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Conformational stability of Cys 45 ‐alkylated and hydrogen peroxide‐oxidised glutathione S ‐transferase
Author(s) -
Sluis-Cremer Nicolas,
Dirr Heini
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)00826-u
Subject(s) - chemistry , iodoacetamide , dithiothreitol , glutathione , guanidinium chloride , cysteine , hydrogen peroxide , dimer , alkylation , stereochemistry , hydrogen bond , medicinal chemistry , enzyme , organic chemistry , catalysis , molecule
A highly reactive cysteine residue in class pi glutathione S ‐transferases enhances their susceptibility to chemical alkylation and oxidative stress. Alkylation of the reactive Cys 45 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 (H 2 O 2 ) also results in a loss of catalytic and gluthathione‐binding function, but these effects are reversed by the addition of 5 mM glutathione or dithiothreitol. Analysis by SDS‐PAGE of the H 2 O 2 ‐oxidised enzyme indicates oxidation‐induced formation of disulphide bonds involving Cys 45 . Equilibrium‐unfolding studies with guanidinium chloride indicate that the unfolding of Cys 45 ‐alkylated and H 2 O 2 ‐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 Cys 45 does not affect the unfolding pathway for pGSTP1‐1. H 2 O 2 ‐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 ( Δ G(H 2 O) = 11.6 kcal/mol) when compared with unmodified and AEDANS‐alkylated enzyme ( Δ G(H 2 O) = 22.5 kcal/mol).