Mechanism of Peroxide-Inactivation of the Sulphydryl Enzyme Glyceraldehyde-3-Phosphate Dehydrogenase

Abstract

The inactivation of glyceraldehyde-3-phosphate dehydrogenase by hydrogen peroxide has (a) I n the absence of catalysts been investigated. The reaction obeyed two rate equations : did not oxidize the "essential" sulphydryl groups of the enzyme to disulphide, but to sulphenic acid residues. Peroxide-inactivated glyceraldehyde-3-phosphate dehydrogenase could be fully reactivated and sulphydryl oxidation fully reversed if the enzyme was treated immediately with excess small thiol. The reversibility of inactivation decreased progressively if thiol treatment was delayed. 4 hours after peroxide treatment, neither enzyme inactivation nor sulphydryl oxidation could be reversed.Although SH-enzymes are normally inactivated by most oxidizing agents [l], relatively little information is available about the precise mechanisms involved. Thus, the powerful oxidizing agent, H,O,, inactivates most SH-enzymes [l] and in the case of papain, the inactivation is caused exclusively by SHoxidation [Z]. The nature of the products formed after H,O,-oxidation of protein thiols is not clear, although they are frequently assumed to be disulphides.The purpose of the present work was to study the mechanisms of inactivation of SH-enzymes by peroxides. For this purpose glyceraldehyde-3-phosphate dehydrogenase was chosen. This enzyme is inactivated by SH-reagents such as mercurials and alkylating activation of glyceraldehyde-3-phosphate dehydrogenase has been published. It would therefore seem that this enzyme is a very suitable one to use for a study of the mechanism of peroxide-inactivation of SH-enzymes. Moreover, our previous work indicated that whereas o-iodosobenzoate caused disulphide formation in the protein monothiol, yeast cytochrome c , peroxides did not [7]. As there is good evidence that o-iodosobenzoate causes intramolecular disulphide formation in glyceraldehyde-3-phosphate dehydrogenase [4] this enzyme is also very suitable for testing out the previous suggestion [7] that peroxide-oxidation of protein thiols rarely produces disulphides. MATERIALS AND METHODS MaterialsNAD, o-iodosobenzoate, glutathione, cysteine, 5,5'-dithiobis(2-nitrobenzoic acid), and horse heart cytochrome c, TypeII, were obtained from Sigma Chemical Co. (St. Louis, Mo.). Sodium borohydride

Glyceraldehyde-3-Phosphate Dehydrogenase Regulates Endothelin-1 Expression by a Novel, Redox-Sensitive Mechanism Involving mRNA Stability

“…The protein undergoes S nitrosylation by NO (22,40), NAD ϩ covalent linkage upon S nitrosylation (40), nitroalkylation by nitrated fatty acids (3), S glutathionylation by glutathione or even by NO (39), and extensive oxidation by peroxynitrite or H 2 O 2 (33,53). Among these modifications, S glutathionylation, the formation of mixed disulfides between glutathione and the cysteine of some proteins, has attracted great interest, as it has recently emerged as a potential mechanism for the regulation of signaling and metabolic pathways under both physiological and pathological conditions (13,36).…”

Yeast glyceraldehyde-3-phosphate dehydrogenase. Evidence that subunit cooperativity in catalysis can be controlled by the formation of a complex with phosphoglycerate kinase

“…For phosphoglycerate kinase immobilization, 20 mg CNBr/ml gel were used. Preparation of immobilized monomeric and dimeric forms of yeast glyceraldehyde-3-phosphate dehydrogenase was as previously described [5, 121. The inactivation of the matrix-bound monomeric species by H 2 0 2 was carried out following the method of Little and O'Brien [13]. Immobilized enzyme was washed with 50 vol.…”

The accessible cysteine residue of human transcortin

“…The occurrence of cysteic acid in native transcortin cannot be assumed since that degree of oxidation cannot be reversed by DTT; the question then arises as to the presence of sulphenic (-SOH) or sulphinic (-SOzH) acid which can be reduced to thiol [ 151. It has been suggested that sulphenic acids, unlike disulphides, may be reduced to thiols by sodium arsenite, a very mild reductant [ 16,171. The effects of arsenite and of another mild reductant, sodium ascorbate, were therefore studied.…”