Characterization of cysteine residues of glutathione S-transferase P: evidence for steric hindrance of substrate binding by a bulky adduct to cysteine 47.
Glutathione S-transferase P (GST-P) lost the enzymatic activity by 7-fluoro-4-sulfamoyl-2, 1, 3-benzodiazole (ABD-F), a thiol-group chemical modifier, but did not by methylmethanethiol-sulfonate. Both ABD-F and methylmethanethiolsulfonate reacted with Cys47 and Cys101. These two cysteine residues were site-directedly mutated with serine residues. Only the Cys101Ser lost the enzymatic activity by the treatment of ABD-F. On carbon 13 NMR experiments, a NMR signal of S-[13C]CH3 adduct to Cys47 did not show any change by the addition of S-hexylglutathione. These facts revealed that Cys47 did not locate at the active site, and a bulky adduct to Cys47 hindered the binding of substrates to the active site.
Nishihira
J
J
Second Department of Biochemistry, School of Medicine, Hokkaido University, Sapporo, Japan.
Ishibashi
T
T
Sakai
M
M
Nishi
S
S
Kumazaki
T
T
Hatanaka
Y
Y
Tsuda
S
S
Hikichi
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002247:Carbon Isotopes; D003001:Cloning, Molecular; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010069:Oxadiazoles / Q000494:pharmacology*; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism
D002247:Carbon Isotopes; D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D010069:Oxadiazoles; D010446:Peptide Fragments; D011994:Recombinant Proteins; C014674:methyl methanethiosulfonate; C046364:4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole; D008741:Methyl Methanesulfonate; D005982:Glutathione Transferase; D003545:Cysteine