Inactivation of NADP(+)-dependent isocitrate dehydrogenase by nitric oxide.
Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. NO donors such as S-nitrosothiols, diethylamine NONOate, spermine NONOate, and 3-morpholinosydnomine N-ethylcarbamide (SIN-1)/superoxide dismutase inactivated ICDH in a dose- and time-dependent manner. The inhibition of ICDH by S-nitrosothiol was partially reversed by thiol, such as dithiothreitol or 2-mercaptoethanol. Loss of enzyme activity was associated with the depletion of the cysteine-reactive 5,5'-dithiobis-(2-nitrobenzoate) and the loss of fluorescent probe N,N'-dimethyl-N(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethyleneamine accessible thiol groups. Using electrospray ionization mass spectrometry with tryptic digestion of protein, we found that nitric oxide forms S-nitrosothiol adducts on Cys305 and Cys387. These results indicate that S-nitrosylation of cysteine residues on ICDH is a mechanism involving the inactivation of ICDH by NO. The structural alterations of modified enzyme were indicated by the changes in protease susceptibility and intrinsic tryptophan fluorescence. When U937 cells were incubated with 200 microM SNAP for 1 h, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed. Furthermore, stimulation with lipopolysaccharide significantly decreased intracellular ICDH activity in RAW 264.7 cells, and this effect was blocked by NO synthase inhibitor N(omega)-methyl-L-arginine. This result indicates that ICDH was also inactivated by endogenous NO. The NO-mediated damage to ICDH may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.
D000818:Animals; D002460:Cell Line; D002942:Circular Dichroism; D010450:Endopeptidases / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology; D051379:Mice; D009249:NADP / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D020298:U937 Cells
D020030:Nitric Oxide Donors; D009569:Nitric Oxide; D009249:NADP; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+); D010450:Endopeptidases; D005978:Glutathione