Mechanism of inhibition of glyceraldehyde-3-phosphate dehydrogenase by nitric oxide and its regulation by the glutathione redox cycle.

摘要

Overproduction of the signaling molecule nitric oxide (NO) can be toxic, resulting in cell and tissue damage. However, the mechanisms by which NO mediates toxicity and the cellular defense mechanisms which protect cells from NO toxicity are not known. To investigate this problem, I chose to study the regulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by NO, since it had been shown in isolated enzyme systems that GAPDH was inhibited by NO and it was proposed that this inhibition contributes to NO-mediated toxicity. NO, generated by chemical NO donors or by the enzyme, NO synthase, inhibited GAPDH activity in both purified enzyme preparations and in intact cells. Incorporation of NO into GAPDH occurred with a stoichiometry which correlated with the degree of inhibition, suggesting that NO inhibits GAPDH by S-nitrosylation. The active site cysteine residue of GAPDH is the target for S-nitrosylation since NO-mediated GAPDH inhibition was blocked by substrate binding and modification of GAPDH by NO protected the enzyme from subsequent modification by iodoacetic acid. Although S-nitrosylation of GAPDH led to the covalent attachment of NAD