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 the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by nitric oxide (NO) and modified enzyme activity was examined. There are 16 free thiols, including 4 active site thiols, in a tetramer of GAPDH molecule. NO donors, sodium nitroprusside (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 activity was also inhibited to 5-10% of control activity. In the presence of 30 mM dithiothreitol (DTT), these effects were completely blocked. Since similar 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 molecule. 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 with 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 reached 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 arsenite, which is known to reduce sulfenic acid to thiol, almost completely restored both thiol groups and enzyme activity. These findings suggest that nitric 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 one type of thiol modification by NO, occurred when GAPDH was treated with SNAP but not SNP. Analysis of thiol modification showed that SNAP preferentially nitrosylated the active site thiols, the nitrosylation of which fully disappeared by DTT posttreatment. It seems that SNAP nitrosylates the active site thiols of GAPDH to prevent these thiols from oxidizing to sulfenic acid.
Department of Veterinary Pharmacology, College of Agriculture, Osaka Prefecture University, Sakai, Japan.
T
T
Ishii
O
O
Sunami
H
H
Nakajima
H
H
Nishio
T
T
Takeuchi
F
F
Hata
D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004794:Enzyme Repression / Q000187:drug effects; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D009243:NAD / Q000378:metabolism; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D010084:Oxidation-Reduction; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D013434:Sulfenic Acids / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D020030:Nitric Oxide Donors; C110881:S-nitro-N-acetylpenicillamine; D013434:Sulfenic Acids; D013438:Sulfhydryl Compounds; D009243:NAD; D009599:Nitroprusside; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010396:Penicillamine; D003545:Cysteine; D004229:Dithiothreitol