Mutation of Gln125 to Asn selectively abolishes the thymidylate kinase activity of herpes simplex virus type 1 thymidine kinase

The broad substrate specificity of herpes simplex virus type 1 (HSV-1) thym idine kinase (TK) has provided the basis for selective antiherpetic therapy and, more recently, suicide gene therapy for the treatment of cancer. We h ave now constructed an HSV-1 TK mutant enzyme, in which an asparagine (N) r esidue is substituted for glutamine (Q) at position 125, and have evaluated the effect of this amino acid change on enzymatic activity. In marked cont rast with wild-type HSV-1 TK, which displays both thymidine kinase and thym idylate kinase activities, the HSV-1 TK(Q125N) mutant was unable to phospho rylate pyrimidine nucleoside monophosphates but retained significant phosph orylation activity for thymidine and a series of antiherpetic pyrimidine an d purine nucleoside analogs. The abrogation of HSV-1 TK-associated thymidyl ate kinase activity resulted in a 100-fold accumulation of the monophosphat e form of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) in osteosarcoma cells transfected with the HSV-1 TK(Q125N) gene compared with osteosarcoma cells expressing wild-type HSV-1 TK. BVDU monophosphate accumulation gave rise t o a much greater inhibition of cellular thymidylate synthase in HSV-1 TK(Q1 25N) gene-transfected cells than wild-type HSV-1 TK gene-transfected osteos arcoma tumor cells without significantly changing the cytostatic potency of BVDU for the HSV-1 TK gene-transfected tumor cells. Accordingly, the prese nce of the Q125N mutation in HSV-1 TK gene-transfected tumor cells was foun d to result in a multilog decrease in the cytostatic activity of those pyri midine nucleoside analogs that in their monophosphate form do not have mark ed affinity for thymidylate synthase [i.e., 1-beta -D-arabinofuranosylthymi ne and (E)-5-(2-bromovinyl)-1-beta -D-arabinofuranosyluracil].