The conversion of glyceraldehyde-3-phosphate dehydrogenase to an acylphosphatase by trinitroglycerin and inactivation of this activity by azide and ascorbate.
Trinitroglycerin oxidizes the essential sulfhydryl group, Cys-149, of pig muscle glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate : NAD+ oxidoreductase(phosphorylating) EC 1.2.1.12) TO A SLUFENIC ACID, NOT TO A DISULFIDE. This conclusion is based on the observation that the inactivation of the dehydrogenase activity of the enzyme by the organic nitrate induces the acylphosphatase activity which is catalyzed by the sulfenic acid form of the enzyme. Inorganic nitrite is released during this process which is stoichiometric with the degree of inactivation of the dehydrogenase. The acylphosphatase activity induced by trinitroglycerin, unlike the dehydrogenase activity, is sensitive to CN-. Treatment of the enzyme oxidized with trinitroglycerin with 14-CN- leads to the incorporation of protein-bound 14-CN-, which is stoichiometric with the degree of inactivation of the dehydrogenase. Treatment of the sulfenic acid form of glyceraldehyde-3-phosphate dehydrogenase at pH 5.3 with a 10-fold molar excess of azide over the concentration of enzyme subunit completely inactivates the acylphosphatase reaction catalyzed by the oxidized enzyme. Concomitantly, the dehydrogenase activity catalyzed by the sulfhydryl form of the enzyme reappears which indicates that excess azide reduces the sulfenic acid which is required for the acylphosphatase. Treatment of the oxidized enzyme with a stoichiometric amount of azide at pH 5.3 stimulates the acylphosphatase activity and does not lead to the reappearance of dehydrogenase activity. When the sulfenic acid form of the enzyme is incubated with 20 mM L-ascorbate at pH 5.3, the acylphosphatase activity is completely inactivated and the dehydrogenase activity catalyzed by the reduced form of the enzyme is recovered. Thus, L-ascorbate also reduces the protein sulfenic acid which is required for the acylphosphatase activity.
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D001386:Azides / Q000494:pharmacology*; D001565:Benzoates / Q000494:pharmacology; D001665:Binding Sites; D003486:Cyanides / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D005996:Nitroglycerin / Q000494:pharmacology*; D010084:Oxidation-Reduction; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D013434:Sulfenic Acids; D013552:Swine
D001386:Azides; D001565:Benzoates; D003486:Cyanides; D013434:Sulfenic Acids; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010744:Phosphoric Monoester Hydrolases; D005996:Nitroglycerin; D001205:Ascorbic Acid; D004229:Dithiothreitol