Human glutathione transferase A4-4 crystal structures and mutagenesis reveal the basis of high catalytic efficiency with toxic lipid peroxidation products

The oxidation of Lipids and cell membranes generates cytotoxic compounds implicated in the etiology of aging, cancer, atherosclerosis, neurodegenerative diseases, and other illnesses. Glutathione transferase (GST) A4-4 is a key component in the defense against the products of this oxidative stress because, unlike other Alpha class GSTs, GST A4-4 shows high catalytic activity with Lipid peroxidation products such as 4-hydroxynon-2-enal (HNE). The crystal structure of human apo GST A4-4 unexpectedly possesses an ordered C-terminal a-helix, despite the absence of any ligand. The structure of human GST A4-4 in complex with the inhibitor S-(2-iodobenzyl) glutathione reveals key features of the electrophilic substrate-binding pocket which confer specificity toward HNE. Three structural modules form the binding site for electrophilic substrates and thereby govern substrate selectivity: the pl-cll loop, the end of the alpha 4 helix, and the C-terminal alpha 9 helix. A few residue changes in GST A4-4 result in a9 taking over a predominant role in ligand specificity from the N-terminal loop region important for GST Al-l. Thus, the C-terminal helix a9 in GST A4-4 provides pre-existing ligand complementarity rather than acting as a flexible cap as observed in other GST structures. Hydrophobic residues in the alpha 9 helix, differing from those in the closely related GST Al-l, delineate a hydrophobic specificity canyon for the binding of lipid peroxidation products. The role of residue Tyr212 as a key catalytic residue, suggested by the crystal structure of the inhibitor complex, is confirmed by mutagenesis results. Tyr212 is positioned to interact with the aldehyde group of the substrate and polarize it for reaction. Tyr212 also coopts part of the binding cleft ordinarily formed by the N-terminal substrate recognition region in the homologous enzyme GST Al-l to reveal an evolutionary swapping of function between different recognition elements. A structural model of catalysis is presented based on these results. (C) 1999 Academic Press.