Conformational states of human placental glutathione transferase as probed by limited proteolysis.
Limited proteolysis experiments have been carried out on human placental glutathione transferase in its different forms. The reduced enzyme, as well as the oxidized form and that inactivated with cystamine were all sensitive to 10% (w/w) trypsin, under nondenaturing conditions. The proteolytic cleavage was accompanied by a concomitant loss of enzymatic activity. On the contrary, the presence of glutathione or glutathione conjugates strongly protected the reduced enzyme against inactivation and from the proteolytic attack. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and peptide sequence analysis showed that only the peptide bond between Lys 44 and Ala 45 was cleaved. Since Lys 44 has been demonstrated to be involved in the glutathione binding, it is suggested that the region surrounding this amino acid residue (alpha B helix) could be more exposed to the solvent, in the absence of glutathione. Crystallographic data also indicated that this region is flexible, supporting the idea that it may be involved in the observed conformational change upon glutathione binding.
Lo Bello
M
M
Department of Biology, University of Rome Tor Vergata, Italy.
Pastore
A
A
Petruzzelli
R
R
Parker
M W
MW
Wilce
M C
MC
Federici
G
G
Ricci
G
G
D000409:Alanine; D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005982:Glutathione Transferase / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D014357:Trypsin / Q000378:metabolism
D046911:Macromolecular Substances; D010446:Peptide Fragments; D005982:Glutathione Transferase; D014357:Trypsin; D008239:Lysine; D000409:Alanine