Benini S, Rypniewski W R, Wilson K S, Ciurli S, Mangani S
EMBL, c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany.
J Biol Inorg Chem. 2001 Oct;6(8):778-90. doi: 10.1007/s007750100254.
The structure of Bacillus pasteurii urease (BPU) inhibited with phosphate was solved and refined using synchrotron X-ray diffraction data from a vitrified crystal (1.85 A resolution, 99.3% completeness, data redundancy 4.6, R-factor 17.3%, PDB code 6UBP). A distance of 3.5 A separates the two Ni ions in the active site. The binding mode of the inhibitor involves the formation of four coordination bonds with the two Ni ions: one phosphate oxygen atom symmetrically bridges the two metal ions (1.9-2.0 A), while two of the remaining phosphate oxygen atoms bind to the Ni atoms at 2.4 A. The fourth phosphate oxygen is directed into the active site channel. Analysis of the H-bonding network around the bound inhibitor indicates that phosphate is bound as the H2PO4- anion, and that an additional proton is present on the Odelta2 atom of Asp(alpha363), an active site residue involved in Ni coordination through Odelta1. The flexible flap flanking the active site cavity is in the open conformation. Analysis of the complex reveals why phosphate is a relatively weak inhibitor and why sulfate does not bind to the nickels in the active site. The implications of the results for the understanding of the urease catalytic mechanism are reviewed. A novel alternative for the proton donor is presented.
利用来自玻璃化晶体的同步辐射X射线衍射数据（分辨率1.85 Å，完整性99.3%，数据冗余度4.6，R因子17.3%，PDB代码6UBP），解析并精修了受磷酸盐抑制的巴氏芽孢杆菌脲酶（BPU）的结构。活性位点中的两个镍离子相距3.5 Å。抑制剂的结合模式涉及与两个镍离子形成四个配位键：一个磷酸氧原子对称地桥接两个金属离子（1.9 - 2.0 Å），而其余三个磷酸氧原子中的两个以2.4 Å的距离与镍原子结合。第四个磷酸氧指向活性位点通道。对结合抑制剂周围氢键网络的分析表明，磷酸盐以H2PO4-阴离子形式结合，并且在天冬氨酸（α363）的Oδ2原子上存在一个额外的质子，天冬氨酸是通过Oδ1参与镍配位的活性位点残基。活性位点腔侧翼的柔性瓣处于开放构象。对该复合物的分析揭示了为什么磷酸盐是一种相对较弱的抑制剂，以及为什么硫酸盐不与活性位点中的镍结合。综述了这些结果对理解脲酶催化机制的意义。提出了质子供体的一种新的替代方式。
