Inactivation of key metabolic enzymes by mixed-function oxidation reactions: Possible implication in protein turnover and ageing

Abstract

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 P450 reductase, and cytochrome P450 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 H202 and reduction of Fe(EI) to Fe(ll) followed by oxidation of enzyme-bound Fe(II) by H202 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(S), and (iii) H202 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 turnover. 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. coli (1-3). Inactivation of glutamine synthetase by all enzyme systems is dependent on 02 and NAD(P)H (except in the case of xanthine oxidase, for which hypoxanthine serves as an electron donor). All systems are stimulated by Fe(III) and inhibited by catalase, Mn(II), EDTA, o-phenanthroline, and histidine (1-3). Inactivation of glutamine synthetase by either the ascorbate system or the NADH oxidase system (other systems not tested) is associated with the modification of a single histidine residue in each glutamine synthetase subunit (4). This inactivation renders the glutamine synthetase more susceptible to proteolytic degradation by subtilisin (5) as well as by proteases in E. coli (1, 2). Thus, the inactivation reaction appears to "mark" glutamine synthetase for proteolytic digestion.We report here that, in addition to the glutamine synthetase from E. coli, the glutamine synthetase from rat liver and a number of other key metabolic-enzymes, many of which possess ahistidine or another easily oxidizable residue at the catalytic site, are inactivated in vitro by the NADH oxidase system and also by the rabbit liver microsomal cytochrome P-450 system. During ageing, inactive or less active forms of severa...

Different selectivities of oxidants during oxidation of methionine residues in the α‐1‐proteinase inhibitor

“…In both enzymes, a site-specific attack of the reactive oxygen species at or near the active center is assumed to be responsible for loss of enzyme activity. Inactivation of Lglutamine synthetase could be correlated to the loss of one His residue out of 16 His residues by mixed-function oxidation with ascorbate, Fe(III) and 02 [41,42].…”

Biochemistry and pathology of radical-mediated protein oxidation

“…They inactivate a wide range of enzymes (e.g. [39][40][41]) and are suggested to be important for both proteolytic turnover [39] and the accumulation of proteins during aging [42]. The inactivation of one of the most studied enzymes, glutamine synthetase, is influenced by its adenylation state [39], which also regulates the enzyme and some multi-enzyme cascades [43,44].…”

Carbonylation of glycolytic proteins is a key response to drug-induced oxidative stress and apoptosis

“…Carbonylation of proteins has been reported to severely impair their function, 21,22 so as a number of glycolytic enzymes were shown to be carbonylated upon VP16 treatment of HL60 cells, it was important to measure the rate of glycolysis in VP16-treated cells. (As a control, we also assayed the percentage of glucose metabolised by the pentose phosphate pathways; as no enzymes from this pathway were seen to be carbonylated in VP16-treated cells.)…”

“…14 Protein carbonylation can modify the rate of protein degradation, with some proteins showing increased and others reduced turnover. 8,[15][16][17][18][19][20] Perhaps most significantly, carbonylation of a protein can also reduce its activity, 21 for example glutamine synthase exposed to metal catalysed oxidation has reduced enzyme activity. 22 Consequently, cells that have large numbers of protein carbonyls may be expected to have impaired function.…”