模拟人脱氧胞苷激酶上Ser-74的磷酸化可选择性提高对dC和dC类似物的催化活性。
Mimicking phosphorylation of Ser-74 on human deoxycytidine kinase selectively increases catalytic activity for dC and dC analogues.
作者信息
McSorley Theresa, Ort Stephan, Hazra Saugata, Lavie Arnon, Konrad Manfred
机构信息
Max-Planck-Institute for Biophysical Chemistry, Research Group Enzyme Biochemistry, Am Fassberg 11, Göttingen, Germany.
出版信息
FEBS Lett. 2008 Mar 5;582(5):720-4. doi: 10.1016/j.febslet.2008.01.048. Epub 2008 Feb 5.
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 k(cat) 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.
摘要
脱氧胞苷激酶（dCK）在丝氨酸74位点的细胞内磷酸化会导致核苷激酶活性增加。我们通过在细菌产生的dCK中引入丝氨酸74突变为谷氨酸（Ser-74-Glu）来模拟这种磷酸化，并使用各种核苷底物研究动力学参数。S74E突变使dC的催化常数（k(cat)）值增加11倍，使抗癌类似物2'-脱氧氟代胞苷（dFdC）和阿糖胞苷（AraC）的k(cat)值增加3倍。相比之下，嘌呤底物的反应速率降低。在人胚肾293（HEK293）细胞中，我们发现通过比较瞬时转染的dCK(S74E)-绿色荧光蛋白（GFP）和野生型dCK-GFP，模拟丝氨酸74的磷酸化对细胞定位没有影响。我们注意到磷酸化可能是一种增强相对较慢的dCK酶催化活性的机制。
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