Characterization of a nitric-oxide-catalysed ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase.
Auto-ADP-ribosylation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GraPDH) has recently been demonstrated to be dramatically stimulated in the presence of nitric oxide. In order to obtain insight into the sequence of events leading to ADP-ribosylation of GraPDH, we studied the target amino acid, the nucleotide cofactor requirement, pH dependency and the stoichiometry of the reaction. Basal as well as stimulated ADP-ribose transfer is inhibited by the SH-group alkylating reagent, N-ethylmaleimide. Furthermore, the radiolabel of auto-[32P]ADP-ribosylated GraPDH is removed by treatment with HgCl2, suggesting an ADP-ribose-cysteine bond. Several indirect and direct mechanistic considerations point to NAD+ as the only cofactor for the ADP-ribosylation reaction, excluding the possibility of a reaction sequence involving a NAD-glycohydrolase(s) followed by nonenzymatic ADP-ribose transfer to GraPDH. Optimal ADP-ribosylations were carried out at alkaline pH values using 10 microM free NAD+ as the sole nucleotide cofactor. Bovine serum albumin with an S-nitrosylated SH group can serve as a model of ADP-ribose transfer from NAD+ and suggests that the nitric-oxide-modified SH group (S-nitrosylated SH group) is a prerequisite for the reaction.
Dimmeler
S
S
University of Konstanz, Faculty of Biology, Federal Republic of Germany.
Brüne
B
B
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D002384:Catalysis; D002478:Cells, Cultured; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009132:Muscles / Q000201:enzymology; D009569:Nitric Oxide / Q000378:metabolism*; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013379:Substrate Specificity
D000246:Adenosine Diphosphate Ribose; D012710:Serum Albumin, Bovine; D009569:Nitric Oxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases