Human deoxycytidine kinase as a deoxyribonucleoside phosphorylase.
Human deoxycytidine kinase (dCK) is a key enzyme in the 5'-phosphorylation of purine and pyrimidine deoxynucleosides with deoxycytidine as the most efficient substrate. The ability of dCK to degrade 2'-deoxyribonucleosides to free nucleobases and 2-deoxy-alpha-d-ribofuranose-1-phosphate was demonstrated by 1H-31P correlation spectroscopy and by isotope enzyme kinetic methods. The reaction depended on inorganic phosphate, and dCK showed maximum cleavage activity between pH 7 and pH 8. In this pH range, [HPO4(2-)] is the dominant phosphate species, most likely being the phosphate donor. All natural deoxyribonucleosides could be cleaved and the Vmax of the phosphorylytic reaction compared to the kinase reaction was about 2-10%. The formation of free nucleobases occurred only with reduced dCK, because the reaction was highly dependent on the presence of reducing agents such as dithiotreitol. Thus, recombinant dCK can act as a phosphorylase, similar to the nucleoside phosphorylase family of enzymes. This catalytic activity is important for the design of in vitro experiments with dCK, such as crystallization and NMR spectroscopy.
Usova
Elena
E
Department of Molecular Biosciences, Section of Veterinary Medical Biochemistry, Swedish University of Agricultural Sciences, Biomedical Center, Box 575, S-751 23 Uppsala, Sweden. elena.usova@vmk.slu.se
Maltseva
Tatiana
T
Földesi
András
A
Chattopadhayaya
Jyoti
J
Eriksson
Staffan
S
D002384:Catalysis / Q000187:drug effects; D003842:Deoxycytidine Kinase / Q000737:chemistry* / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009708:Nucleotidases / Q000737:chemistry* / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry; D010710:Phosphates / Q000494:pharmacology; D011687:Purines / Q000378:metabolism; D011743:Pyrimidines / Q000378:metabolism; D013379:Substrate Specificity
D003854:Deoxyribonucleotides; D009711:Nucleotides; D010710:Phosphates; D011687:Purines; D011743:Pyrimidines; D003842:Deoxycytidine Kinase; D009708:Nucleotidases; C020014:deoxynucleoside-5'-monophosphatase; C030986:pyrimidine; D004229:Dithiothreitol; C030985:purine