Active sites of diacylglycerol kinase from Escherichia coli are shared between subunits
Active sites of diacylglycerol kinase from Escherichia coli are shared between subunits
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DOI：
10.1021/bi982763t
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发表时间：
1999-04-27
期刊：
影响因子：
2.9
通讯作者：
Bowie, JU
中科院分区：
文献类型：
--
作者：
Lau, FW;Chen, X;Bowie, JU
我们发现来自不同亚基的残基参与形成大肠杆菌三聚体膜蛋白二酰基甘油激酶（DGK）的活性位点。鉴定出5个可能的活性位点突变体：A14Q、N72S、E76L、K94L和D95N。这五种突变体的催化功能都明显受损，但从近紫外和远紫外圆二色光谱来看，没有明显的结构改变。我们发现A14Q或E76L与N72S或K94L的混合物具有比突变蛋白本身更大的活性，这表明Ala14和Glu76可能在一个半位点上，而Asn72和Lys94在另一个半位点上。与共享位点模型一致，我们还发现(1)A14Q和N72S亚基混合物的活性峰值出现在等摩尔浓度；(2) A14Q和N72S混合酶的最大活性为野生型酶的20%，与理论最大值25%基本一致；(3)突变亚基的活性不能通过与野生型亚基混合而恢复；(4)发现双突变体A14Q/N72S在两个假定的半位点上都有突变，使野生型亚基失活；(5)三聚体蛋白的共享位点模型可以很好地描述A14Q/N72S突变体失活的浓度依赖性。出乎意料的是，我们发现单突变体D95N的行为方式与双突变体A14Q/N72S相似，使野生型亚基失活。我们认为Asp95在多个活性位点起作用。
We show that residues from different subunits participate in forming the active site of the trimeric membrane protein diacylglycerol kinase (DGK) from Escherichia coli. Five likely active-site mutants were identified: A14Q, N72S, E76L, K94L, and D95N. All five of these mutants possessed significantly impaired catalytic function, without evidence of gross structural alterations as judged by their similar near-UV and far-UV circular dichroism spectra. We found that mixtures of either A14Q or E76L with N72S or K94L possessed much greater activity than the mutant proteins by themselves, suggesting that Ala14 and Glu76 may be on one half-site while Asn72 and Lys94 are on another half-site. Consistent with the shared site model, we also found that (1) peak activity of A14Q and N72S subunit mixtures occur at equimolar concentrations; (2) the maximum activity of the A14Q and N72S mixture was 20% of the wild-type enzyme, in good agreement with the theoretical maximum of 25%; (3) the activity of mutant subunits could not be recovered by mixing with the wild-type subunits; (4) a double mutant, A14Q/N72S, bearing mutations in both putative half-sites was found to inactivate wild-type subunits; (5) the concentration dependence of inactivation by the A14Q/N72S mutant could be well described by a shared site model for a trimeric protein. Unexpectedly, we found that the single mutant D95N behaved in a manner similar to the double mutant, A14Q/N72S, inactivating wild-type subunits. We propose that Asp95 plays a role in more than one active site.