The A167Y mutation converts the herpes simplex virus type 1 thymidine kinase into a guanosine analogue kinase.
The thymidine (dThd) kinase (TK) encoded by herpes simplex virus type 1 (HSV-1) is not only endowed with dThd kinase, but also with thymidylate (dTMP) kinase and 2'-deoxycytidine (dCyd) kinase (dCK) activity. HSV-1 TK also recognizes a variety of antiherpetic guanine nucleoside analogues such as acyclovir (ACV), ganciclovir (GCV), lobucavir (LBV), penciclovir (PCV), and others (i.e., A5021). Site-directed mutagenesis of the highly conserved Ala-167 to Tyr in HSV-1 TK completely abolished TK, dTMP-K, and dCK activity, but maintained ACV-, GCV-, LBV-, PCV-, and A5021-phosphorylating capacity. A variety of 5-substituted pyrimidine nucleoside substrates, but also a number of selective HSV-1 TK inhibitors structurally related to thymine lost significant binding affinity for the mutant enzyme and did not markedly compete with GCV phosphorylation by the mutant enzyme. These findings could be explained by computer-assisted modeling data that revealed steric hindrance of the pyrimidine ring in the HSV-1 TK active site by the large 4-hydroxybenzyl ring of 167-Tyr, while the positioning of the purine ring of guanine-based HIV-1 TK substrates in the active site was kept virtually unaltered. Surprisingly, the efficiency of conversion the antiherpetic 2'-deoxyguanosine analogues ACV, GCV, LBV, PCV, and A5021 to their phosphorylated forms by the A167Y mutant HSV-1 TK was far more pronounced than for the wild-type enzyme. Therefore, the single A167Y mutation converts the wild-type HSV-1 TK from a predominantly pyrimidine nucleos(t)ide kinase into a virtually exclusive purine (guanine) nucleoside analogue kinase.
Balzarini
Jan
J
Rega Institute for Medical Research, K. U. Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. jan.balzarini@rega.kuleuven.ac.be
Liekens
Sandra
S
Esnouf
Robert
R
De Clercq
Erik
E
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics*; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology; D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018259:Herpesvirus 1, Human / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011684:Purine Nucleosides / Q000378:metabolism / Q000494:pharmacology; D011685:Purine Nucleotides / Q000378:metabolism / Q000494:pharmacology; D011741:Pyrimidine Nucleosides / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics
D000970:Antineoplastic Agents; D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D011684:Purine Nucleosides; D011685:Purine Nucleotides; D011741:Pyrimidine Nucleosides; D014443:Tyrosine; D017853:Phosphotransferases (Alcohol Group Acceptor); C055032:guanosine kinase; D013937:Thymidine Kinase; D000409:Alanine