Structures of the Klebsiella aerogenes urease apoenzyme and two active-site mutants
Structures of the Klebsiella aerogenes urease apoenzyme and two active-site mutants
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DOI：
10.1021/bi960424z
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发表时间：
1996-08-20
期刊：
影响因子：
2.9
通讯作者：
Karplus, PA
中科院分区：
文献类型：
--
作者：
Jabri, E;Karplus, PA
来自产气克雷伯氏菌的尿素酶[Jabri等人（1995）Science 268，998-1004]是一种（α β γ）（3）三聚体，其中每个α-亚基具有一个含有二镍活性中心的（α β）（8）-桶结构域。在这里，我们通过在2.3埃分辨率下对尿素酶脱辅基酶进行结构分析，并在2.5埃分辨率下对两个关键催化残基（H219 A和H320 A）的突变体进行结构分析，更详细地研究了尿素酶的结构-功能关系。除了活性位点，其中一个水分子取代了缺失的氨基甲酸酯和镍原子，脱辅基酶的结构几乎与全酶的结构相同，这表明高度的预组织有助于解释镍的紧密结合。在H219 A的结构中，主要的变化涉及活性位点瓣的构象移位和排序，但Asp（α 211）侧链的小位移可能导致H219 A活性降低。在H320 A结构中，催化水，主要是全酶中的Ni-2配体，转移到桥接位置。这种变化表明镍连接对环境相当敏感，连接的变化可能导致H320 A活性降低10（5）倍。此外，这些结果表明，尿素酶是弹性的镍离子和突变的损失，尿素酶的三级/四级结构的分析表明，这种酶的稳定性可能在很大程度上是由于其埋葬了一个不寻常的大部分的残基：50%的γ-亚基，30%的β-亚基，和60%的α-亚基。
Urease from Klebsiella aerogenes [Jabri et al. (1995) Science 268, 998-1004] is an (alpha beta gamma)(3) trimer with each alpha-subunit having an (alpha beta)(8)-barrel domain containing a binickel active center. Here we examine structure-function relations for urease in more detail through structural analysis of the urease apoenzyme at 2.3 Angstrom resolution and mutants of two key catalytic residues (H219A and H320A) at 2.5 Angstrom resolution. With the exception of the active site, in which a water molecule takes the place of the missing carbamate and nickel atoms, the structure of the apoenzyme is nearly identical to that of the holoenzyme, suggesting a high degree of preorganization which helps explain the tight binding of nickel, In the structure of H219A, the major change involves a conformational shift and ordering of the active site flap, but a small shift in the side chain of Asp(alpha 211) could contribute to the lower activity of H219A. In the H320A structure, the catalytic water, primarily a Ni-2 ligand in the holoenzyme, shifts into a bridging position. This shift shows that the nickel ligation is rather sensitive to the environment and the change in ligation may contribute to the 10(5)-fold lower activity of H320A. In addition, these results show that urease is resilient to the loss of nickel ions and mutations, Analysis of the urease tertiary/quaternary structure suggests that the stability of this enzyme may be largely due to its burial of an unusually large fraction of its residues: 50% in the gamma-subunit, 30% in the beta-subunit, and 60% in the alpha-subunit.