果蠅多巴胺乙醯轉移酶催化循環與結構關聯性之研究

The Relationship between the Catalytic Cycle and Structure of Dopamine N-Acetyltransferase from Drosophila melanogaster

楊伊琛(Yang, Yi Chen)

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

果蠅多巴胺乙醯轉移酶（Dopamine N-acetyltransferase, Dat）是一種苯烷基胺乙醯轉移酶（AANAT, EC 2.3.1.87），屬於GCN5相關的胺基端乙醯化轉移酶（GCN5-related N-acetyltransferase, GNAT）超家族中的一類酵素。果蠅多巴胺乙醯轉移酶能催化乙醯化（N-acetylation）反應的進行，將乙醯輔酶A（Acetyl-CoA, Ac-CoA）上的乙醯基（Acetyl group）轉移至苯烷基胺（Arylalkylamine）這類受質的胺基，最終產生乙醯苯烷基胺（N-acetyl-arylalkylamine）和輔酶A。在過去的文獻裡，經由酵素動力學抑制實驗證實苯烷基胺乙醯轉移酶具有定序二二連續型機制（Ordered bi bi sequential mechanism）的酵素特性。在先前的研究中，我們也已經利用等溫滴定量熱儀（Isothermal titration calorimetry, ITC）與酵素動力學抑制實驗（Enzyme inhibition kinetics）證實果蠅多巴胺乙醯轉移酶具有定序二二連續型機制的酵素特性：果蠅多巴胺乙醯轉移酶必須先和乙醯輔酶A結合，才能與受質結合，最終發生乙醯化反應。然而，此機制其中的蛋白質結構資訊尚有不明。此外，在之前實驗室利用浸泡式結晶（Soaking）的方式取得果蠅多巴胺乙醯轉移酶三元複合體（Ternary form）的結構中，發現在輔因子與受質結合位中存在乙醯苯烷基胺和輔酶A的電子雲；在ITC結果，我們發現當果蠅多巴胺乙醯轉移酶結合輔酶A時，會與受質的產物發生結合。由此看來，這兩種產物似乎無法自動釋出酵素外。因此，我們好奇果蠅多巴胺乙醯轉移酶是如何進行酵素回收和催化反應。
在本研究中，我們利用共結晶（Co-crystallization）的方式解出解析度為1.20 Å的果蠅多巴胺乙醯轉移酶三元複合體（tDat/CoA/Ac-PEA complex）結構。比較之前實驗室解出的果蠅多巴胺乙醯轉移酶（Apo form）、二元複合體（tDat/Ac-CoA complex）與本研究解出的三元複合體（tDat/CoA/Ac-PEA complex）結構，我們發現果蠅多巴胺乙醯轉移酶的構型不同於二元複合體與三元複合體；二元複合體與三元複合體的構型是非常相像的。然後，我們進一步發現當乙醯輔酶A結合果蠅多巴胺乙醯轉移酶之後，會引發此酵素構型上的轉變。其中，果蠅多巴胺乙醯轉移酶受質結合位構型的轉變可能是決定受質是否可以結合到此酵素的關鍵因素。因此，根據本研究的結構分析結果提供關於定序二二連續型酵素機制在蛋白質結構上的資訊。此外，比較共結晶與浸泡式結晶所解出的果蠅多巴胺乙醯轉移酶三元複合體結構，可以發現大體結構並無太大的差異，但是在共結晶的三元複合體結構中，蛋白質表面會額外多出一個乙醯苯乙胺（N-acetyl-2-phenylethylamine, Ac-PEA）。這個現象暗示也許有一些因子可以協助果蠅多巴胺乙醯轉移酶產物釋出與酵素循環。透過等溫滴定量熱儀與共結晶所設計的競爭實驗，我們可以發現對於果蠅多巴胺乙醯轉移酶而言，乙醯輔酶A對於輔酶A有較強勢的競爭關係。而這樣一個不對等的競爭關係，可能是導致果蠅多巴胺乙醯轉移酶能夠回收再利用的主要因素。最後，為了瞭解果蠅多巴胺乙醯轉移酶的過渡態（Transition state），我們根據之前實驗室所提出催化三聯體（Catalytic triad）的催化機制中，設計了三個與穀胺酸47（E47）相關的點突變體（E47D、E47Q與E47N）。經由等溫滴定量熱儀與酵素活性實驗，我們可以發現對穀胺酸47進行點突變的三個點突變體相對於野生型（Wild-type）果蠅多巴胺乙醯轉移酶，在與受質的結合親和力與催化活性上都有明顯下降的趨勢。
根據本研究的實驗結果，我們提出了關於果蠅多巴胺乙醯轉移酶的催化循環（Catalytic cycle）假說：果蠅多巴胺乙醯轉移酶需要先與乙醯輔酶A結合引發構型轉變後，才可以與受質發生結合進行乙醯化反應；反應結束後的產物並不會從酵素中自動釋出，而是需要乙醯輔酶A的幫助，將輔酶A與乙醯苯烷基胺從酵素中競爭下來，使酵素回到二元複合體（tDat/Ac-CoA complex）的型態，以利酵素進行下一輪的催化反應。

