Site-directed mutagenesis of human glutathione transferase P1-1. Mutation of Cys-47 induces a positive cooperativity in glutathione transferase P1-1.
Glutathione transferase P1-1 (EC 2.5.1.18) is a dimeric enzyme composed of identical subunits each containing one binding site for GSH and a second for the co-substrate e.g. 1-chloro-2,4-dinitrobenzene. Steady-state kinetics are strictly hyperbolic toward both these substrates. Replacement of Cys-47 with alanine or serine decreases the affinity for GSH and triggers a positive kinetic cooperativity with respect to the substrate. Hill coefficients were 1.31 and 1.43 for the C47A and C47S mutants. C47A/C101S and C47S/C101S double mutants display lower affinity for GSH and higher Hill coefficients (1.57 and 1.56, respectively) when compared with C47A and C47S single mutants. Conversely, replacement of Cys-101 with alanine or serine does not yield any cooperativity and any marked change of kinetic parameters. Fluorometric experiments gave sigmoidal isothermic GSH binding curves for all the Cys-47 mutants, with Hill coefficients similar to that obtained by the kinetic approach. These data, together with the activation experiments performed in the presence of S-hexylglutathione, suggest that the substitution of Cys-47 yields a dimeric low-affinity enzyme which may be revealed by the lack of a peculiar electrostatic bond between the thiolate form of Cys-47 and the protonated amino group of Lys-54.
Ricci
G
G
Department of Biology, University of Rome Torr Vergata, Italy.
Lo Bello
M
M
Caccurri
A M
AM
Pastore
A
A
Nuccetelli
M
M
Parker
M W
MW
Federici
G
G
D003545:Cysteine / Q000378:metabolism*; D004789:Enzyme Activation; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013696:Temperature
D007527:Isoenzymes; D005982:Glutathione Transferase; D005978:Glutathione; D003545:Cysteine