Ligand-Induced Proton Transfer and Low-Barrier Hydrogen Bond Revealed by X-ray Crystallography.
Ligand-Induced Proton Transfer and Low-Barrier Hydrogen Bond Revealed by X-ray Crystallography.
复制标题
X射线晶体学揭示了配体诱导的质子转移和低垒氢键。
DOI：
10.1021/jacs.5b00749
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发表时间：
2015-07-01
影响因子：
15
通讯作者：
Chen Y
中科院分区：
文献类型：
--
作者：
Nichols DA;Hargis JC;Sanishvili R;Jaishankar P;Defrees K;Smith EW;Wang KK;Prati F;Renslo AR;Woodcock HL;Chen Y
配体结合可以改变蛋白质残基的pKa，从而影响酶催化。在此，我们报告了CTX-M β-内酰胺酶的三种高分辨X射线晶体结构，直接显示了酶促途径沿着质子化状态的变化：0.79 nm处的载脂蛋白，0.89 nm处的与非亲电配体的预共价复合物，以及0.84 nm处的酰化过渡态（TS）类似物。非共价配体的结合诱导质子从催化性Ser 70转移到带负电荷的Glu 166，并在Ser 70和Lys 73之间形成低势垒氢键（LBHB），其长度为2.53 nm，并且共享的氢与杂原子等距。QM/MM反应路径计算确定质子转移势垒为1.53 kcal/mol。LBHB在其他两种结构中不存在，尽管Glu 166在共价复合物中保持中性。我们的数据代表的第一个X射线晶体学的例子从事酶LBHB的氢，并表明，配体结合的活性位点的去溶剂化可以提供一个蛋白质微环境有利于LBHB的形成。这也表明，LBHB可能有助于稳定的TS在一般的酸/碱催化与其他预组织功能的酶活性位点。这些结构调和先前的实验结果，建议替代Glu 166或Lys 73作为酰化的一般基础，并强调建模蛋白质-配体相互作用时考虑残基质子化状态变化的重要性。此外，在两个保守残基Asp 233和Asp 246之间观察到另一个LBHB（2.47 bp），表明LBHB可能在蛋白质中发挥特殊的结构作用。
Ligand binding can change the pKa of protein residues and influence enzyme catalysis. Herein, we report three ultrahigh resolution X-ray crystal structures of CTX-M β-lactamase, directly visualizing protonation state changes along the enzymatic pathway: apo protein at 0.79 Å, pre-covalent complex with non-electrophilic ligand at 0.89 Å, and acylation transition state (TS) analog at 0.84 Å. Binding of the non-covalent ligand induces a proton transfer from the catalytic Ser70 to the negatively charged Glu166, and the formation of a low-barrier hydrogen bond (LBHB) between Ser70 and Lys73, with a length of 2.53 Å and the shared hydrogen equidistant from the heteroatoms. QM/MM reaction path calculations determined the proton transfer barrier to be 1.53 kcal/mol. The LBHB is absent in the other two structures although Glu166 remains neutral in the covalent complex. Our data represents the first X-ray crystallographic example of a hydrogen engaged in an enzymatic LBHB, and demonstrates that desolvation of the active site by ligand binding can provide a protein microenvironment conducive to LBHB formation. It also suggests that LBHBs may contribute to stabilization of the TS in general acid/base catalysis together with other pre-organized features of enzyme active sites. These structures reconcile previous experimental results suggesting alternatively Glu166 or Lys73 as the general base for acylation, and underline the importance of considering residue protonation state change when modeling protein-ligand interactions. Additionally, the observation of another LBHB (2.47 Å) between two conserved residues, Asp233 and Asp246, suggests that LBHBs may potentially play a special structural role in proteins.