Heterodimeric deoxyguanosine kinase/deoxyadenosine kinase of Lactobacillus acidophilus R-26: heterotropic activation of deoxyadenosine kinase subunit implicated by limited proteolysis and affinity labeling.
Heterodimeric deoxyguanosine kinase/deoxyadenosine kinase of Lactobacillus acidophilus R-26: heterotropic activation of deoxyadenosine kinase subunit implicated by limited proteolysis and affinity labeling.
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嗜酸乳杆菌 R-26 的异二聚脱氧鸟苷激酶/脱氧腺苷激酶：脱氧腺苷激酶亚基的异向激活涉及有限的蛋白水解和亲和标记。
DOI：
10.1021/bi00249a025
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Ives,DH
中科院分区：
文献类型：
--
作者：
Ikeda,S;Ives,DH
乳酸杆菌的脱氧鸟苷（dGuo）激酶/脱氧腺苷（dAdo）激酶复合物已通过使用新构建的dATP-Sepharose柱作为最终步骤（2700倍纯化）纯化至均一。最近已经建立了复合物的异二聚体结构[Ikeda et al.（1994）Biochemistry 33，5328-5334]。基于迄今为止积累的动力学和结构数据，提出了dGuo或dGTP异向性活化dAdo激酶亚基的模型：（1）两个亚基的酶构象存在内在差异，其中dAdo激酶亚基处于约束（闭合）状态，而对应的dGuo激酶亚基处于松弛（开放）状态，如它们的相对Vmax值以及在异嗜性活化的存在或不存在下所反映的，和（2）dGuo或dGTP与dGuo激酶亚基活性位点结合引起的构象变化通过亚基-亚基相互作用引起dAdo激酶亚基的激活。这些机制得到了以下新发现的有力支持：（1）低浓度的离液剂如盐酸胍能使dAdo激酶的Vmax增加2倍，其动力学方式与dGuo激酶激活时相同，但对dGuo激酶无影响;（2）胰蛋白酶对dAdo激酶的蛋白水解失活速度明显慢于dGuo激酶，但dGTP能使其失活速度达到与dGuo激酶相同的水平;（3）dGuo对dAdo激酶的激活作用在dATP存在下胰蛋白酶对dGuo激酶的差异性蛋白水解失活过程中被消除;（4）用[8- 14 C]-8-叠氮基-Ade光亲和标记产生一种新的激酶异二聚体，由于dGuo激酶活性位点的特异性标记，dAdo激酶亚基被永久激活。
The deoxyguanosine (dGuo) kinase/deoxyadenosine (dAdo) kinase complex of Lactobacillus has been purified to homogeneity by using a newly constructed dATP-Sepharose column as a final step (2700-fold purification). A heterodimeric structure for the complex has recently been established [Ikeda et al.(1994) Biochemistry 33, 5328—5334], On the basis of the kinetic and structural data accumulated so far, a model for the heterotropicactivation of the dAdo kinase subunit by dGuo or dGTP is proposed:(1) there is an intrinsic difference in the enzyme conformation of the two subunits, with the dAdo kinase subunit being in a constrained (closed) state and the counterpart dGuo kinase subunit being in a relaxed (open) state, as reflected in their relative Vmax values and in the presence or absence of heterotropic activation, and (2) the conformational change inducedby the binding of dGuo or dGTP to the active site of the dGuo kinase subunit causes the activation of the dAdo kinase subunit through subunit—subunit interactions. These proposed mechanisms are strongly supported by the following new findings made in this work:(1) low concentrations of chaotropic agents such as guanidine—HC1 were found to increase the Vmax of dAdo kinase upto 2-fold-in the same kinetic fashion, apparently, as the activation by dGuo-while showing no effect on dGuo kinase;(2) the proteolytic inactivation of dAdo kinase by trypsin is significantly slower than that of dGuo kinase, but its rate of inactivation is stimulatedby dGTP to the same level as for dGuo kinase;(3) the activating effect of dGuo on dAdo kinase was abolished in the course of differential proteolytic inactivation of the dGuo kinase by trypsin in thepresence of dATP; and (4) photoaffinity labeling with [8-14C]-8-azido-Ade produces a new species of kinase heterodimerin which the dAdo kinase subunit is permanently activated as a result of specific labeling of the dGuo kinase active site.