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 (D162) present within a highly con- served tri-peptide motif that is thought to function in nucleoside binding. In this study we generated a mutant, D162Q, by site-directed mutagenesis, puri- fied both the wild-type and mutant TKs to near homogeneity by single-step affinity chromato- graphy and determined the kinetic parameters for thymidine, ATP, dTHP and d)-FP interactions. A 12- fold increase in K m for thymidine by D162Q TK (K m = 6.67 pH) relative to wild-type enzyme (K m = 0"56 ltH) was observed and the absence of any alteration in K m for ATP suggests that D162 parti- cipates in nucleoside binding. Furthermore, the K~ for dTHP is significantly higher for D162Q TK than for HSV-1 TK which is indicative of a shared or overlapping binding site with thymidine. This as- sessment is further supported by the different inhibition patterns of D162Q and wild-type TKs observed using (~_32p)5.NsdUHP photoaffinity label- ling in the presence of thymidine, ganciclovir or dTHP. Interestingly, the K~ for dl-rP was 30-fold lower for D162Q TK (K i = 2.2 llH) than for the wild- type enzyme (K i = 65.8 pM) which provides further evidence of the importance of D162 in TK function.