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during oxidative stress or if an appropriate electron acceptor is missing, the
reduced forms of the abovementioned enzymes are able to react with O 2 to
form ROS (29). Often in the following redox cycle, Fe 2+ is involved and highly
reactive species are formed ( OH) that are able to oxidize amino acids directly
at the metal-binding site of the enzyme, therefore inhibiting the enzymatic
activity and often rendering the inactivated enzyme to preferential degrada-
tion (255).
Therefore, virus NAD(P)H oxidases and reductases, including CYP450s,
might lead to the oxidation of numerous proteins. In addition to that, xanthine
oxidase, horseradish peroxidase, and glucose oxidase are also able to do so
(256). GS of E. coli was demonstrated to be inactivated by MCO systems,
including nonenzymatic systems comprised of either ascorbate, O 2 and Fe 3+ ,
or Fe 2+ and O 2 ; and enzymatic systems such as rabbit liver microsomal CYP450
reductase together with CYP450 isozyme 2 [P-450(LM2)], microbial NADH
oxidase, putidaredoxin reductase together with putidaredoxin with or without
CYP450, xanthine oxidase together with ferredoxin or putidaredoxin, and
partially purified enzymes (NADH oxidase) from Klebsiella aerogenes or E.
coli . Inactivation of GS by all enzyme systems was shown to be dependent on
O 2 and NAD(P)H (except in the case of xanthine oxidase, for which hypoxan-
thine serves as an electron donor). All systems are stimulated by Fe 3+ and
inhibited by catalase, Mn(II), EDTA, o-phenanthroline, and histidine (257).
Inactivation of GS by either the ascorbate system or the NADH oxidase
system is associated with the modification of a single histidine residue in each
GS subunit.
Fucci et al. (253) found that 10 enzymes (alcohol dehydrogenase, Asparto-
kinase III, creatine kinase, enolase, GS, glyceraldehyde-3-phosphate dehydro-
genase, lactate dehydrogenase, phosphoglycerate kinase (PGK), and pyruvate
kinase) were inactivated by both MFO systems NADH oxidase and CYP450.
All of the inactivation reactions required NAD(P)H and were inhibited by
catalase. It is noteworthy that most of the susceptible enzymes are either
synthetases, kinases, or NAD(P)-dependent dehydrogenases; that is, they
possess a nucleotide-binding site at the catalytic center. In addition, they
require divalent metal cations for activity and contain a histidine residue at or
near the catalytic site. The inactivation of enzymes by MFO systems could
occur by the mechanism demonstrated in Figure 1.17 (reactions 39-42).
A central role of hydrogen peroxide was often demonstrated by the block-
ing effect of catalase. The involvement of iron was demonstrated by the inhibit-
ing action of chelators, whereas a role of Fe 2+ is underlined by the fact that
Fe 2+ and O 2 also inactivates the GS (257). In contrast, Fe 3+ has no such effect
regardless of the presence of oxygen. The fact that the inactivation of GS is
associated with the loss of just 1 of 16 histidine residues in each subunit is a
clear indication of a site-specific event. In addition to that, also the inactivation
of other enzymes, as PGK or mammalian SOD, is due to modification of a
single histidine. A site-specific binding of Fe 2+ that attacks a histidine in the
catalytic center or nearby was suggested (253).
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