The C-terminal region, Arg201-Gln209, of glutathione S-transferase P contributes to stability of the active-site conformation.
The C-terminal region of rat glutathione S-transferase P (GST-P) was deleted by either carboxypeptidase (CPase) A and B or site-specific truncation to evaluate the role of the region in the catalytic mechanism. The C-terminal sequence from the 201st to 209th amino-acid residues is Arg-Pro-Ile-Asn-Gly-Asn-Gly-Lys-Gln. When seven of the C-terminal amino-acid residues from the C-terminus were removed by the CPases, the catalytic activity decreased in parallel with the amino-acid removal, amounting to less than 5% of that of the wild-type GST-P. On the other hand, a decrease of the catalytic activity was observed in a different manner when the C-terminal sequence was site-specifically truncated. The VmaxGSH/KmGSH values of the mutants withthree (GSTd207-209), four (GSTd206-209) and seven (GSTd203-209) C-terminal amino-acid residues deleted, were comparable or similar to that of the wild-type GST-P, whereas those of five (GSTd205-209), six (GSTd204-209), and eight (GSTd202-209) amino-acid residue-truncated mutants decreased to 43%, 40%, and 19% of that of the wild-type GST-P, respectively. Similar results were obtained as for VmaxCDNB/KmCDNB. The nine amino-acid residue-truncated mutant showed no catalytic activity. Heat treatment at 50 degrees C for 5 min had little effect on the catalytic activities of the wild-type GST-P and GSTd204-209, whereas those of GSTd207-209, GSTd206-209, GSTd203-209 and GSTd202-209 decreased to 22%, 27%, 18% and 10%, respectively, compared to the catalytic activity of the non-treated enzymes. Considering these results, it is concluded that the C-terminal region, Arg201-Gln209, has an important role in stabilizing the active-site conformation.
Nishihira
J
J
Central Research Institute, Hokkaido University, Sapporo, Japan.
Hibiya
Y
Y
Sakai
M
M
Nishi
S
S
Kumazaki
T
T
Ohki
S
S
Sakamoto
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001665:Binding Sites; D002268:Carboxypeptidases; D002384:Catalysis; D004795:Enzyme Stability; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics; D005998:Glycine; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry*; D011487:Protein Conformation; D051381:Rats
D010446:Peptide Fragments; D001120:Arginine; D005982:Glutathione Transferase; D002268:Carboxypeptidases; D005998:Glycine