Usova E V, Eriksson S
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, Uppsala.
Eur J Biochem. 1997 Sep 15;248(3):762-6. doi: 10.1111/j.1432-1033.1997.t01-2-00762.x.
Deoxycytidine kinase (dCK) is a salvage pathway enzyme with broad substrate specificity that can phosphorylate both pyrimidine and purine deoxynucleosides, including important antiviral and cytostatic agents. The kinetic behaviour of dCK is complex with saturation curves showing negative cooperativity. In this study, we have expressed and affinity purified recombinant dCK, using the pET 9d vector system with a histidine tag-sequence and a thrombin cleavage site fused to the N-terminus of the dCK coding sequence. The His-tagged protein showed essentially the same kinetic properties as the protease cleaved protein and the purified protein isolated from human spleen. However, addition of 0.2-0.4 M NaCl during the dCK reaction caused a stimulation of 2'-deoxycytidine (dCyd), and the antileukemic analog 2-chlorodeoxyadenosine (CldAdo) phosphorylation, but an inhibition of the 2'-deoxyguanosine (dGuo) phosphorylation, both with His-tagged and protease cleaved dCK. The negative cooperativity observed with dCyd was eliminated by the presence of 0.4 M NaCl so that the Hill coefficient changed from 0.6 to 1.4. In contrast, dGuo phosphorylation that initially followed Michaelis-Menten kinetics showed negative cooperativity after addition of 0.4 NaCl. The alterations in kinetic properties were not accompanied by any apparent changes in subunit structure as revealed by gel filtration. The major form of dCK eluted in the position corresponding to a dimer in the presence or absence of salt, but a minor fraction of dCK, eluting in the position of a tetramer, was diminished in the presence of salt. The mechanism for the effects of 0.4 M NaCl on dCK kinetic behaviour is not known but it is most likely due to alterations in the conformation of the active site of the enzyme. The effects described here also may explain some of the discrepancies reported in the literature on the substrate specificity of this complex enzyme.
脱氧胞苷激酶（dCK）是一种补救途径酶，具有广泛的底物特异性，能够磷酸化嘧啶和嘌呤脱氧核苷，包括重要的抗病毒和细胞生长抑制剂。dCK的动力学行为很复杂，其饱和曲线显示出负协同性。在本研究中，我们使用pET 9d载体系统表达并亲和纯化了重组dCK，该载体系统带有组氨酸标签序列和凝血酶切割位点，融合在dCK编码序列的N端。带有组氨酸标签的蛋白质表现出与蛋白酶切割后的蛋白质以及从人脾脏中分离出的纯化蛋白质基本相同的动力学性质。然而，在dCK反应过程中添加0.2 - 0.4 M NaCl会刺激2'-脱氧胞苷（dCyd）和抗白血病类似物2-氯脱氧腺苷（CldAdo）的磷酸化，但会抑制2'-脱氧鸟苷（dGuo）的磷酸化，无论是带有组氨酸标签的还是蛋白酶切割后的dCK均如此。0.4 M NaCl的存在消除了dCyd观察到的负协同性，使得希尔系数从0.6变为1.4。相反，最初遵循米氏动力学的dGuo磷酸化在添加0.4 NaCl后显示出负协同性。凝胶过滤显示，动力学性质的改变并未伴随着亚基结构的任何明显变化。在有盐或无盐存在的情况下，dCK的主要形式在对应二聚体的位置洗脱，但在有盐存在时，在四聚体位置洗脱的一小部分dCK减少了。0.4 M NaCl对dCK动力学行为产生影响的机制尚不清楚，但很可能是由于酶活性位点构象的改变。这里描述的这些影响也可能解释了文献中关于这种复杂酶底物特异性报道的一些差异。
