基轉移酶之過程，並以艾爾曼的試劑 (Ellman's reagent, DTNB) 測定是否有新的產物生成。結果顯示乙醯輔酶A可取代在多巴胺乙醯基轉移酶內的輔酶A (coenzyme A)，並讓乙醯基化基質離開。於是，一個新的多巴胺乙醯基轉移酶-乙醯輔酶A複合物 (Dat-AcCoA complex) 產生，可結合新的基質，並進行乙醯基轉移反應。
為了瞭解多巴胺乙醯基轉移酶上與輔助因子或基質結合有關的胺基酸殘基，軟體Ligplot+ 被用來分析多巴胺乙醯基轉移酶的二元結構 (蛋白質資料庫編碼：3TE4) 和三元體 (未發表)。在Ligplot+ 分析報告中，精胺酸153和離胺酸192與輔助因子的結合有關，而甲硫胺酸121可能會參與基質的結合。在CAVER軟體的分析顯示，甲硫胺酸121和天門冬胺酸142位於反應通道的瓶頸處。這四個胺基酸被取代成丙氨酸，並用等溫滴定微量熱儀和酵素活性測定來確認它們的角色。出乎意料地，在等溫滴定微量熱儀試驗中，丙氨酸121突變株 (M121A) 完全失去乙醯輔酶A結合能力，並且在活性檢驗中，僅殘存13%的活性。考慮丙氨酸121突變株 (M121A)二級結構的顯著改變及丙氨酸121與輔助因子的遙遠距離，所以丙氨酸121突變株 (M121A) 喪失輔助因子結合能力的原因應該是結構改變而不是與輔助因子交互作用改變。對於乙醯輔酶A結合，精胺酸153為重要殘基，因為在丙氨酸153突變株 (R153A) 的等溫滴定微量熱儀試驗中，沒有偵測到輔助因子結合。在滴定微量熱儀試驗中，丙氨酸192突變株 (K192A) 對乙醯輔酶A和輔酶A的結合親和力減少，但仍保有催化的能力。丙氨酸142突變株 (D142A) 對輔助因子或基質的結合以及催化能力則沒有明顯受影響。
總結，多巴胺乙醯基轉移酶遵守次序連續性機制，先與乙醯輔酶結合，再結合基質，之後進行乙醯基轉移。為了進行下一輪反應，乙醯輔酶A可取代在多巴胺乙醯基轉移酶內的輔酶A，並讓乙醯基化基質離開。於是，一個新的多巴胺乙醯基轉移酶-乙醯輔酶A複合物產生，可結合新的基質，並進行乙醯基轉移。此外，甲硫胺酸121會影響蛋白質結構，而精胺酸153也被確認是參與多巴胺乙醯基轉移酶結合輔助因子的重要胺基酸殘基。

關鍵字

多巴胺乙醯基轉移酶 ； 芳烴基烷基胺乙醯基轉移酶 ； 乙醯化作用 ； 乙醯輔酶A ； 次序連續性機制 ； 酵素再循環 ； 等溫滴定微量熱儀 ； 核磁共振儀 ； 熱力學 ； 結合親和力 ； 化學位移 ； 艾爾曼的試劑 ； 構型變化 ； 結合位 ； 定位突變 ； 酵素動力學 ； 輔助因子結合殘基 ； 蛋白質結構

並列摘要

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.

並列關鍵字

Dopamine N-acetyltransferase ； Arylalkylamine N-acetyltransferase ； Acetylation ； Acetyl-coenzyme A ； Ordered sequential mechanism ； Enzyme recycling ； Isothermal titration calorimetry ； Nuclear magnetic resonance ； Thermodynamics ； Binding affinity ； Chemical shift ； Ellman's reagent ； Conformational change ； Binding site ； Site-directed mutagenesis ； Enzyme kinetics ； Cofactor binding residue ； Protein structure

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