Pearson M A, Michel L O, Hausinger R P, Karplus P A
Section of Biochemistry, Cornell University, Ithaca, New York 14853, USA.
Biochemistry. 1997 Jul 1;36(26):8164-72. doi: 10.1021/bi970514j.
Cys319 is located on a mobile flap covering the active site of Klebsiella aerogenes urease but does not play an essential role in catalysis. Four urease variants altered at position C319 range from having high activity (C319A) to no measurable activity (C319Y), indicating Cys is not required at this position, but its presence is highly influential [Martin, P. R., & Hausinger, R. P. (1992) J. Biol. Chem. 267, 20024-20027]. Here, we present 2.0 A resolution crystal structures of C319A, C319S, C319D, and C319Y proteins and the C319A variant inhibited by acetohydroxamic acid. These structures show changes in the hydration of the active site nickel ions and in the position and flexibility of the active site flap. The C319Y protein exhibits an alternate conformation of the flap, explaining its lack of activity. The changes in hydration and conformation suggest that there are suboptimal protein-solvent and protein-protein interactions in the empty urease active site which contribute to urease catalysis. Specifically, we hypothesize that the suboptimal interactions may provide a significant source of substrate binding energy, and such hidden energy may be a common phenomenon for enzymes that contain mobile active site loops and undergo an induced fit. The acetohydroxamic acid-bound structure reveals a chelate interaction similar to those seen in other metalloenzymes and in a small molecule nickel complex. The inhibitor binding mode supports the proposed mode of urea binding. We complement these structural studies with extended functional studies of C319A urease to show that it has enhanced stability and resistance to inhibition by buffers containing nickel ions. The near wild-type activity and enhanced stability of the C319A variant make it useful for further studies of urease structure-function relationships.
半胱氨酸319（Cys319）位于覆盖产气克雷伯菌脲酶活性位点的一个可移动的侧翼上，但在催化过程中不发挥关键作用。在C319位点发生改变的四种脲酶变体，活性从高（C319A）到无法检测到（C319Y）不等，这表明该位置不需要半胱氨酸，但它的存在具有很大影响[马丁，P. R.，& 豪辛格，R. P.（1992年）《生物化学杂志》267卷，20024 - 20027页]。在此，我们展示了C319A、C319S、C319D和C319Y蛋白以及被乙酰氧肟酸抑制的C319A变体的2.0埃分辨率晶体结构。这些结构显示了活性位点镍离子水合作用的变化以及活性位点侧翼的位置和灵活性的变化。C319Y蛋白表现出侧翼的另一种构象，这解释了其缺乏活性的原因。水合作用和构象的变化表明，在空的脲酶活性位点存在次优的蛋白质 - 溶剂和蛋白质 - 蛋白质相互作用，这些相互作用有助于脲酶催化。具体而言，我们推测次优相互作用可能提供了底物结合能量的重要来源，并且这种隐藏能量可能是含有可移动活性位点环并经历诱导契合的酶的常见现象。乙酰氧肟酸结合的结构揭示了一种与其他金属酶以及小分子镍配合物中所见类似的螯合相互作用。抑制剂结合模式支持所提出的尿素结合模式。我们通过对C319A脲酶进行扩展的功能研究来补充这些结构研究，以表明它具有增强的稳定性以及对含镍离子缓冲液抑制的抗性。C319A变体接近野生型的活性和增强的稳定性使其可用于进一步研究脲酶的结构 - 功能关系。
