Characterization of Three Essential Residues in the Conserved ATP-Binding Region of Epstein-Barr Virus Thymidine Kinase
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- 作者：Chung-Chun Wu ; Tsuey-Ying Hsu ; and Jen-Yang Chen
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：March 29, 2005
- 年：2005
- 卷：44
- 期：12
- 页码：4785 - 4793
- 全文大小：248K
- 年卷期：v.44,no.12(March 29, 2005)
- ISSN：1520-4995
文摘
The thymidine kinase encoded by Epstein-Barr virus (EBV TK) is an important target forantiviral therapy and the treatment of EBV-associated malignancies. Through computer-assisted alignmentwith other human herpesviral TK proteins, EBV TK was shown to contain a conserved ATP-bindingmotif as for 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 andcompared with EBV wild-type TK (wtTK). Mutant G294V lost its ATP-binding ability and was inactivein enzyme activity assay. As the enzyme activity of G294A was reduced to 20% of that of wtTK, the Kmfor ATP binding of G294A was 48.7 
M as compared with 30.0 M of EBV wtTK. These results suggestedthat G294 participates in ATP binding and contributes to maintenance of structure. EBV TK mutantsK297E, K297Q, and K297R lost their ATP-binding ability and enzyme activity. However, K297R wasshown to have a preference for usage of GTP (Km: 43.0 M) instead of ATP (Km: 87.6 M) as thephosphate donor. This implies that, in addition to nucleotide binding, K297 was involved in the selectionof 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 theenzyme activity. Interestingly, T298A retained its ATP-binding ability, suggesting a role of T298 in thecatalytic process but not in the coordination of ATP. This study demonstrated that amino acid residuesG294, K297, and T298 in the ATP-binding motif of EBV TK enzyme are essential for the enzymaticactivity but are involved in different aspects of its action.
M as compared with 30.0 M of EBV wtTK. These results suggestedthat G294 participates in ATP binding and contributes to maintenance of structure. EBV TK mutantsK297E, K297Q, and K297R lost their ATP-binding ability and enzyme activity. However, K297R wasshown to have a preference for usage of GTP (Km: 43.0 M) instead of ATP (Km: 87.6 M) as thephosphate donor. This implies that, in addition to nucleotide binding, K297 was involved in the selectionof 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 theenzyme activity. Interestingly, T298A retained its ATP-binding ability, suggesting a role of T298 in thecatalytic process but not in the coordination of ATP. This study demonstrated that amino acid residuesG294, K297, and T298 in the ATP-binding motif of EBV TK enzyme are essential for the enzymaticactivity but are involved in different aspects of its action.