SITE-DIRECTED MUTAGENESIS CLARIFIES THE SUBSTRATE POSITION WITHIN THE 3-DIMENSIONAL MODEL OF THE ACTIVE-SITE OF HERPES-SIMPLEX VIRUS TYPE-1 THYMIDINE KINASE

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 K-m value for thymidine as the recombinant wildtype enzyme (0.2 mu M), V-max 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 K-i of 146 mu M. 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.