Modifying human thymidylate kinase to potentiate azidothymidine activation

Based on the knowledge of the crystal structures of yeast and Escherichia c oli thymidylate kinases (TmpKs) and the observation that TmpK from E. coli can phosphorylate azidothymidine monophosphate (AZT-MP) much more efficient ly than either the yeast or the highly homologous human enzyme, we have eng ineered yeast and human TmpKs to obtain enzymes that have dramatically impr oved AZT-MP phosphorylation properties. These modified enzymes have propert ies that make them attractive candidates for gene therapeutic approaches to potentiating the action of AZT as an inhibitor of human immunodeficiency v irus (HIV) replication. In particular, insertion of the lid domain of the b acterial TmpK into the human enzyme results in a pronounced change of the a cceptance of AZT-MP such that it is now phosphorylated even faster than TMP .