%0 Journal Article %A Suzutani, Tatsuo %A Davies, Lawrence C. %A Honess, Robert W. %T Kinetic studies of the predicted substrate-binding site of varicella-zoster virus thymidine kinase %D 1993 %J Journal of General Virology, %V 74 %N 6 %P 1011-1016 %@ 1465-2099 %R https://doi.org/10.1099/0022-1317-74-6-1011 %I Microbiology Society, %X 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. %U https://www.microbiologyresearch.org/content/journal/jgv/10.1099/0022-1317-74-6-1011