ATP and phosphate reciprocally affect subunit association of human recombinant High Km 5′-nucleotidase -: Role for the C-terminal polyglutamic acid tract in subunit association and catalytic activity
ATP and phosphate reciprocally affect subunit association of human recombinant High Km 5′-nucleotidase -: Role for the C-terminal polyglutamic acid tract in subunit association and catalytic activity
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DOI：
10.1046/j.1432-1327.1999.00099.x
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发表时间：
1999-02-01
期刊：
影响因子：
--
通讯作者：
Mitchell, BS
中科院分区：
文献类型：
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作者：
Spychala, J;Chen, V;Mitchell, BS
IMP特异性高Km 5 '-核苷酸酶（EC 3.1.3.5）是一种普遍存在的酶，其活性受底物、ATP和无机磷酸盐高度调节。编码这种酶的cDNA最近已被克隆，并发现在C-末端含有一个独特的9个谷氨酸和4个天冬氨酸残基的延伸。为了研究这种酸性尾部以及ATP和无机磷酸盐对酶功能的影响，我们对5 '-核苷酸酶cDNA进行了几种结构修饰，在大肠杆菌中表达了相应的蛋白质，并比较了它们的分子和动力学性质。与从人胎盘中纯化的酶一样，所有重组蛋白都被ATP激活，被无机磷酸盐抑制。虽然S-0.5值较高，但纯化蛋白变体的比活性（除了在C-末端截短的）相似。全长酶亚基的分子量估计为57.3 kDa，天然蛋白质的分子量估计为195 kDa，通过凝胶过滤色谱法测定。增加NaCl的浓度至0.3 M促进蛋白质的寡聚化和332 kDa的聚集体的形成。ATP诱导进一步低聚到715 kDa，而无机磷酸盐减少估计分子量为226 kDa。相反，在N-末端的30个氨基酸的截短，这并没有改变酶的性质，在C-末端的聚谷氨酸/天冬氨酸尾的13个残基的去除引起深刻的动力学和结构的变化，包括比活性降低29倍，并在AMP的存在下，由无机磷酸盐抑制的敏感性显着增加。在结构上，有一个戏剧性的损失的能力，形成低聚物在生理盐浓度，这是只有部分恢复的NaCl或ATP的加入。这些数据表明聚谷氨酸束在5 '-核苷酸酶亚基的缔合和解离过程中具有重要功能。
IMP-specific, High Km 5'-nucleotidase (EC 3.1.3.5) is an ubiquitous enzyme, the activity of which is highly regulated by substrate, ATP, and inorganic phosphate. The cDNA encoding this enzyme has recently been cloned and found to contain a unique stretch of nine glutamic and four aspartic acid residues at the C-terminus. To study the effects of this acidic tail, and of ATP and inorganic phosphate on enzyme function, we generated several structural modifications of the 5'-nucleotidase cDNA, expressed the corresponding proteins in Escherichia coli and compared their molecular and kinetic properties. As with the enzyme purified from human placenta, all recombinant proteins were activated by ATP and inhibited by inorganic phosphate. Although the S-0.5-values were higher, the specific activities of the purified protein variants (except that truncated at the C-terminus) were similar. The molecular mass of the full-length enzyme subunit has been estimated at 57.3 kDa and the molecular mass of the native protein, as determined by gel-filtration chromatography, was estimated to be 195 kDa. Increasing the concentration of NaCl to 0.3 M promoted oligomerization of the protein and the formation of aggregates of 332 kDa. ATP induced further oligomerization to 715 kDa, while inorganic phosphate reduced the estimated molecular mass to 226 kDa. In contrast to the truncation of 30 amino acids at the N-terminus, which did not alter enzyme properties, the removal of the polyglutamic/aspartic acid tail of 13 residues at the C-terminus caused profound kinetic and structural changes, including a 29-fold decrease in specific activity and a significant increase in the sensitivity to inhibition by inorganic phosphate in the presence of AMP. Structurally, there was a dramatic loss of the ability to form oligomers at physiological salt concentration which was only partially restored by the addition of NaCl or ATP. These data suggest an important function of the polyglutamic acid tract in the process of association and dissociation of 5'-nucleotidase subunits.