Site‐Directed Mutagenesis Clarifies the Substrate Position within the Three‐Dimensional Model of the Active Site of Herpes Simplex Virus Type‐1 Thymidine Kinase

Abstract

Site‐directed mutagenesis was used to experimentally verify the 3D model of the active site of herpes simplex virus type‐1 thymidine kinase (HSV 1 TK) obtained by homology modelling. For this purpose, D215 and K317 were replaced by R and G, respectively, at homologous positions in the aciclovir‐insensitive bovine herpes virus type‐1 thymidine kinase (BHV 1 TK). Wild‐type and mutated enzymes were expressed in Escherichia coli using a gene fusion vector and purified to homogeneity. While both mutants had the same Km value for thymidine as the recombinant wild‐type enzyme (0.2 μM), Vmax was decreased to 20–25% of the original wild‐type value. The recombinant wild‐type enzyme was inhibited by the substrate analogue aciclovir with a Ki of 146 μM. Both mutants were able to phosphorylate aciclovir to about the same extent as the wild‐type enzyme. These findings suggest that neither D215 nor K317 are directly involved in substrate binding. Therefore, a rearrangement of the 3D model is suggested, concerning the assignment of the substrate‐binding site and co‐substrate‐binding site at the right and left side of the phosphate‐binding loop, respectively.