A single amino acid substitution abolishes feedback inhibition of vaccinia virus thymidine kinase.
Exploitation of differences in the substrate specificity of the type I and type II thymidine kinases (EC 2.7.1.21, TK) expressed by the Herpesviridae and Poxviridae (and human cells), respectively, has lead to the development of effective antiherpetic drugs such as acyclovir and gancyclovir. Analysis of type I TK protein sequences reveals a consensus sequence which corresponds to domain IV of type II TK proteins such as that encoded by vaccinia virus (VV). The type I descriptor (Xpho - + + Xpho) differs at the second position from the type II consensus sequence (Xpho Xphi + + Xpho) by having an aspartic acid residue (D) substituted for a glutamine (Q). To test the hypothesis that this substitution may be responsible for the observed differences in substrate specificity of these enzymes and as a approach to identify the nucleoside binding site of the type II VV TK, site-directed mutagenesis was employed to alter glutamine 114 (Q114) within domain IV of VV TK to a histidine (Q114H) or an aspartic acid (Q114D). All of the mutant enzymes retained full enzymatic activity as compared to wild-type VV TK when thymidine or bromodeoxyuridine were used as substrates. However, unlike the wild-type herpes simplex (type 1) TK enzyme, neither wild-type nor domain IV VV TK mutants were able to phosphorylate acyclovir or cytidine substrates. Surprisingly, the domain IV VVTK mutants displayed a dramatic loss of feedback inhibition by dTTP. Mutations of the Q114 position also lead to a difference in ATP binding as demonstrated by an altered elution pattern of Q114H and Q114D from an ATP-agarose affinity column with dTTP. Taken together, these results suggest that domain IV of VV TK is not involved directly in substrate discrimination but instead participates in feedback inhibition by dTTP.
Black
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ME
Department of Microbiology, Oregon State University, Corvallis 97331-3804.
Hruby
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DE
D000212:Acyclovir / Q000378:metabolism; D000595:Amino Acid Sequence; D001973:Bromodeoxyuridine / Q000378:metabolism; D002846:Chromatography, Affinity; D003062:Codon; D003562:Cytidine / Q000378:metabolism; D005246:Feedback; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008715:Methionine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D013379:Substrate Specificity; D013936:Thymidine / Q000378:metabolism; D013937:Thymidine Kinase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics
D003062:Codon; D011994:Recombinant Proteins; D003562:Cytidine; D008715:Methionine; D013937:Thymidine Kinase; D001973:Bromodeoxyuridine; D013936:Thymidine; D000212:Acyclovir