Winayanuwattikun Pakorn, Ketterman Albert J
Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakhon Pathom 73170, Thailand.
J Biol Chem. 2005 Sep 9;280(36):31776-82. doi: 10.1074/jbc.M502612200. Epub 2005 Jul 12.
In Anopheles dirus glutathione transferase D3-3, there are electrostatic interactions between the negatively charged glutamyl alpha-carboxylate group of glutathione, the positively charged Arg-66, and the negatively charged Asp-100. This ionic interaction is stabilized by a network of hydrogen bonds from Ser-65, Thr-158, Thr-162, and a conserved water-mediated contact. This alternating ionic bridge interaction between negatively and positively charged residues stabilized by a network of hydrogen bonding we have named an electron-sharing network. We show that the electron-sharing network assists the glutamyl alpha-carboxylate of glutathione to function as a catalytic base accepting the proton from the thiol group forming an anionic glutathione, which is a crucial step in the glutathione transferase (GST) catalysis. Kinetic studies demonstrate that the mutation of electron-sharing network residues results in a decreased ability to lower the pKa of the thiol group of glutathione. Although the residues that contribute to the electron-sharing network are not conserved in the primary sequence, structural characterizations indicate that the presence of the network can be mapped to the same region in all GST classes. A structural diversification but functional conservation suggests a significant role for the electron-sharing network in catalysis as the purpose was maintained during the divergent evolution of GSTs. This network appears to be a functionally conserved motif that contributes to the "base-assisted deprotonation" model suggested to be essential for the glutathione ionization step of the catalytic mechanism.
在中华按蚊谷胱甘肽转移酶D3-3中，谷胱甘肽带负电荷的谷氨酰α-羧基、带正电荷的Arg-66和带负电荷的Asp-100之间存在静电相互作用。这种离子相互作用通过Ser-65、Thr-158、Thr-162形成的氢键网络以及保守的水介导接触得以稳定。我们将这种由正负电荷残基之间交替的离子桥相互作用通过氢键网络稳定的结构称为电子共享网络。我们发现，电子共享网络有助于谷胱甘肽的谷氨酰α-羧基作为催化碱，从硫醇基团接受质子形成阴离子型谷胱甘肽，这是谷胱甘肽转移酶（GST）催化过程中的关键步骤。动力学研究表明，电子共享网络残基的突变导致降低谷胱甘肽硫醇基团pKa的能力下降。尽管构成电子共享网络的残基在一级序列中并不保守，但结构表征表明该网络的存在可映射到所有GST类别的同一区域。结构多样化但功能保守表明电子共享网络在催化中具有重要作用，因为其在GSTs的分化进化过程中保持了这一功能。该网络似乎是一个功能保守的基序，有助于“碱辅助去质子化”模型，该模型被认为对催化机制的谷胱甘肽电离步骤至关重要。
