Critical role of sulfenic acid formation of thiols in the inactivation of glyceraldehyde-3-phosphate dehydrogenase by nitric oxide

The relationship between possible modifications of thr thiol groups of glyc eraldehyde-3-phosphate dehydrogenase (GAPDH) by nitric oxide (NO) and modif ied enzyme activity was examined. There are 16 free thiols, including 4 act ive site thiols, in a tetramer of GAPDH molecule. NO donors, sodium nitropr usside (SNP), and S-nitroso-N-acetyl-DL-penicillamine (SNAP) decreased the number of free thiols with a concomitant inhibition of GAPDH activity in a concentration- and time-dependent manner. After treatment for 30 min, free thiols were maximally decreased to 8-10 per GAPDH tetramer and enzyme activ ity was also inhibited to 5-10% of control activity. In the presence of 30 mM dithiothreitol (DTT), these effects were completely blocked. Since simil ar results were obtained in the case of hydrogen peroxide (H2O2) treatment, which is known to oxidize the thiols, these effects of nitric oxide donors were probably due to modification of thiol groups present in a GAPDH molec ule. On the other hand, DTT posttreatment after the treatment of GAPDH with SNP, SNAP, or H2O2 did not completely restore the modified thiols and the inhibited enzyme activity. DTT posttreatment after the 30 min-treatment wit h these agents restored free thiols to 14 in all treatments. In the case of SNAP treatment, all 4 active sites were restored and enzyme activity reach ed more than 80% of the control activity, but in two other cases one active site remained modified and enzyme activity was restored to about only 20%. Therefore, all 4 free thiols in the active site seem to be very important for full enzyme activity. DTT posttreatment in the presence of sodium arsen ite, which is known to reduce sulfenic acid to thiol, almost completely res tored both thiol groups and enzyme activity. These findings suggest that ni tric oxide inhibits GAPDH activity by modifications of the thiols which are essential for this activity, and that the modification includes formation of sulfenic acid, which is not restored by DTT. S-nitrosylation, which is o ne type of thiol modification by NO, occurred when GAPDH was treated with S NAP hut not SNP. Analysis of thiol modification showed that SNAP preferenti ally nitrosylated the active site thiols, the nitrosylation of which fully disappeared by DTT posttreatment. It seems that SNAP nitrosylates the activ e site thiols of GAPDH to prevent these thiols from oxidizing to sulfenic a cid. BIOCHEM PHARMACOL 58;1:133-143, 1999. (C) 1999 Elsevier Science Inc.