Vararattanavech Ardcharaporn, Ketterman Albert J
Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, 25/25 Putthamonthol Road 4, Salaya, Nakon Pathom 73170, Thailand.
Biochem J. 2007 Sep 1;406(2):247-56. doi: 10.1042/BJ20070422.
The present study characterized conserved residues in a GST (glutathione transferase) in the active-site region that interacts with glutathione. This region of the active site is near the glycine moiety of glutathione and consists of a hydrogen bond network. In the GSTD (Delta class GST) studied, adGSTD4-4, the network consisted of His(38), Met(39), Asn(47), Gln(49), His(50) and Cys(51). In addition to contributing to glutathione binding, this region also had major effects on enzyme catalysis, as shown by changes in kinetic parameters and substrate-specific activity. The results also suggest that the electron distribution of this network plays a role in stabilization of the ionized thiol of glutathione as well as impacting on the catalytic rate-limiting step. This area constitutes a second glutathione active-site network involved in glutathione ionization distinct from a network previously observed interacting with the glutamyl end of glutathione. This second network also appears to be functionally conserved in GSTs. In the present study, His(50) is the key basic residue stabilized by this network, as shown by up to a 300-fold decrease in k(cat) and 5200-fold decrease in k(cat)/K(m) for glutathione. Although these network residues have a minor role in structural integrity, the replaced residues induced changes in active-site topography as well as generating positive co-operativity towards glutathione. Moreover, this network at the glycine moiety of GSH (glutathione) also contributed to the 'base-assisted deprotonation model' for GSH ionization. Taken together, the results indicate a critical role for the functionally conserved basic residue His(50) and this hydrogen bond network in the active site.
本研究对谷胱甘肽转移酶（GST）活性位点区域中与谷胱甘肽相互作用的保守残基进行了表征。该活性位点区域靠近谷胱甘肽的甘氨酸部分，由一个氢键网络组成。在所研究的GSTD（δ类GST）即adGSTD4-4中，该网络由His(38)、Met(39)、Asn(47)、Gln(49)、His(50)和Cys(51)组成。除了有助于谷胱甘肽结合外，该区域对酶催化也有主要影响，动力学参数和底物比活性的变化表明了这一点。结果还表明，该网络的电子分布在谷胱甘肽离子化硫醇的稳定中发挥作用，同时也影响催化限速步骤。该区域构成了第二个参与谷胱甘肽离子化的谷胱甘肽活性位点网络，不同于先前观察到的与谷胱甘肽谷氨酰末端相互作用的网络。这第二个网络在GST中似乎在功能上也是保守的。在本研究中，His(50)是由该网络稳定的关键碱性残基，谷胱甘肽的k(cat)降低了高达300倍，k(cat)/K(m)降低了5200倍就表明了这一点。尽管这些网络残基在结构完整性方面作用较小，但被取代的残基引起了活性位点拓扑结构的变化，并对谷胱甘肽产生了正协同性。此外，GSH（谷胱甘肽）甘氨酸部分的这个网络也促成了GSH离子化的“碱辅助去质子化模型”。综上所述，结果表明功能保守的碱性残基His(50)和活性位点中的这个氢键网络起着关键作用。
