Mimicking phosphorylation of Ser-74 on human deoxycytidine kinase selectively increases catalytic activity for dC and dC analogues |
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Authors: | McSorley Theresa Ort Stephan Hazra Saugata Lavie Arnon Konrad Manfred |
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Institution: | a Max-Planck-Institute for Biophysical Chemistry, Research Group Enzyme Biochemistry, Am Fassberg 11, D-37077 Göttingen, Germany b University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics, 900 South Ashland Avenue, Chicago, IL 60607, USA |
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Abstract: | Intracellular phosphorylation of dCK on Ser-74 results in increased nucleoside kinase activity. We mimicked this phosphorylation by a Ser-74-Glu mutation in bacterially produced dCK and investigated kinetic parameters using various nucleoside substrates. The S74E mutation increases the kcat values 11-fold for dC, and 3-fold for the anti-cancer analogues dFdC and AraC. In contrast, the rate is decreased for the purine substrates. In HEK293 cells, we found that by comparing transiently transfected dCK(S74E)-GFP and wild-type dCK-GFP, mimicking the phosphorylation of Ser-74 has no effect on cellular localisation. We note that phosphorylation may represent a mechanism to enhance the catalytic activity of the relatively slow dCK enzyme. |
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Keywords: | Deoxycytidine kinase Nucleoside analogues Prodrug phosphorylation Protein phosphorylation Subcellular localisation |
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