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

1 Division of Virology, National Institute for Medical Research, Mill Hill, London NW7 1AA and 2 Drug Development Section, Cancer Research Campaign Laboratories, The Institute of Cancer Research, Belmont, Sutton, Surrey SM2 5NG, U.K. To investigate the mechanism of kinetic action and substrate recog...

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Bibliographic Details
Published in:Journal of general virology 1993-06, Vol.74 (6), p.1011-1016
Main Authors: Suzutani, Tatsuo, Davies, Lawrence C, Honess, Robert W
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Base Sequence
Binding Sites
Biological and medical sciences
Escherichia coli - genetics
Fundamental and applied biological sciences. Psychology
Herpesvirus 3, Human - enzymology
Herpesvirus 3, Human - genetics
Kinetics
Microbiology
Molecular Sequence Data
Morphology, structure, chemical composition, physicochemical properties
Mutagenesis, Site-Directed
Mutation
Nucleotides - metabolism
Nucleotides - pharmacology
Recombinant Proteins - metabolism
Sequence Homology, Amino Acid
Substrate Specificity
Thymidine Kinase - antagonists & inhibitors
Thymidine Kinase - genetics
Thymidine Kinase - isolation & purification
Thymidine Kinase - metabolism
Uridine - analogs & derivatives
varicella-zoster virus
Virology
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Summary:1 Division of Virology, National Institute for Medical Research, Mill Hill, London NW7 1AA and 2 Drug Development Section, Cancer Research Campaign Laboratories, The Institute of Cancer Research, Belmont, Sutton, Surrey SM2 5NG, U.K. 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, 136 threonine to leucine and 137 isoleucine 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. Present address: Department of Microbiology, Asahikawa Medical College, 4-5 Nishikagura, Asahikawa 078, Japan. Received 10 August 1992; accepted 25 January 1993.
ISSN:0022-1317
1465-2099
DOI:10.1099/0022-1317-74-6-1011