關鍵字

多巴胺乙醯轉移酶 ； 苯烷基胺乙醯轉移酶 ； 定序二二連續型機制 ； 等溫滴定量熱儀 ； X射線晶體學 ； 共結晶 ； 催化循環

並列摘要

Dopamine N-acetyltransferase (Dat) found in Drosophila melanogaster belongs to arylalkylamine N-acetyltransferase (AANAT, EC 2.3.1.87) family, which is a member of GCN5-related N-acetyltransferase (GNAT) superfamily. Dat catalyzes arylalkylamine N-acetylation which transfers acetyl group of acetyl-CoA (Ac-CoA) to arylalkylamine to generate N-acetyl-arylalkylamine and CoA. AANAT had been reported the ordered bi bi sequential mechanism by enzyme inhibition analysis as well. In our previous study, we had also determined Dat is ordered bi bi sequential mechanism using isothermal titration calorimetry (ITC) and enzyme inhibition kinetics. Dat has to bind cofactor (Ac-CoA) first and then followed by substrate (Arylalkylamine). Nevertheless, the underlying structural mechanism still remains ambiguous. Furthermore, we had found the electron density map of products, N-acetyl-arylalkylamine and CoA, on substrate and cofactor binding site in ternary structure by soaking. It seemed products cannot auto-release. Thus, we interested in how Dat conducts enzymatic recycling and the catalytic process.
In this study, we solved 1.20 Å resolution ternary structure (Dat/N-acetyl-arylalkylamine/CoA) of Dat by co-crystallization. Comparing apo form, binary form (tDat/Ac-CoA complex) and ternary form (tDat/CoA/Ac-PEA complex) of tDat, we found conformation of apo form Dat was different from binary and ternary form among them; structures of binary and ternary form were similar with each other. Then, we found the conformational change after Ac-CoA binding with tDat. The conformational change of substrate binding site may decide whether tDat can binding substrate or not. Thus, we elucidated ordered bi bi sequential mechanism of Dat by x-ray structural analysis. Additionally, the overall ternary structure of co-crystallization was similar with soaking except for an additional Ac-PEA outside the protein surface. The phenomenon implied may exist some factors to facilitate product release and enzyme recycle of Dat. Using isothermal titration calorimetry (ITC) and x-ray co-crystallization to carry out the competitive experiments, we found Ac-CoA showed dominantly competitive relation with respect to CoA. The dominantly competitive relation between Ac-CoA and CoA may resulted in enzymatic recycle of Dat. Finally, we based on catalytic triad to generate three variants, E47D, E47Q and E47N, for approaching transition state. All of them lost their substrate binding affinity and led to dramatic catalytic activity decrease.
According to our results, we suggested Ac-CoA priorly binds to Dat providing a conformation change which facilitates substrate binding and forms ternary form. Ac-CoA may serve as driving force in catalyzation process of Dat. Our results implied Ac-CoA drives CoA and N-acetyl-arylalkylamine in Dat (ternary form) away, and occupies the cofactor binding site of Dat. Then Dat returns to state of binary form and accomplishes enzymatic recycling.

並列關鍵字

Dopamine N-acetyltransferase ； arylalkylamine N-acetyltransferase ； Ordered bi bi sequential mechanism ； Isothermal titration calorimetry ； X-ray crystallography ； Co-crystallization ； Catalytic cycle

參考文獻

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