Residue 234 is a master switch of the alternative-substrate activity profile of human and rodent theta class glutathione transferase T1-1.
BACKGROUND
The Theta class glutathione transferase GST T1-1 is a ubiquitously occurring detoxication enzyme. The rat and mouse enzymes have high catalytic activities with numerous electrophilic compounds, but the homologous human GST T1-1 has comparatively low activity with the same substrates. A major structural determinant of substrate recognition is the H-site, which binds the electrophile in proximity to the nucleophilic sulfur of the second substrate glutathione. The H-site is formed by several segments of amino acid residues located in separate regions of the primary structure. The C-terminal helix of the protein serves as a lid over the active site, and contributes several residues to the H-site.


METHODS
Site-directed mutagenesis of the H-site in GST T1-1 was used to create the mouse Arg234Trp for comparison with the human Trp234Arg mutant and the wild-type rat, mouse, and human enzymes. The kinetic properties were investigated with an array of alternative electrophilic substrates to establish substrate selectivity profiles for the different GST T1-1 variants.


RESULTS
The characteristic activity profile of the rat and mouse enzymes is dependent on Arg in position 234, whereas the human enzyme features Trp. Reciprocal mutations of residue 234 between the rodent and human enzymes transform the substrate-selectivity profiles from one to the other.


CONCLUSIONS
H-site residue 234 has a key role in governing the activity and substrate selectivity profile of GST T1-1.


CONCLUSIONS
The functional divergence between human and rodent Theta class GST demonstrates that a single point mutation can enable or suppress enzyme activities with different substrates.
Shokeer
Abeer
A
Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, SE-75123 Uppsala, Sweden.
Mannervik
Bengt
B
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000378:metabolism; D017931:DNA Primers; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D051381:Rats; D013379:Substrate Specificity; D014364:Tryptophan / Q000378:metabolism
D017931:DNA Primers; D014364:Tryptophan; D001120:Arginine; D005982:Glutathione Transferase; C495790:glutathione transferase T1-1, human