Epsilon Open Archive

Abstract

The major pathway for biosynthesis of DNA precursors is via the reduction of ribonucleosides to deoxyribonucleosides by the ribonucleotide reductase enzyme system which occurs in all proliferating cells. As a complement to this is de novo synthesis pathway where most cells express deoxyribonucleoside kinases. Deoxycytidine kinase is a cytosolic enzyme and one of four deoxynucleoside kinases in mammalian cells. Human deoxycytidine kinase (dCK) have been cloned and expressed using bacterial vector systems in Escherichia coli. A method to prepare active and stable enzyme for biochemical and structural studies was developed and the effects of high monovalent salt ions were studied. The series of pre-steady-state kinetic experiment were performed with recombinant dCK and different substrates analogues based on intrinsic fluorescence spectroscopy. UTP has been shown to be more efficient phosphate donor as compared to ATP probably by allowing a tighter complex formation between dCK and dCyd. Therefore, the higher fluorescence amplitudes observed for dCyd binding to dCK in the presence of UTP are most likely a consequence of a different conformational change induced by dCyd binding to the UTP-dCK complex. These studies lead to new information regarding the steps involved in interactions between dCK and its most important ligands and provide a possible general model for nucleoside kinase action. There are important differences in the efficiency of dCK from human and mouse cells in the capacity to phosphorylate deoxyribonucleosides and this fact make drug testing in rodent models unreliable. To help to establish better animal models for future drug testing we used a model of human dCK based on the known structure of the human deoxyguanosine kinase to try to explain why certain substitutions in the mouse dCK give differences in substrate specificity. A series of mutations were introduced in the cDNA for human dCK leading to substitutions mimicking the mouse dCK sequence and three amino acid substitutions (Q179R-T184K-H187N) were shown to be most important. To investigate the mechanism of substrate binding the complexes formed between dCK and ligands, such as dCyd, dAdo and dThd, have characterised for the first time by NMR spectroscopy. We showed that ligands, such as dCyd or dAdo, adopt a South-type sugar conformation when bound to dCK. The bound South-type 2'-deoxynucleosides are likely to be in a "near transition state" such that they can be phosphorylate into their 5'-monophosphates by the phosphate donor, ATP. It was found that dCK has ability to degrade deoxyribonucleosides to bases and most likely deoxyribose-1 phosphate. The reaction depended on inorganic phosphate and relative high concentration of enzyme. All natural deoxyribonucleosides could be cleaved but the specific activity of the phosphorylitic reaction compared to the kinase reaction was low, about 2-10%.