The Inactivation of the Acyl Phosphatase Activity Catalyzed by the Sulfenic Acid Form of Glyceraldehyde 3-Phosphate Dehydrogenase by Dimedone and Olefins

Abstract

Abstract Treatment of the sulfenic acid form of glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) with a 2-fold molar excess of dimedone over the concentration of enzyme subunit completely inactivates the acyl phosphatase reaction catalyzed by the oxidized enzyme. The dehydrogenase activity catalyzed by the reduced form of the enzyme is not reactivated when the dimedone-inactivated enzyme is treated with dithiothreitol. When the acyl phosphatase is inactivated by [14C]dimedone ∼1 µg atom of 14C is incorporated per µeq of oxidized enzyme subunit which is not removed by gel filtration on Sephadex G-25. A 14C-labeled peptide from a tryptic digest of the acyl phosphatase which was inactivated with [14C]dimedone has been isolated in pure form and has been subjected to sequence analysis. This analysis has shown that [14C]dimedone forms a thioether derivative of Cys-149 by reacting with the sulfenic acid at the active site of the acyl phosphatase. The dehydrogenase activity is unaffected by [14C]dimedone when reduced glyceraldehyde 3-phosphate dehydrogenase is treated with [14C]dimedone and 14C radioactivity is not incorporated into the reduced protein. The acyl phosphatase activity catalyzed by the sulfenic acid form of glyceraldehyde 3-phosphate dehydrogenase isolated from pig muscle is also inactivated by 25 mm 3-cyclohexene-1-carboxylate at pH 5.1 in the presence of 0.1 m (NH4)2SO4 and other salts. Treatment of the inactivated acyl phosphatase with dithiothreitol does not reactivate the dehydrogenase reaction catalyzed by the reduced form of the enzyme. This indicates that treatment of the sulfenic acid form of the enzyme with the olefin leads to the covalent modification of Cys-149, the catalytically essentially sulfhydryl group for the dehydrogenase. Treatment of reduced glyceraldehyde 3-phosphate dehydrogenase with 3-cyclohexene-1-carboxylate under identical conditions has no effect on the dehydrogenase activity. The acyl phosphatase is also inactivated by 5.0 mm dihydropyran at pH 7.6 and by 30 mm tetrahydrophthalimide at pH 6.0. Treatment of the acyl phosphatase inactivated by these reagents with dithiothreitol does not reactivate the dehydrogenase activity catalyzed by the reduced form of the enzyme. Under identical conditions used to inactivate the acyl phosphatase, these two olefins do not inactivate the dehydrogenase activity catalyzed by native glyceraldehyde 3-phosphate dehydrogenase when added to it directly. Amino acid sequence analysis of the tryptic peptide which contains Cys-149 after the acyl phosphatase was inactivated with tetrahydrophthalimide suggests that an adduct between the sulfenic acid at the active site of the acyl phosphatase and tetrahydrophthalimide is formed during the inactivation.