Oksanen Esko, Ahonen Anna-Karoliina, Tuominen Heidi, Tuominen Vesa, Lahti Reijo, Goldman Adrian, Heikinheimo Pirkko
Structural Biology and Biophysics, Institute of Biotechnology, P.O. Box 65, University of Helsinki, FIN-00014 Helsinki, Finland.
Biochemistry. 2007 Feb 6;46(5):1228-39. doi: 10.1021/bi0619977.
We have determined the structures of the wild type and seven active site variants of yeast inorganic pyrophosphatase (PPase) in the presence of Mg2+ and phosphate, providing the first complete structural description of its catalytic cycle. PPases catalyze the hydrolysis of pyrophosphate and require four divalent metal cations for catalysis; magnesium provides the highest activity. The crystal form chosen contains two monomers in the asymmetric unit, corresponding to distinct catalytic intermediates. In the "closed" wild-type active site, one of the two product phosphates has already dissociated, while the D115E variant "open" conformation is of the hitherto unobserved two-phosphate and two-"bridging" water active site. The mutations affect metal binding and the hydrogen bonding network in the active site, allowing us to explain the effects of mutations. For instance, in Y93F, F93 binds in a cryptic hydrophobic pocket in the absence of substrate, preserving hydrogen bonding in the active site and leading to relatively small changes in solution properties. This is not true in the presence of substrate, when F93 is forced back into the active site. Such subtle changes underline how low the energy barriers are between thermodynamically favorable conformations of the enzyme. The structures also allow us to associate metal binding constants to specific sites. Finally, the wild type and the D152E variant contain a phosphate ion adjacent to the active site, showing for the first time how product is released through a channel of flexible cationic side chains.
我们已经确定了在存在Mg2+和磷酸盐的情况下酵母无机焦磷酸酶（PPase）的野生型和七个活性位点变体的结构，首次完整描述了其催化循环。PPases催化焦磷酸水解，催化过程需要四个二价金属阳离子；镁的催化活性最高。所选择的晶体形式在不对称单元中包含两个单体，对应于不同的催化中间体。在“封闭”的野生型活性位点中，两个产物磷酸盐之一已经解离，而D115E变体的“开放”构象是迄今未观察到的双磷酸盐和两个“桥连”水的活性位点。这些突变影响金属结合以及活性位点中的氢键网络，使我们能够解释突变的影响。例如，在Y93F中，F93在没有底物的情况下结合在一个隐蔽的疏水口袋中，保持活性位点中的氢键，导致溶液性质的相对较小变化。在有底物存在时情况并非如此，此时F93被迫回到活性位点。这种细微的变化突显了该酶热力学有利构象之间的能量屏障有多低。这些结构还使我们能够将金属结合常数与特定位点联系起来。最后，野生型和D152E变体在活性位点附近含有一个磷酸根离子，首次展示了产物如何通过柔性阳离子侧链通道释放。
