Nitric oxide-induced covalent modification of glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase.

Publisher Summary This chapter describes the experiments that allow the identification of the target amino acid of the substrate. Both nitric oxide (NO) donors and active NO synthases (NOS) cause [ 32 P]NAD(H)-dependent automodification of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This cyclic guanosine monophospahte (cGMP)-independent action of NO involves a protein S -nitrosothiol intermediate, which is followed by the covalent modification of GAPDH by [ 32 P]NAD + or [ 32 P]NADH. The successful labeling of proteins with [ 32 P]NAD + suggests that an adenosine diphosphate (ADP)-ribosylation (like) mechanism operates. The covalent binding of the whole molecule NAD + is reported, as the ability of GAPDH to transfer the ADP-ribose moiety of NAD + to free cysteine, resulting in a true thioglycosidic linkage. Nitric oxide-induced modification of GAPDH is achieved in vitro and in intact cells, substantiating the role of NO group transfer chemistry and protein thiol modification in NO signaling. The function of GAPDH is not restricted to glycolysis, so it is intriguing to determine which other functions of GAPDH are affected by nitric oxide in this posttranslational protein modification mechanism.