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

Abstract

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.