Inactivation of key metabolic enzymes by mixed-function oxidation reactions: possible implication in protein turnover and ageing.
Several mixed-function oxidation systems catalyze the inactivation of Escherichia coli glutamine synthetase. Inactivation involves modification of a single histidine residue in each enzyme subunit and makes the enzyme susceptible to proteolytic degradation. We show here that 10 key enzymes in metabolism are inactivated by a bacterial NADH oxidase and by an oxidase system comprised of NADPH, cytochrome P-450 reductase, and cytochrome P-450 isozyme 2 from rabbit liver microsomes. Most of the inactivatable enzymes require a divalent cation for activity and all but one (enolase) possess a nucleotide binding site. Glutamine synthetase, pyruvate kinase, and phosphoglycerate kinase are protected from inactivation by their substrates; substrate protection of other enzymes was not tested. We propose that inactivation involves mixed-function oxidization system-catalyzed synthesis of H(2)O(2) and reduction of Fe(III) to Fe(II) followed by oxidation of enzyme-bound Fe(II) by H(2)O(2) to generate oxygen radicals that attack a histidine (or other oxidizable amino acid) at the metal binding site of the enzyme. This is supported by the following: (i) most of the inactivation reactions are inhibited by EDTA and by catalase, (ii) both mixed-function oxidation systems reduce Fe(III), and (iii) H(2)O(2) together with Fe(II) catalyzes nonenzymic inactivation of glutamine synthetase. In view of the fact that inactivation of glutamine synthetase makes it susceptible to proteolytic degradation, it is possible that mixed-function oxidation system-catalyzed inactivation of enzymes is a regulatory step in enzyme turn-over. In addition, the implication of oxidative inactivation reactions in ageing is suggested by the fact that many of the enzymes inactivated by mixed-function oxidation systems are known to accumulate as inactive forms during ageing.
D000375:Aging; D000818:Animals; D002374:Catalase / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*; D011489:Protein Denaturation; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits
D009097:Multienzyme Complexes; D011506:Proteins; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D002374:Catalase; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases