Deoxynucleoside analogues are prodrugs that canfunction as inhibitors of both viral and cellular DNA replication processes. They are important in anti-cancer therapy because they hinder DNA synthesis and cellular mitosis. Within the cell, deoxyribonucleotides are synthesized using the salvage pathways by converting the unphosphorylated nucleosides to their mono, di- and tri-phosphate forms using a phosphoryl donor: ATP or UTP. Human deoxycytidine kinase (dCK) is the first and rate-limiting enzyme in this process. The dCK protein uses nucleotide triphosphates to phosphorylate several clinically important nucleoside analogue prodrugs in addition to its natural substrates. The preferred physiological phosphoryl donor for dCK is UTP although it is less prevalent in the human body than ATP. Our objective is to improve the understanding of the phosphate-donor binding loop of dCK by kinetic analysis of a series of mutants of Asp241 and Phe242. These mutants were designed in an attempt to improve the activity of dCK with phosphate donors. Results show several mutants with improved kinetics and some with an ATP donor preference over UTP
