Conformational stability of Cys45-alkylated and hydrogen peroxide-oxidised glutathione S-transferase.
A highly reactive cysteine residue in class pi glutathione S-transferases enhances their susceptibility to chemical alkylation and oxidative stress. Alkylation of the reactive Cys45 in the porcine class pi enzyme (pGSTP1-1) with either N-iodoacetyl-N'-(5-sulpho-1-naphthyl)ethylenediamine or iodoacetamide results in a loss of enzyme activity and glutathione-binding function. Similarly, oxidation of pGSTP1-1 with hydrogen peroxide (H2O2) also results in a loss of catalytic and glutathione-binding function, but these effects are reversed by the addition of 5 mM glutathione or dithiothreitol. Analysis by SDS-PAGE of the H2O2-oxidised enzyme indicates oxidation-induced formation of disulphide bonds involving Cys45. Equilibrium-unfolding studies with guanidinium chloride indicate that the unfolding of Cys45-alkylated and H2O2-oxidised pGSTP1-1 can be described by a two-state model in which the predominant thermodynamically stable species are the folded dimer and unfolded monomer. Unfolding transition curves suggest that the introduction of a large and bulky AEDANS at Cys45 does not affect the unfolding pathway for pGSTP1-1. H2O2-oxidised pGSTP1-1, on the other hand, appears to follow a different unfolding pathway. This appears not to be a result of the introduction of disulphide bonds since the reduction of these bonds in the oxidised protein with dithiothreitol does not affect the unfolding transition. Furthermore, the conformational stability of the oxidised protein is significantly diminished (delta G(H2O) = 11.6 kcal/mol) when compared with unmodified and AEDANS-alkylated enzyme (delta G(H2O) = 22.5 kcal/mol).
Sluis-Cremer
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N
Department of Biochemistry, University of the Witwatersrand, Johannesburg, South Africa.
Dirr
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H
D000478:Alkylation; D000818:Animals; D003545:Cysteine / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D004795:Enzyme Stability; D005978:Glutathione / Q000378:metabolism / Q000494:pharmacology; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000737:chemistry*; D009282:Naphthalenesulfonates / Q000737:chemistry; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013552:Swine; D013816:Thermodynamics
D006146:Guanidines; D009282:Naphthalenesulfonates; C006421:1,5-I-AEDANS; D006861:Hydrogen Peroxide; D005982:Glutathione Transferase; D005978:Glutathione; D019791:Guanidine; D003545:Cysteine; D004229:Dithiothreitol