Clarification of the role of key active site residues of glutathione transferase zeta/maleylacetoacetate isomerase by a new spectrophotometric technique.
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 K(m) 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 alpha-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 alpha-halo acid substrates.
Board
Philip G
PG
Molecular Genetics Group, John Curtin School of Medical Research, Australian National University, Canberra ACT 2601, Australia. philip.board@anu.edu.au
Taylor
Matthew C
MC
Coggan
Marjorie
M
Parker
Michael W
MW
Lantum
Hoffman B
HB
Anders
M W
MW
D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D005978:Glutathione / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D013053:Spectrophotometry / Q000379:methods; D019745:cis-trans-Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology
D000596:Amino Acids; C407705:GSTZ1 protein, human; D005982:Glutathione Transferase; D019745:cis-trans-Isomerases; C021531:maleylacetoacetate isomerase; D005978:Glutathione