Varicella-zoster virus thymidine kinase. Characterization and substrate specificity.
The varicella-zoster virus (VZV) thymidine kinase (TK) EC 2.7.2.21) catalyzes the phosphorylation of many anti-VZV nucleosides. Purified, bacterially expressed VZV TK was characterized with regard to N-terminal amino acid sequence, pI value, pH optimum, metal ion requirement, phosphate donor and acceptor specificity, and inhibition by dTTP. Initial velocities of thymidine phosphorylation with variable MgATP concentrations fit a two-site model with apparent Km values for MgATP of 0.10 and 900 microM. dTTP was a noncompetitive inhibitor of thymidine phosphorylation but was competitive with MgATP. Phosphate donor and acceptor specificities of the bacterially expressed enzyme were indistinguishable from those of VZV TK purified from infected cells. Detailed studies of the nucleoside specificity with the bacterially expressed enzyme showed that, for a given sugar moiety, thymine nucleosides were the most efficient substrates followed by nucleosides of cytosine, uracil, adenine, and with some exceptions, guanine. For a given pyrimidine or purine (except guanine), 2'-deoxyribonucleosides were the most efficient substrates, followed by arabinosides, ribonucleosides, 2',3'-dideoxyribonucleosides, and the acyclic moiety of acyclovir.
Roberts
G B
GB
Wellcome Research Laboratories, Research Triangle Park, NC 27709.
Fyfe
J A
JA
McKee
S A
SA
Rahim
S G
SG
Daluge
S M
SM
Almond
M R
MR
Rideout
J L
JL
Koszalka
G W
GW
Krenitsky
T A
TA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D014645:Herpesvirus 3, Human / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008670:Metals; D009943:Organophosphorus Compounds / Q000378:metabolism; D010766:Phosphorylation; D011741:Pyrimidine Nucleosides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013937:Thymidine Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D002241:Carbohydrates; D008670:Metals; D009943:Organophosphorus Compounds; D011741:Pyrimidine Nucleosides; D000255:Adenosine Triphosphate; D013937:Thymidine Kinase