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Low-molecular-weight S-nitrosothiols (RSNOs) are well known for their capability to transnitrosate cysteine residues of enzymes thereby altering their catalytic activity. It is unknown, however, whether N-nitrosomelatonin (NOMela) which is highly effective in transnitrosating low-molecular-weight thiols (RSHs) can also alter protein function. In the present study, we report on such a capability with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a target enzyme. Reaction of NOMela with GAPDH resulted in an increase of RSNOs at the expense of RSHs. Somewhat surprisingly, NOMela was about 10-fold more effective than S-nitrosocysteine in inhibiting GAPDH. Vitamin C and glutathione increased the NOMela-dependent inhibition of the enzyme by accelerating the intermediacy of nitroxyl which is also highly effective in nitrosating RSHs. The occurrence of this intermediate during the NOMela–vitamin C reaction was verified by using Mn(III)-tetrakis(1-methyl-4-pyridyl)porphorin pentachloride as nitroxyl scavenger. The NOMela-dependent inactivation of GAPDH was so effective that this reaction can be used to quantify NOMela with high sensitivity.