Characterization of the glutathione binding site of aldose reductase

Despite extensive investigations, the physiological role of the polyol path way enzyme-aldose reductase (AR) remains obscure. While the enzyme reduces glucose in vivo and in vitro, kinetic and structural studies indicate ineff icient carbohydrate binding to the active site of the enzyme. The active si te is lined by hydrophobic residues and appears more compatible with the bi nding of medium- to long-chain aliphatic aldehydes or hydrophobic aromatic aldehydes. In addition, our recent studies show that glutathione (GS) conju gates are also reduced efficiently by the enzyme. For instance, the GS conj ugate of acrolein is reduced with a catalytic efficiency 1000-fold higher t han the parent aldehyde, indicating specific recognition of glutathione by the active site residues of AR. An increase in the catalytic efficiency upo n glutathiolation was also observed with trans-2-nonenal, trans-2-hexenal a nd trans, trans-2,4-decadienal, establishing that enhancement of catalytic efficiency was specifically due to the glutathione backbone and not specifi c to the aldehyde. Structure-activity relationships with substitution or de letion of amino acids of GSH indicated specific interactions of the active site with gamma -Glu1 and Cys of GSH. Molecular modeling revealed that the glutathione-propanal conjugate could bind in two distinct orientations. In orientation I, gamma -Glu1 of the of AR is compatible with binding and redu ction of various hydrophobic aldehydes, generated during the peroxidation o f biological membrane lipids under oxidative stress. However, due to their electrophilicity, lipid-derived aldehydes may not be accessible to AR; inst ead, conjugates of these aldehydes with cellular thiols may be better subst rates [7,8,14]. Thus, in thiol-competent cells, the formyl thiols, rather t han free aldehydes, are more likely to be the endogenous substrates of AR. With this view, the present study was designed to model the possible orient ations in which the GSH-aldehyde can efficiently and specifically bind to t he active site of AR, and can identify the amino acid residues that are pre ferentially involved in the interaction of GS-aldehyde conjugates to the AR enzyme active site.