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

Abstract

The inactivation of glyceraldehyde-3-phosphate dehydrogenase by hydrogen peroxide has been investigated. The reaction obeyed two rate equations: (a) In the absence of catalysts (b) In the presence of catalysts These Kinetics are identical to those previously observed during glutathione oxidation by peroxides. Enzyme inactivation was shown to be strictly related to sulphydryl group modification and this modification can account for the inactivation. For total inactivation H2O2, like iodoacetate, modified about 3.5 sulphydryl groups in the enzyme. o-Iodosobenzoate, however, modified approximately 8.5 sulphydryl groups and it was concluded that H2O2, unlike o-iodosobenzoate, 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.