Jabri E, Karplus P A
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.
Biochemistry. 1996 Aug 20;35(33):10616-26. doi: 10.1021/bi960424z.
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 A resolution and mutants of two key catalytic residues (H219A and H320A) at 2.5 A 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 221 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.
产气克雷伯菌的脲酶[贾布里等人（1995年），《科学》268卷，998 - 1004页]是一种(αβγ)3三聚体，每个α亚基都有一个(αβ)8桶状结构域，其中包含一个双镍活性中心。在这里，我们通过对分辨率为2.3 Å的脲酶脱辅基酶的结构分析以及对两个关键催化残基（H219A和H320A）的突变体在分辨率为2.5 Å时的结构分析，更详细地研究了脲酶的结构 - 功能关系。除了活性位点外，脱辅基酶的结构与全酶几乎相同，在活性位点中，一个水分子取代了缺失的氨基甲酸酯和镍原子，这表明存在高度的预组织，这有助于解释镍的紧密结合。在H219A的结构中，主要变化涉及活性位点侧翼的构象转变和有序化，但Aspα221侧链的小位移可能导致H219A活性较低。在H320A结构中，催化水（主要是全酶中的Ni - 2配体）转移到桥连位置。这种转移表明镍配位对环境相当敏感，配位的变化可能导致H320A活性降低10^5倍。此外，这些结果表明脲酶对镍离子的损失和突变具有弹性。对脲酶三级/四级结构的分析表明，这种酶的稳定性可能很大程度上归因于其大量残基的埋藏：γ亚基中为50%，β亚基中为30%，α亚基中为60%。
