Aldose reductase is inactivated by physiological disulfides such as GSSG and cystine. To study the mechanism of disulfide-induced enzyme inactivation, we examined the rate and extent of enzyme inactivation using wild-type human aldose reductase and mutants containing cysteine-to-serine substitutions at positions 80 (C80S), 298 (C298S), and 303 (C303S). The wild-type, C80S, and C303S enzymes lost >80% activity following incubation with GSSG, whereas the C298S mutant was not affected. Loss of activity correlated with enzyme thiolation. The binary enzyme-NADP ؉ complex was less susceptible to enzyme thiolation than the apoenzyme. These results suggest that thiolation of human aldose reductase occurs predominantly at Cys-298. Energy minimization of a hypothetical enzyme complex modified by glutathione at Cys-298 revealed that the glycyl carboxylate of glutathione may participate in a charged interaction with His-110 in a manner strikingly similar to that involving the carboxylate group of the potent aldose reductase inhibitor Zopolrestat. In contrast to what was observed with GSSG and cystine, cystamine inactivated the wild-type enzyme as well as all three cysteine mutants. This suggests that cystamine-induced inactivation of aldose reductase does not involve modification of cysteines exclusively at position 80, 298, or 303.Aldose reductase (alditol:NADP oxidoreductase, EC 1.1.1.21) (ALR2) 1 catalyzes with a broad catalytic efficiency the NADPHdependent reduction of aldo-sugars and a variety of aromatic and aliphatic aldehydes to their corresponding alcohols. This enzyme is the first in a pathway that results in the transformation of glucose to fructose using sorbitol as a metabolic intermediate. This so-called "polyol pathway" is not a "high flux" metabolic route except in hyperglycemic conditions such as diabetes mellitus and galactosemia, where elevated concentrations of glucose and galactose, respectively, result in enhanced accumulation of their corresponding polyols in various tissues such as the eye lens (1, 2). Since these polyols do not readily permeate cell membranes, their intracellular accumulation is thought to create an osmotic imbalance, resulting ultimately in sugar cataract formation (3-5). Intensive effort has been mounted to identify inhibitors of aldose reductase for use as therapeutic tools against diabetic complications such as cataract and retinopathy (6 -9).Aldose reductase is subject to modifications leading to enzyme forms with an altered sensitivity to various inhibitors. Thus, the so-called "activated" ALR2 generated through apparently different processes such as isomerization (10), glycosylation (11), and thiol-dependent oxidation (12-14), besides displaying differences in substrate specificity, has a greatly reduced sensitivity to different aldose reductase inhibitors. Indeed, others recently reported the purification of human ALR2 with kinetic properties consistent with those described for an oxidized form of the enzyme (15). The potential involvement of cysteine residues in catalysis ...

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