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P. Nagy et al. / Free Radical Biology & Medicine 49 (2010) 792–799
occurred because chloride and analogues of tyrosine have different
rate-determining steps in their oxidation by the enzyme. The catalytic
cycle of MPO starts by the reaction of the native enzyme with H2O2 to
form Compound I. Compound I has an oxy-ferryl heme center and a
porphyrin radical and it can engage in both one- and two-electron
reactions. One-electron donors (such as Tyr) reduce Compound I to
Compound II, which is the dominant state of the enzyme during
catalytic turnover in most systems. Compound II also has an oxy-ferryl
heme center and is reduced via one-electron reactions in a rate-
determining step to regenerate the native enzyme [41]. In vivo
numerous one-electron reductants will reduce Compound I to
Compound II. Turnover of Compound II, however, is much more
restricted so that it is likely that enkephalins can compete with other
substrates at this stage of the enzymatic cycle for oxidation [42].
Interestingly, in contrast to HRP, the rate of oxidation of the various
Tyr-containing peptides by MPO was relatively independent of their
size. This suggests that they had similar access to the active site of
Compound II. The one exception was the small peptide YG, which was a
relatively poor substrate. A possible explanation for its tardy oxidation is
that the negative charge on the carboxylate group of this substrate is
optimally located to prevent access of the dipeptide to the active site. In
support of this proposal, it has been reported that eliminating the
negative charge on cysteine by removing its carboxylate group or
esterification dramatically increases the rate constants for reaction of
aliphatic thiols with both Compound I and Compound II [30].
alcohol) and sulfoxide formation on the Tyr and Met residues,
respectively. In these reactions the Tyr residue is modified by the
addition of a hydroperoxide (or alcohol) group, and cyclization through
conjugate addition of the amine nitrogen destroys its aromatic character
(Scheme 1, Reaction 3b). Oxidation of the Met residue to its sulfoxide
does not compromise the analgesic activity of Met-Enk [49]. However,
because the Tyr residue and its terminal amine group are pivotal for the
activity of both Met-Enk and Leu-Enk [7,49], these oxidative Tyr
modifications could dampen their opiate effect. Moreover, hydroper-
oxides are highly reactive species that could have adverse biological
effects. If enkephalins are released locally by neutrophils together with a
large amount of O•2−, such inactivation may be possible and warrants
further investigation.
Acknowledgments
This work was supported by the Marsden Fund and the Health
Research Council of New Zealand and used equipment provided by the
National Research Centre for Growth and Development.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
Previous studies have shown that neutrophils can oxidize Met-Enk
[19,20]. We have extended these studies and characterized new
products. We have shown that neutrophils use MPO, H2O2, and O•2− to
oxidize the enkephalins via O•2− addition to their Tyr radicals. Although
most of the H2O2 released by neutrophils is converted to HOCl, Met-Enk
and Leu-Enk competed to some extent for MPO and were directly
oxidized to their Tyr radicals at physiological chloride concentrations.
Leu-Enk was converted to its hydroperoxide as the major oxidation
product (via Reactions 1–3). The Met residue of Met-Enk reacts with a
substantial amount of the generated HOCl to give MetS=O. However,
HOCl is a promiscuous oxidant that is likely to be quenched by proteins
and other two-electron-scavenging small molecules (such as GSH,
urate, or ascorbate) under physiological conditions [43]. We demon-
strated this effect by showing that, in the presence of added methionine
or albumin, Met enkephalin oxidation was largely inhibited. Met (or
albumin) had no effect on the yield of Tyr-OH–Met=O, which is
generated by a radical-mediated pathway (Reactions 1–5). Although
radical scavengers such as ascorbate have the potential to scavenge Tyr
radicals [44,45], the physiological relevance of Tyr radical formation is
demonstrated by the fact that dityrosine formation is used as a clinical
biomarker for oxidative stress [35,46,47]. Because Tyr radicals react
preferentially with superoxide over dimerization [10,13] neutrophils
might oxidize enkephalins via superoxide-mediated radical mechanism
in vivo. We have shown that this mechanism operates even when most
of the Met-Enk was converted to its sulfoxide (by neutrophil-generated
HOCl), to give in this case a hydroperoxide–sulfoxide derivative.
A novel finding from this work is that neutrophils can oxidize
methionine residues independent of hypohalous acids by using the
peroxidation activity of myeloperoxidase in conjunction with O•2−. Such
reactions may not be restricted to enkephalins and could occur through
protein-bound Tyr radicals reacting with O•2− to form hydroperoxides
(as in [15]) and then oxidizing neighboring methionine residues.
Previous data suggest that neutrophils are not only contributors to
pain in inflamed tissue, but could also be a major source of enkephalins
that protect against neuropathic pain [6,17]. Enkephalins may also have
a priming effect on neutrophil responses at physiological concentrations
[18,48]. Based on our results, enkephalin oxidation by neutrophils could
also occur at sites of inflammation. In fact when neutrophils are
activated under inflammatory conditions, locally high concentrations of
neutrophil-generated opioid peptides and oxidants could favor their
biochemical interactions. Oxidation results in hydroperoxide (or
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