Modification of the active site of aldose reductase by nitric oxide

Aldose reductase, the first enzyme of the polyol pathway of glucose metabolism, has been implicated in the etiology of secondary diabetic complications. However, its in vivo mechanisms of regulation arc not known. In this study we show that nitric oxide (NO) donors, sodium nitroprusside (SNP), S-nitroso-N-acetylpencillamine (SNAP), and 3-morpholinosydonemine (SIN-1) cause a time- and concentration-dependent loss of catalytic activity of wild type recombinant aldose reductase. The enzyme activity was partially recovered by dithiothreitol and sodium horohydride. NADPH and NADP protected the enzyme from NO-induced inactivation. Since in a complete system with both substrates, NADPH and glyceraldehyde, the enzyme was not protected against inactivation by NO-donors, it is concluded that the enzyme is modified during the catalytic cycle. In contrast to the wild type enzyme and mutant forms C303S and C80S the mutant C298S was unaffected by NO donors, indicating selective modification of the enzyme at cys-298. These studies suggest that by specific modification of the active site thiol (cys-298), NO may be an endogenous regulator of the polyol pathway and cellular metabolism associated with aldose reductase.