Conformational stability of Cys45‐alkylated and hydrogen peroxide‐oxidised glutathione S‐transferase

FEBS Letters

Sluis-Cremer, Nicolas¹  Dirr, Heini¹ 

[1] Protein Structure-Function Research Programme, Department of Biochemistry, University of the Witwatersrand, Johannesburg 2050, South Africa

关键词: Conformational stability;    Unfolding;    Glutathione S-transferase;    Oxidative stress;    Hydrogen peroxide;    pGSTP1-1;    porcine glutathione S-transferase class pi with two 1-type subunits;    IAEDANS;    N-iodocetyl-N′-(5-sulpho-1-naphthyl)ethylenediamine;    SEC-HPLC;    size-exclusion-high performance liquid chromatography;    DTNB;    5;    5′-dithiobis(2-nitrobenzoic acid);    GSH;    reduced glutathione;    DTT;    dithiothreitol;   

DOI  :  10.1016/0014-5793(95)00826-U

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 gluthathione-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 (ΔG(H₂O) = 11.6 kcal/mol) when compared with unmodified and AEDANS-alkylated enzyme (ΔG(H₂O) = 22.5 kcal/mol).

【 授权许可】

Unknown