hGSTZ1-1 (human glutathione transferase Zeta 1-1) catalyses a range of glutathione-dependent reactions and plays an important role in the metabolism of tyrosine via its maleylacetoacetate isomerase activity. The crystal structure and sequence alignment of hGSTZ1 with other GSTs (glutathione transferases) focused attention on three highly conserved residues (Ser-14, Ser-15, Cys-16) as candidates for an important role in catalysis. Progress in the investigation of these residues has been limited by the absence of a convenient assay for kinetic analysis. In this study we have developed a new spectrophotometric assay with a novel substrate [(±)-2-bromo-3-(4-nitrophenyl)propionic acid]. The assay has been used to rapidly assess the potential catalytic role of several residues in the active site. Despite its less favourable orientation in the crystal structure, Ser-14 was the only residue found to be essential for catalysis. It is proposed that a conformational change may favourably reposition the hydroxyl of Ser-14 during the catalytic cycle. The Cys16→Ala (Cys-16 mutated to Ala) mutation caused a dramatic increase in the Km for glutathione, indicating that Cys-16 plays an important role in the binding and orientation of glutathione in the active site. Previous structural studies implicated Arg-175 in the orientation of α-halo acid substrates in the active site of hGSTZ1-1. Mutation of Arg-175 to Lys or Ala resulted in a significant lowering of the kcat in the Ala-175 variant. This result is consistent with the proposal that the charged side chain of Arg-175 forms a salt bridge with the carboxylate of the α-halo acid substrates.
Abbreviations used: BCPP, 2-bromo-3-(4-chlorophenyl)propanoic acid; BNPP, (±)-2-bromo-3-(4-nitrophenyl)propanoic acid; CFA, chlorofluoroacetic acid; Cys16→Ala, Cys-16 mutated to Ala etc.; DCA, dichloroacetic acid; FA, fumarylacetone; FAA, fumarylacetoacetate; GS-CPP, 2-(glutathion-S-yl)-3-(4-chlorophenyl)propanoic acid; GS-NPP, 2-(glutathion-S-yl)-3-(4-nitrophenyl)propanoic acid; GST, glutathione transferase; hGSTZ1-1, human GST Zeta 1-1; MA, maleylacetone; SA, salicylic acid; TCHQ, tetrachlorohydroquinone.