Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin.
Glutathione S-transferase pi has been shown to reactivate 1-cysteine peroxiredoxin (1-Cys Prx) by formation of a complex [L.A. Ralat, Y. Manevich, A.B. Fisher, R.F. Colman, Biochemistry 45 (2006) 360-372]. A model of the complex was proposed based on the crystal structures of the two enzymes. We have now characterized the complex of GST pi/1-Cys Prx by determining the M(w) of the complex, by measuring the catalytic activity of the GST pi monomer, and by identifying the interaction sites between GST pi and 1-Cys Prx. The M(w) of the purified GST pi/1-Cys Prx complex is 50,200 at pH 8.0 in the presence of 2.5mM glutathione, as measured by light scattering, providing direct evidence that the active complex is a heterodimer composed of equimolar amounts of the two proteins. In the presence of 4M KBr, GST pi is dissociated to monomer and retains catalytic activity, but the K(m) value for GSH is increased substantially. To identify the peptides of GST pi that interact with 1-Cys Prx, GST pi was digested with V8 protease and the peptides were purified. The binding by 1-Cys Prx of each of four pure GST pi peptides (residues 41-85, 115-124, 131-163, and 164-197) was investigated by protein fluorescence titration. An apparent stoichiometry of 1mol/subunit 1-Cys Prx was measured for each peptide and the formation of the heterodimer is decreased when these peptides are included in the incubation mixture. These results support our proposed model of the heterodimer.
Ralat
Luis A
LA
Department of Chemistry and Biochemistry, Brown Laboratory, University of Delaware, Academy Street, Newark, DE 19716, USA.
Misquitta
Stephanie A
SA
Manevich
Yefim
Y
Fisher
Aron B
AB
Colman
Roberta F
RF
D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D019281:Dimerization; D051549:Glutathione S-Transferase pi / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D054464:Peroxiredoxins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D054464:Peroxiredoxins; D051549:Glutathione S-Transferase pi