A functionally conserved basic residue in glutathione transferases interacts with the glycine moiety of glutathione and is pivotal for enzyme catalysis

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 His³⁸, Met³⁹, Asn⁴⁷, Gln⁴⁹, His⁵⁰ and Cys⁵¹. 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, His⁵⁰ 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 His⁵⁰ and this hydrogen bond network in the active site.