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Abstract

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 H-1-P-31 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, [HPO42-] is the dominant phosphate species, most likely being the phosphate donor. All natural deoxyribonucleosides could be cleaved and the V-max 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 3 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. (C) 2004 Elsevier Ltd. All rights reserved.

Keywords

deoxycytidine kinase; nucleoside analogs; phosphorylation; phosphorolysis reaction; nucleoside phosphorylase

Published in

Journal of Molecular Biology
2004, volume: 344, number: 5, pages: 1347-1358
Publisher: ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD

SLU Authors

  • Usova, Elena

    • Department of Molecular Biosciences, Swedish University of Agricultural Sciences

  • Eriksson, Staffan

    • Department of Molecular Biosciences, Swedish University of Agricultural Sciences

UKÄ Subject classification

Biochemistry
Molecular Biology

Publication identifier

  • DOI: https://doi.org/10.1016/j.jmb.2004.10.016

Permanent link to this page (URI)

https://res.slu.se/id/publ/3548