Zinc chelation and structural stability of adenylate kinase from Bacillus subtilis.
Zinc chelation and structural stability of adenylate kinase from Bacillus subtilis.
复制标题
枯草芽孢杆菌腺苷酸激酶的锌螯合和结构稳定性。
DOI：
10.1021/bi00199a019
复制
发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
A. Gilles
中科院分区：
文献类型：
--
作者：
V. Perrier;W. Surewicz;P. Glaser;L. Martineau;C. Craescu;H. Fabian;H. Mantsch;O. Bārzu;A. Gilles
来自枯草芽孢杆菌的腺苷酸激酶，像来自嗜热脂肪芽孢杆菌的酶一样，含有结构性锌原子。来自B的酶中的Cys153。参与锌配位的嗜热脂肪菌在腺苷酸激酶中被B取代。枯草杆菌的天冬氨酸残基。因此，我们有兴趣确定这种差异是否对这些蛋白质的结构，金属螯合和整体稳定性产生影响。我们还感兴趣的是，确定是否His138，这是保守的许多腺苷酸激酶，可以作为第四个合作伙伴的金属螯合，并在一般情况下，他是否可以成功地取代半胱氨酸或天冬氨酸在协调锌的腺苷酸激酶从B。枯草芽孢杆菌来自B的adk基因。通过聚合酶链反应克隆了枯草芽孢杆菌的cDNA。野生型蛋白质，以及通过定点诱变获得的几个变体，在大肠杆菌中表达，并通过生化和物理化学方法进行分析。来自B的腺苷酸激酶的H138 N和D153 C突变体。枯草芽孢杆菌表现出类似于野生型蛋白质的性质，表明His138不参与金属配位，Asp153，就像来自B的酶中类似位置的Cys。嗜热脂肪菌，可参与锌螯合作用。这是第一个实验证据表明，天冬氨酸可以参与结构锌原子的配位。另一方面，D153H和D153T变体在其锌结合性质上显示出显著变化。后者的蛋白质对缓冲液透析（在存在和不存在2 mM EDTA的情况下）导致金属离子的去除和酶活性的损失。（250字处删节）
Adenylate kinase from Bacillus subtilis, like the enzyme from Bacillus stearothermophilus, contains a structural zinc atom. Cys153 in the enzyme from B. stearothermophilus, which is involved in the zinc coordination, is replaced in the adenylate kinase from B. subtilis by an aspartic acid residue. Therefore, we were interested in establishing whether this difference has an impact on the structure, the metal chelation, and the overall stability of these proteins. We also were interested in determining whether His138, which is conserved in many adenylate kinases, can act as a fourth partner in the metal chelation and, in general, whether His can successfully replace Cys or Asp in coordinating zinc in the adenylate kinase from B. subtilis. The adk gene from B. subtilis was cloned by polymerase chain reaction. The wild-type protein, together with several variants obtained by site-directed mutagenesis, were expressed in Escherichia coli and analyzed by biochemical and physicochemical methods. The H138N and D153C mutants of adenylate kinase from B. subtilis exhibited properties similar to those of the wild-type protein, indicating that His138 is not involved in metal coordination and that Asp153, just like Cys in the analogous position in the enzyme from B. stearothermophilus, can participate in zinc chelation. This is the first experimental evidence indicating that aspartic acid can be involved in the coordination of a structural zinc atom. On the other hand, the D153H and D153T variants showed significant changes in their zinc-binding properties. Dialysis of the latter proteins against buffer (in both the presence and the absence of 2 mM EDTA) resulted in removal of the metal ion and loss of enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
影响因子：
2.9
作者：
B. Vallée;D. Auld
通讯作者：
B. Vallée;D. Auld