Mutations of Gly to Ala in human glutathione transferase P1-1 affect helix 2 (G-site) and induce positive cooperativity in the binding of glutathione.
Previous kinetic studies on human glutathione transferase P1-1 have indicated that the motions of an irregular alpha-helix (helix 2) lining the glutathione (GSH) binding site are viscosity dependent and may modulate the affinity of GSH binding. The effect of single amino acid residue substitutions (Gly to Ala) in this region is investigated here by site-directed mutagenesis. Three mutants (Gly41Ala, Gly50Ala and Gly41Ala/Gly50Ala) were overexpressed in Escherichia coli, purified, and characterized by kinetic, structural, and spectroscopic studies. All these mutant enzymes show kcat values similar to that of the wild-type enzyme, while the [S]0.5 for GSH increases about eight-fold in the Gly41Ala mutant and more than 100-fold in the Gly41Ala/Gly50Ala double mutant. This change in affinity towards GSH is accompanied by an induced positive cooperativity as reflected by Hill coefficients of 1.4 (Gly41Ala) and 1.7 (Gly41Ala/Gly50Ala) upon substrate binding. Taken together, these data suggest that the region around helix 2 is markedly altered leading to the observed intersubunit communication. Molecular modeling of the Gly41Ala/Gly50Ala mutant and of the inactive oxidized form of the native enzyme provides a structural explanation of our results.
Lo Bello
M
M
Department of Biology, University of Rome "Tor Vergata", Italy. Lobello@uniroma2.it
Nuccetelli
M
M
Chiessi
E
E
Lahm
A
A
Mazzetti
A P
AP
Battistoni
A
A
Caccuri
A M
AM
Oakley
A J
AJ
Parker
M W
MW
Tramontano
A
A
Federici
G
G
Ricci
G
G
D000409:Alanine; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005978:Glutathione / Q000378:metabolism*; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005998:Glycine; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry
D007527:Isoenzymes; D011994:Recombinant Proteins; D014364:Tryptophan; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase; D005978:Glutathione; D003545:Cysteine; D000409:Alanine; D005998:Glycine