Kinetic studies with N2-phenylguanines and with L-thymidine indicate that herpes simplex virus type-1 thymidine kinase and thymidylate kinase share a common active site.
It is known that the Herpes simplex virus type 1 (HSV-1)-encoded thymidine kinase (TK) co-purifies with an associated thymidylate kinase (TMPK) activity and that thymidylate (TMP) inhibits the phosphorylation of thymidine by the HSV-1 TK. Here we demonstrate that: (i) TMP phosphorylation catalysed by the viral TMPK is competitively inhibited by thymidine (TdR) with a Ki equal to its Km as substrate for the viral TK; (ii) L-thymidine (L-TdR), the enantiomer of the naturally occurring D-TdR and a substrate for the HSV-1 TK [Spadari, Maga, Focher, Ciarrocchi, Manservigi, Arcamone, Capobianco, Caruso, Colonna, Iotti and Garbesi (1992) J. Med. Chem. 35, 4214-4220], is a powerful inhibitor of the HSV-1 TMPK activity with a Ki value identical with its Km as a substrate for the viral TK; (iii) both viral TK and TMPK activities are inhibited, in a competitive way and with identical Ki values, by novel, non-substrate inhibitors of HSV-1 TK, N2-phenylguanines; (iv) L-TdR is phosphorylated to L-TMP by the viral TK, but L-TMP is not phosphorylated to L-TDP by the viral TMPK activity; and (v) L-TMP inhibits competitively and with identical potencies the phosphorylation of TdR and TMP catalysed respectively by the HSV-1 TK and TMPK activities. In conclusion, our data demonstrate that both TK and TMPK activities encoded by HSV-1 share a common active site which is very tolerant in accepting modified nucleosides, but cannot readily accommodate modified nucleoside monophosphates.
Maga
G
G
Istituto di Genetica Biochimica ed Evoluzionistica, CNR, Pavia, Italy.
Focher
F
F
Wright
G E
GE
Capobianco
M
M
Garbesi
A
A
Bendiscioli
A
A
Spadari
S
S
D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D007700:Kinetics; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism*; D010766:Phosphorylation; D013237:Stereoisomerism; D013379:Substrate Specificity; D013936:Thymidine / Q000378:metabolism*; D013937:Thymidine Kinase / Q000378:metabolism*
C088966:N(2)-phenylguanine; D006147:Guanine; D013937:Thymidine Kinase; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase; D013936:Thymidine