Chemical modification of GSH transferase P1-1 confirms the presence of Arg-13, Lys-44 and one carboxylate group in the GSH-binding domain of the active site.
GSH transferase P1-1 (GSTP1-1) was modified with group-specific reagents. Kinetic experiments demonstrated that inactivation of GSTP1-1 occurred upon reaction of one arginine residue per subunit with diacetyl, one lysine residue per subunit with 2,4,6-trinitrobenzene sulphonate, or one carboxylate group per subunit with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. All three inactivation reactions were inhibited by compounds known to bind at the GSH site of the enzyme but were unaffected by the electrophile 1-chloro-2,4-dinitrobenzene. N-terminal sequence analysis showed that Arg-13 was modified by diacetyl and that this modification was inhibited by GSH. Arg-11 was not modified. The lysine residue modified by 2,4,6-trinitrobenzene sulphonate and protected by S-octylglutathione was identified as Lys-44 by sequencing of tryptic peptides. The findings are in agreement with the involvement of Arg-13 and Lys-44 in binding of GSH, as determined from the crystal structure [Reinemer, Dirr, Ladenstein, Huber, Lo Bello, Frederici and Parker (1992) J. Mol. Biol. 227, 214-226]. The present data also implicate a single carboxylate in GSH binding, consistent with the involvement of Asp-98 of subunit B determined from the crystallographic study. The GSH-binding determinants of GSTP1-1 are compared using sequence similarity with those of GSTs of Alpha, Mu and Theta classes.
Xia
C
C
Department of Biochemistry and Molecular Biology, University College London, U.K.
Meyer
D J
DJ
Chen
H
H
Reinemer
P
P
Huber
R
R
Ketterer
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000032:analysis*; D001665:Binding Sites; D002264:Carboxylic Acids / Q000032:analysis*; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000032:analysis*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D002264:Carboxylic Acids; D010446:Peptide Fragments; D001120:Arginine; D005982:Glutathione Transferase; D005978:Glutathione; D008239:Lysine