Kinetic studies of the predicted substrate-binding site of varicella-zoster virus thymidine kinase.

To investigate the mechanism of kinetic action and substrate recognition of varicella-zoster virus (VZV) thymidine kinase (TK), we designed and isolated a site-directed mutant VZV TK which has double amino acid substitutions, 136threonine to leucine and 137isoleucine to leucine (SDM TK). This mutant was designed to alter the substrate-binding site of the VZV TK to duplicate that of the herpes simplex virus type 2 enzyme. Kinetic studies of the activity of wild-type TK indicated that the binding order of ATP and thymidine is random and that wild-type VZV TK possessed high thymidylate kinase (TM-K) activity. The sensitivity of VZV TK to bisubstrate analogues, dinucleotides of adenosine and thymidine, showed that the optimum distance between the ATP- and substrate-binding sites is two phosphoryl groups greater than with the natural substrate for TK activity. SDM TK lost deoxycytidine kinase activity and had reduced TK and TM-K activities. Inhibition studies on both WT and SDM TK by 5-halogenovinyluridine analogues and their 5' monophosphate derivatives revealed that amino acids at positions 136 and 137 are involved in substrate binding, probably through a role in the formation of the binding pocket for bulky substrates.