Conservative Mutations of Glutamine-125 in Herpes Simplex Virus Type 1 Thymidine Kinase Result in a Ganciclovir Kinase with Minimal Deoxypyrimidine Kinase Activities
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- 作者：Trenton A. Hinds ; Cesar Compadre ; Barry K. Hurlburt ; and Richard R. Drake
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：April 11, 2000
- 年：2000
- 卷：39
- 期：14
- 页码：4105 - 4111
- 全文大小：103K
- 年卷期：v.39,no.14(April 11, 2000)
- ISSN：1520-4995
文摘
The herpes simplex virus type 1 thymidine kinase (HSV-1 TK) is the major anti-herpes viruspharmacological target, and it is being utilized in combination with the prodrug ganciclovir as a toxingene therapeutic for cancer. One active-site amino acid, glutamine-125 (Gln-125), has been shown toform hydrogen bonds with bound thymidine, thymidylate, and ganciclovir in multiple X-ray crystalstructures. To examine the role of Gln-125 in HSV-1 TK activity, three site-specific mutations of thisresidue to an aspartic acid, an asparagine, or a glutamic acid were introduced. These three mutants andwild-type HSV-1 TK were expressed in E. coli and partially purified and their enzymatic propertiescompared. In comparison to the Gln-125 HSV-1 TK, thymidylate kinase activity of all three mutants wasdecreased by over 90%. For thymidine kinase activity relative to Gln-125 enzyme, the Km of thymidineincreased from 0.9 
M for the parent Gln-125 enzyme to 3 M for the Glu-125 mutant, to 6000 M forthe Asp-125 mutant, and to 20 M for the Asn-125 mutant. In contrast, the Km of ganciclovir decreasedfrom 69 M for the parent Gln-125 enzyme to 50 M for the Asn-125 mutant and increased to 473 Mfor the Glu-125 mutant. The Asp-125 enzyme was able to poorly phosphorylate ganciclovir, but withnonlinear kinetics. Molecular simulations of the wild-type and mutant HSV-1 TK active sites predict thatthe observed activities are due to loss of hydrogen bonding between thymidine and the mutant aminoacids, while the potential for hydrogen bonding remains intact for ganciclovir binding. When expressedin two mammalian cell lines, the Glu-125 mutant led to GCV-mediated killing of one cell line, while theAsn-125 mutant was equally as effective as wild-type HSV-1 TK in metabolizing GCV and causing celldeath in both cell lines.
M for the parent Gln-125 enzyme to 3 M for the Glu-125 mutant, to 6000 M forthe Asp-125 mutant, and to 20 M for the Asn-125 mutant. In contrast, the Km of ganciclovir decreasedfrom 69 M for the parent Gln-125 enzyme to 50 M for the Asn-125 mutant and increased to 473 Mfor the Glu-125 mutant. The Asp-125 enzyme was able to poorly phosphorylate ganciclovir, but withnonlinear kinetics. Molecular simulations of the wild-type and mutant HSV-1 TK active sites predict thatthe observed activities are due to loss of hydrogen bonding between thymidine and the mutant aminoacids, while the potential for hydrogen bonding remains intact for ganciclovir binding. When expressedin two mammalian cell lines, the Glu-125 mutant led to GCV-mediated killing of one cell line, while theAsn-125 mutant was equally as effective as wild-type HSV-1 TK in metabolizing GCV and causing celldeath in both cell lines.