Modification of aldose reductase (AR) by the nitrosothiolsS-nitroso-N-acetyl penicillamine (SNAP) and N-(β-glucopyranosyl)-N2-acetyl-S-nitrosopenicillamide (glyco-SNAP) resulted in a 3–7-fold increase in its kcat and a 25–40-fold increase in its Km for glyceraldehyde. In comparison with the native protein, the modified enzyme was less sensitive to inhibition by sorbinil and was not activated by SO2−4 anions. The active-site residue, Cys-298, was identified as the main site of modification, because the site-directed mutant in which Cys-298 was replaced by serine was insensitive to glyco-SNAP. The extent of modification was not affected by Pi or O2, indicating that it was not due to spontaneous release of nitric oxide (NO) by the nitrosothiols. Electrospray ionization MS revealed that the modification reaction proceeds via the formation of an N-hydroxysulphenamide-like adduct between glyco-SNAP and AR. In time, the adduct dissociates into either nitrosated AR (AR-NO) or a mixed disulphide between AR and glyco-N-acetylpenicillamine (AR-S-S-X). Removal of the mixed-disulphide form of the protein by lectin-column chromatography enriched the preparation in the high-Km–high-kcat form of the enzyme, suggesting that the kinetic changes are due to the formation of AR-NO, and that the AR-S-S-X form of the enzyme is catalytically inactive. Modification of AR by the non-thiol NO donor diethylamine NONOate (DEANO) increased enzyme activity and resulted in the formation of AR-NO. However, no adducts between AR and DEANO were formed. These results show that nitrosothiols cause multiple structural and functional changes in AR. Our observations also suggest the general possibility that transnitrosation reactions can generate both nitrosated and thiolated products, leading to non-unique changes in protein structure and function.
Structural and kinetic modifications of aldose reductase by S-nitrosothiols
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Sanjay SRIVASTAVA, Bharat L. DIXIT, Kota V. RAMANA, Animesh CHANDRA, Deepak CHANDRA, Albert ZACARIAS, J. Mark PETRASH, Aruni BHATNAGAR, Satish K. SRIVASTAVA; Structural and kinetic modifications of aldose reductase by S-nitrosothiols. Biochem J 15 August 2001; 358 (1): 111–118. doi: https://doi.org/10.1042/bj3580111
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