Conservative mutations of glutamine-125 in herpes simplex virus type 1 thymidine kinase result in a ganciclovir kinase with minimal deoxypyrimidine kinase activities.
The herpes simplex virus type 1 thymidine kinase (HSV-1 TK) is the major anti-herpes virus pharmacological target, and it is being utilized in combination with the prodrug ganciclovir as a toxin gene therapeutic for cancer. One active-site amino acid, glutamine-125 (Gln-125), has been shown to form hydrogen bonds with bound thymidine, thymidylate, and ganciclovir in multiple X-ray crystal structures. To examine the role of Gln-125 in HSV-1 TK activity, three site-specific mutations of this residue to an aspartic acid, an asparagine, or a glutamic acid were introduced. These three mutants and wild-type HSV-1 TK were expressed in E. coli and partially purified and their enzymatic properties compared. In comparison to the Gln-125 HSV-1 TK, thymidylate kinase activity of all three mutants was decreased by over 90%. For thymidine kinase activity relative to Gln-125 enzyme, the K(m) of thymidine increased from 0.9 microM for the parent Gln-125 enzyme to 3 microM for the Glu-125 mutant, to 6000 microM for the Asp-125 mutant, and to 20 microM for the Asn-125 mutant. In contrast, the K(m) of ganciclovir decreased from 69 microM for the parent Gln-125 enzyme to 50 microM for the Asn-125 mutant and increased to 473 microM for the Glu-125 mutant. The Asp-125 enzyme was able to poorly phosphorylate ganciclovir, but with nonlinear kinetics. Molecular simulations of the wild-type and mutant HSV-1 TK active sites predict that the observed activities are due to loss of hydrogen bonding between thymidine and the mutant amino acids, while the potential for hydrogen bonding remains intact for ganciclovir binding. When expressed in two mammalian cell lines, the Glu-125 mutant led to GCV-mediated killing of one cell line, while the Asn-125 mutant was equally as effective as wild-type HSV-1 TK in metabolizing GCV and causing cell death in both cell lines.
Hinds
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Department of Biochemistry, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Compadre
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Hurlburt
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Drake
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D015774:Ganciclovir; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics; D009154:Mutation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism
D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase; D013937:Thymidine Kinase; D015774:Ganciclovir