1e:T51e,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-54.1f:T54b,The mercapturic acid pathway is a major route for the biotransformation of xenobiotic and endobiotic electrophilic compounds and their metabolites. Mercapturic acids (N-acetyl-l-cysteine S-conjugates) are formed by the sequential action of the glutathione transferases, γ-glutamyltransferases, dipeptidases, and cysteine S-conjugate N-acetyltransferase to yield glutathione S-conjugates, l-cysteinylglycine S-conjugates, l-cysteine S-conjugates, and mercapturic acids; these metabolites constitute a "mercapturomic" profile. Aminoacylases catalyze the hydrolysis of mercapturic acids to form cysteine S-conjugates. Several renal transport systems facilitate the urinary elimination of mercapturic acids; urinary mercapturic acids may serve as biomarkers for exposure to chemicals. Although mercapturic acid formation and elimination is a detoxication reaction, l-cysteine S-conjugates may undergo bioactivation by cysteine S-conjugate β-lyase. Moreover, some l-cysteine S-conjugates, particularly l-cysteinyl-leukotrienes, exert significant pathophysiological effects. Finally, some enzymes of the mercapturic acid pathway are described as the so-called "moonlighting proteins," catalytic proteins that exert multiple biochemical or biophysical functions apart from catalysis.20:T6e6,

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