The thymidine kinase encoded by Epstein-Barr virus (EBV TK) is an important target for antiviral therapy and the treatment of EBV-associated malignancies. Through computer-assisted alignment with other human herpesviral TK proteins, EBV TK was shown to contain a conserved ATP-binding motif 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 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 K,,, for ATP binding of G294A was 48.7 mu M as compared with 30.0 mu M of EBV wtTK. These results suggested that G294 participates in ATP binding and contributes to maintenance of structure. EBV TK mutants K297E. K297Q, and K297R lost their ATP-binding ability and enzyme activity. However, K297R was shown to have a preference for usage of GTP (K-m: 43.0 mu M) instead of ATP (K-m: 87.6 mu M) as the phosphate donor. This 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 coordination of ATP. This study 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 its action.
