TY - JOUR
T1 - Site-directed mutagenesis of human glutathione transferase P1-1
T2 - Mutation of Cys-47 induces a positive cooperativity in glutathione transferase P1-1
AU - Ricci, Giorgio
AU - Lo Bello, Mario
AU - Caccuri, Anna Maria
AU - Pastore, Anna
AU - Nuccetelli, Marzia
AU - Parker, Michael W.
AU - Federici, G.
PY - 1995/1/20
Y1 - 1995/1/20
N2 - 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 this substrate. Hill coefficients were 1.31 and 1.43 for the C47A and C47S mutants. C47A/C101A 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 can be converted to a higher affinity state upon binding of GSH on one subunit. These findings indicate a structural communication between subunits 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.
AB - 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 this substrate. Hill coefficients were 1.31 and 1.43 for the C47A and C47S mutants. C47A/C101A 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 can be converted to a higher affinity state upon binding of GSH on one subunit. These findings indicate a structural communication between subunits 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.
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M3 - Article
C2 - 7836386
AN - SCOPUS:0028809193
VL - 270
SP - 1243
EP - 1248
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 3
ER -