Epstein-Barr 病毒胸腺核苷激酶保守區域之鑑定及其生化特性之研究

Identification and Characterization of Conserved Regions in the Epstein-Barr Virus Thymidine Kinase

吳仲峻(Chung-Chun Wu)

指導教授 ： 陳振陽

國立臺灣大學/醫學院/微生物學研究所/博士(2004年)

https://doi.org/10.6342/NTU.2004.00482

摘要

Epstein-Barr 病毒(EBV)所具有的胸線核苷激酶(TK)在對抗病毒或是在治療和EBV相關的腫瘤疾病時常被拿來做為一個很有效的目標。儘管EBV TK 結合各種核苷藥物用來治療病毒和腫瘤疾病已經被廣泛應用，但是EBV TK的蛋白結構及其胺基酸所扮演的生化特性之間的關連並不是非常清楚。本論文在缺乏詳細蛋白結構的情況下，利用基因重組的方式，在EBV TK的幾個保守區域進行點突變以獲得各種點突變株，並對各種點突變株進行一系列的活性和生化特性的測試。本研究通過和另外四株人類疱疹病毒的TK蛋白序列的比對分析，預測出EBV TK可能的核苷和核苷酸的結合區域，經由實驗發現，位於site 1的突變株其酵素活性及與ATP結合親合力的大幅改變顯示此保守區域是EBV TK的ATP結合區域。位於site 3和 site 4 的突變株其酵素活性及與胸線核苷(thymidine)結合親和力的顯著改變則顯示此兩個保守區域是負責EBV TK核苷結合的工作。
接著，進一步的仔細探討這些保守區域中的幾個保守胺基酸所扮演的生化角色。在site 1的部分，如果拿EBV TK的蛋白序列進一步和其他會利用ATP的酵素蛋白序列來比對，可以發現它們會具有一段非常相似於site 1的序列。在site 1中，甘胺酸294(G294)、離胺酸297(K297) 和酥胺酸298(T298) 被選擇來進行點突變以進行進一步的生化特性測試。G294突變成纈胺酸(G294V)時，此突變株將會失去其所有的ATP結合能力以及其酵素活性。如果突變成丙胺酸(G294A) 時，則仍殘存20%相對於野生株EBV TK的活性，且其對ATP結合的Km值也從野生株TK的30.0μM 上升成48.7μM 。這些結果顯示G294這位置對於EBV TK和ATP結合非常重要且其弁酮O負責二級結構的維持。若將K297突變成麩胺酸(K297E)、醯胺麩胺酸(K297Q) 和精胺酸(K297R)時，這些突變株均失去了它們的ATP結合能力和酵素活性。然而，K297R突變株對於磷酸供給來源的喜好從ATP(87.6μM)變成GTP(43.0μM)，這暗示K297除了在核苷酸結合外，也有在EBV TK選擇磷酸供給來源上扮演某個角色。當T298突變成絲胺酸(T298S)時，此突變株會殘存80%相對於野生株的酵素活性，但是如果突變成丙胺酸(T298A)，將會失去大部分的酵素活性，這樣的結果顯示氫氧基在這位置對於酵素活性是重要的。很有趣的是，T298A還存留有ATP結合能力，這樣的結果顯示T298對於EBV TK的重要性可能在於催化過程而不是直接參與ATP的結合。
在site 3的部分，本實驗選擇了天門冬酸392(D392)、精胺酸393(R393)和組織胺酸394(H394) 三個胺基酸進行點突變。所有的突變株中只有D392突變成麩胺酸(D392E)及R393突變成組織胺酸(R393H)兩個突變株仍然有酵素活性，顯示負電荷在D392這位置很重要以及正電荷在R393這位置是需要的。而這兩個突變株對於thymidine的Km值分別為22.9μM 及17.9μM ，相對於野生株EBV TK的4.8μM 來講，均有顯著的上升。另外，這兩個突變株對於抗人類後天免疫不全症病毒(anti-HIV)核苷藥物D4T的Km值分別為5.67μM 及4.57M 相對於野生株的6.97μM 有些釭滬飢C，顯示這兩個位置對於EBV TK和D4T的結合扮演某種程度的角色。而D392E這突變株在金屬離子的喜好測試和需求測試中其結果和野生株的EBV TK有顯著不同，顯示D392在TK和金屬離子結合中是重要的。H394所有的突變株都失去酵素活性，顯示這個胺基酸扮演一個非常重要的角色。
在site 4，纈胺酸401突變成半胱胺酸(V401C)時，此突變株對於thymidine結合的能力有變差，顯示V401在核苷的結合上扮演某種角色。苯丙胺酸402突變成酪胺酸(F402Y)時，保有完全的酵素活性，但是絲胺酸突變株(F402S)則只剩下60%的相對活性。但是F402S突變株顯示了和野生株完全不一樣的基質喜好，原本野生株會利用的AZT、IdU和L-I-OddU，F402S的基質競爭測試顯示這幾種核苷藥物對thymidine的競爭性變差，顯示F402在EBV TK決定基質的利用種類上扮演著重要的角色，而他的苯環結構對於這弁鄐]相當重要。
本論文的實驗結果鑑定了EBV TK的核苷、核苷酸和金屬離子的結合區域，並且也定位出各結合區域中保守胺基酸所扮演的生化角色。

