Structural contributions of Delta class glutathione transferase active-site residues to catalysis

GST (glutathione transferase) is a dimeric enzyme recognized for biotransformation of xenobiotics and endogenous toxic compounds. In the present study, residues forming the hydrophobic substrate-binding site (H-site) of a Delta class enzyme were investigated in detail for the first time by site-directed mutagenesis and crystallographic studies. Enzyme kinetics reveal that Tyr¹¹¹ indirectly stabilizes GSH binding, Tyr¹¹⁹ modulates hydrophobic substrate binding and Phe¹²³ indirectly modulates catalysis. Mutations at Tyr¹¹¹ and Phe¹²³ also showed evidence for positive co-operativity for GSH and 1-chloro-2,4- dinitrobenzene respectively, strongly suggesting a role for these residues in manipulating subunit-subunit communication. In the present paper we report crystal structures of the wild-type enzyme, and two mutants, in complex with S-hexylglutathione. This study has identified an aromatic 'zipper' in the H-site contributing a network of aromatic π-π interactions. Several residues of the cluster directly interact with the hydrophobic substrate, whereas others indirectly maintain conformational stability of the dimeric structure through the C-terminal domain (domain II). The Y119E mutant structure shows major main-chain rearrangement of domain II. This reorganization is moderated through the 'zipper' that contributes to the H-site remodelling, thus illustrating a role in co-substrate binding modulation. The F123A structure shows molecular rearrangement of the H-site in one subunit, but not the other, explaining weakened hydrophobic substrate binding and kinetic co-operativity effects of Phe¹²³ mutations. The three crystal structures provide comprehensive evidence of the aromatic 'zipper' residues having an impact upon protein stability, catalysis and specificity.Consequently, 'zipper' residues appear to modulate and co-ordinate substrate processing through permissive flexing.