Structural and functional properties of the 34-kDa fragment produced by the N-terminal chymotryptic cleavage of glutathione transferase P1-1.
Limited proteolysis of glutathione transferase P1-1 (GSTP1-1) by chymotrypsin generates a 34-kDa GSTP1-1 fragment (a dimer of the 17-kDa subunit composed by residues 48-207) containing the whole C-terminal domain and a part (about 15%) of the N-terminal domain (residues 48-76, i.e., the structural elements beta 3, beta 4, and alpha C). The structural and functional properties of this large fragment have been investigated by analyzing its binding properties to 2-p-toluidinylnaphthalene-6-sulfonate (TNS) extrinsic probe, the TNS displacement technique, and the molecular modeling approach. The results obtained indicated that the 34-kDa GSTP1-1 fragment maintains an hydrophobic pocket with the same structural properties of the corresponding GSTP1-1 hydrophobic binding site. In addition, the 34-kDa GSTP1-1 binds a number of hydrophobic compounds such as 1-chloro-2,4-dinitrobenzene, hemin, and bilirubin with the same affinity of the native enzyme. Being structurally and functionally autonomous, this fragment, mostly constituted by domain II, appears as an independent folding unit in the protein. Nevertheless, in the entire native protein, interdomain interactions occur and are responsible for the major solvent exposure of the H-site in the presence of glutathione.
Aceto
A
A
Istituto di Scienze Biochimiche, Università G. D'Annunzio, Chieti, Italy.
Sacchetta
P
P
Bucciarelli
T
T
Dragani
B
B
Angelucci
S
S
Radatti
G L
GL
Di Ilio
C
C
D001663:Bilirubin / Q000378:metabolism; D001667:Binding, Competitive; D002918:Chymotrypsin / Q000378:metabolism; D004137:Dinitrochlorobenzene / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005982:Glutathione Transferase / Q000302:isolation & purification / Q000378:metabolism*; D006427:Hemin / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D009282:Naphthalenesulfonates / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology
D004137:Dinitrochlorobenzene; D005456:Fluorescent Dyes; D008024:Ligands; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; D006427:Hemin; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D005982:Glutathione Transferase; D002918:Chymotrypsin; D001663:Bilirubin