Effect on substrate binding of an alteration at the conserved aspartic acid-162 in herpes simplex virus type 1 thymidine kinase.
Despite the extensive use of antiviral drugs for the treatment of herpesvirus infections and as pro-drugs for ablative gene therapy of cancer, little structural information about the drug activating enzyme, herpes simplex virus type 1 thymidine kinase (TK), was available until recently. In the absence of the three-dimensional structure we sought to elucidate the function of the key aspartic acid residue (Dl62) present within a highly conserved tri-peptide motif that is thought to function in nucleoside binding. In this study we generated a mutant, D162Q, by site-directed mutagenesis, purified both the wild-type and mutant TKs to near homogeneity by single-step affinity chromatography and determined the kinetic parameters for thymidine, ATP, dTMP and dTTP interactions. A 12-fold increase in Km for thymidine by D162Q TK (Km = 6.67 microM) relative to wild-type enzyme (Km = 0.56 microM) was observed and the absence of any alteration in Km for ATP suggests that D162 participates in nucleoside binding. Furthermore, the Ki for dTMP is significantly higher for D162Q TK than for HSV-1 TK which is indicative of a shared or overlapping binding site with thymidine. This assessment is further supported by the different inhibition patterns of D162Q and wild-type TKs observed using [alpha-32P]5-N3dUMP photoaffinity labelling in the presence of thymidine, ganciclovir or dTMP. Interestingly, the Ki for dTTP was 30-fold lower for D162Q TK (Ki = 2.2 microM) than for the wild-type enzyme (Ki = 65.8 microM) which provides further evidence of the importance of D162 in TK function.
Black
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Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, USA.
Rechtin
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Drake
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D000255:Adenosine Triphosphate / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004279:DNA, Viral; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013379:Substrate Specificity; D013936:Thymidine / Q000378:metabolism; D013938:Thymidine Monophosphate / Q000378:metabolism; D013942:Thymine Nucleotides / Q000378:metabolism; D014764:Viral Proteins
D004279:DNA, Viral; D013942:Thymine Nucleotides; D014764:Viral Proteins; D001224:Aspartic Acid; D013938:Thymidine Monophosphate; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; C095207:US3 protein, Human herpesvirus 1; D008274:Magnesium; C024157:thymidine 5'-triphosphate; D013936:Thymidine