MODIFICATION OF ALDOSE REDUCTASE BY S-NITROSOGLUTATHIONE

Kinetic and structural changes in recombinant human aldose reductase ( AR) due to modification by S-nitrosoglutathione (GSNO) were investigat ed. Incubation of the enzyme with 10-50 mu M GSNO led to a time-and co ncentration-dependent inactivation of the enzyme, with a second-order rate constant of 0.087 +/- 0.009 M-1 min(-1). However, upon exhaustive modification, 30-40% of the enzyme activity was retained. The non-ina ctivated enzyme displayed a 2-3-fold change in K-m for NADPH and K-m f or DL-glyceraldehyde, whereas the K-m for the lipid peroxidation produ ct, 4-hydroxy-2-trans nonenal (HNE), was comparable to that of the unt reated enzyme. The residual activity of the enzyme after GSNO treatmen t was less sensitive to inhibition by the active site inhibitor sorbin il or to activation by sulfate. Significantly higher catalytic activit y was retained when the enzyme was modified in the presence of NADPH, suggesting relatively low reactivity of the E-NADPH complex with GSNO, The modification site was identified using site-directed mutants in w hich each of the solvent-exposed cysteines of the enzyme was replaced individually by serine. The mutant C298S was insensitive to GSNO, wher eas the sensitivity of the mutants C303S and C80S was comparable to th at of the wild-type enzyme. Electrospray ionization mass spectroscopy of the GSNO-modified enzyme revealed a major modified species (70% of the protein) with a molecular mass that was 306 Da higher than that of the untreated enzyme, which is consistent with the addition of a sing le glutathione molecule to the enzyme, The remaining 30% of the protei n displayed a molecular mass that was not significantly different from that of the native enzyme, No nitrosated forms of the enzyme were obs erved. These results suggest that inactivation of AR by GSNO is due to the selective formation of a single mixed disulfide between glutathio ne and Cys-298 located at the NADP-(H)-binding site of the enzyme.