關鍵字

保守區域 ； 胸線核苷EB病毒 ； 激酶

並列摘要

The thymidine kinase (TK) encoded by Epstein-Barr virus (EBV) is an attractive target for antiviral therapy and provides a novel approach to the treatment of EBV-associated malignances. Despite the extensive use of nucleoside analogues for the treatment of viral infections and cancer, the structure-function relationship of EBV TK has been addressed rarely. In the absence of any structural information, we sought to identify and elucidate the functional roles of amino acids in the nucleoside-binding site using site-directed mutagenesis. Through alignment with other human herpesviral TK protein sequences, we predicted that certain conserved regions comprise the nucleotide and nucleoside-binding site of EBV TK and, through site-directed mutagenesis, showed dramatic changes in activity and binding affinity for ATP of site 1 (-GAPGVGKT-) and for thymidine of site 3 (-DRH-) and 4 (-VFP-) mutants. These results identified that site 1 is the ATP-binding site and site 3 and 4 are nucleoside- binding sites of EBV TK.
For site 1, through computer–assisted alignment with other human herpesviral TK proteins, it was shown to share a similar conserved ATP binding motif as the other TK enzymes. To investigate functional roles of three highly conserved residues (G294, K297, T298) within this region, site-directed mutagenesis was employed to generate various mutants. The TK enzyme activity and ATP binding ability of these mutant TK enzymes were determined and compared with EBV wild type TK (wtTK). Mutant G294V lost its ATP binding ability and was inactive in enzyme activity assay. As the enzyme activity of G294A was reduced to 20% of that of wtTK, the Km for ATP binding of G294A was 48.7 μM as compared with 30.0μM of EBV wtTK. These results suggested that G294 participates in ATP binding and contributes for structure maintenance. EBV TK mutants K297E, K297Q, K297R lost their ATP binding ability and enzyme activity. However, K297R was shown to have a preference usage of GTP (Km: 43.0μM) instead of ATP (Km: 87.6μM) as the phosphate donor. It implies that, in addition to nucleotide binding, K297 was involved in the selection of phosphate donor. While EBV TK mutant T298S retained approximately 80% of wtTK enzyme activity, T298A lost its enzyme activity, suggesting that a hydroxyl group at this position is important for the enzyme activity. Interestingly, T298A retained its ATP binding ability, suggesting a role of T298 in the catalytic process but not in the ATP coordination. The results demonstrated that amino acid residues G294, K297 and T298 in the ATP binding motif of EBV TK enzyme are essential for the enzymatic activity but are involved in different aspects of ways.
For site 3, only mutants D392E and R393H retain activity, indicating that a negative charge is important for D392 and a positive charge is required for R393. The Kms for thymidine binding of D392E and R393H were 22.9μM and 17.9μM as compared with 4.8μM of EBV wtTK. The Kms for D4T binding were 5.67μM and 4.57μM as compared with 6.97μM of wt TK. The increased binding affinities of these two mutants for D4T suggest that the two residues also are important for substrate selection. Interestingly, the changed metal ion usage pattern and metal ion requirements of D392E reveal that D392 plays the important role in metal ion binding. H394 cannot be compensated by other amino acids, also indicating a crucial role. These results reveal that site 3 has multiple functions, including metal ion coordination, nucleoside binding and substrate selection.
In site 4, the decreased enzyme activity and thymidine binding affinity of V401 mutants imply that V401 plays a certain role in the nucleoside binding. The F402Y mutant retains full activity, however, F402S retains only 60% relative TK activity. Strikingly, when F402 is substituted by serine, the original preferred pyrimidine substrates, such as AZT, IdU, and L-I-O-ddU (L-form substrate), have decreased competitiveness with thymidine, suggesting that F402 plays a crucial role in substrate specificity and that the aromatic ring is important for function.
Taken together, the nucleotide, nucleoside and metal ion binding sites of EBV TK were identified and the conserved residues in these regions were characterized in this study.

並列關鍵字

conserved region ； Epstein-Barr virus ； thymidine kinase

參考文獻

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