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INTRODUCTION |
Creatine kinases (CK)1
are key enzymes in energy metabolism by catalyzing the reversible
transphosphorylation of creatine by ATP to yield phosphocreatine
(PCr) and ADP. The CK system is present in cells with high and
fluctuating energy demands, such as skeletal and cardiac muscle, brain,
and other neuronal tissues, where a mitochondrial and a cytosolic
isoform are coexpressed (for reviews, see Refs. 1-4). In striated
muscle, cytosolic muscle-type CK is expressed together with
sarcomeric mitochondrial CK (sMtCK), whereas in brain and many other
tissues, the cytosolic brain-type CK is found together with ubiquitous
mitochondrial CK (uMtCK). In contrast to the exclusively dimeric
cytosolic CK, the mitochondrial isoenzymes (MtCK) can form cube-like
octameric molecules (5, 6). MtCK is bound to the outer leaflet of the
inner mitochondrial membrane and located in the intermembrane space,
where it cross-links both mitochondrial membranes, forming contact
sites (7, 8), as well as along the cristae membranes (9). In contact
sites, MtCK forms functional complexes with the adenylate translocator of the inner and porin of the outer mitochondrial membrane, as evidenced, for example, by creatine stimulation of mitochondrial respiration (10) or plasmon resonance spectroscopy (11). In mainly
oxidative tissue, this microcompartment facilitates the vectorial
transport of high-energy phosphate from the sites of energy production
in the mitochondrial matrix to cytosolic sites of energy consumption.
PCr is generated by MtCK using mitochondrial ATP (for review see Ref.
12) and transported into the cytosol, where ATP pools are replenished
by the reverse reaction of cytosolic CK, which is partially located in
the vicinity of cellular ATPases. The octameric structure of MtCK was
shown to be crucial for this transport function of the
"PCr-shuttle," because reduced octamer stability of N-terminally
mutated MtCK (13) transfected into rat neonatal cardiomyocytes resulted
in decreased creatine-stimulated mitochondrial respiration (14).
Mitochondrial sMtCK and uMtCK isoenzymes are highly homologous, sharing
about 85% of identical amino acids, a further 5% of conservative
replacements (15) and the same overall fold in their molecular
structure (6, 16). The active site in particular is highly similar in
all CKs and even in the other related guanidino kinases (17, 18).
Despite this homology, human MtCK isoenzymes differ in many properties,
including substrate affinity, substrate binding synergism, membrane
interaction, and octamer stability (19, 20).
MtCK is known to be very susceptible to oxygen radical damage (21).
Nitric oxide inhibits creatine kinase activity in solution as well as
in adult rat ventricular myocytes. This inhibition can be reversed by
the addition of dithiothreitol (22). In contrast, the inhibition of CK
by superoxide anions (O2
) (23, 24) and
peroxynitrite (ONOO
) is irreversible (25, 26).
Inactivation of cytosolic and mitochondrial CK would interrupt the
"PCr-shuttle" and would have severe effects on the energetics of
work performance and Ca2+ homeostasis in muscle similar to
those seen in double-knockout mice lacking both the cytosolic and
mitochondrial CK (27). ONOO
is the product of the nearly
diffusion-controlled reaction between NO and
O2
, both of these compounds being produced by
mitochondria (28, 29). Because MtCK is located near the production site
of ONOO
, a powerful oxidant, MtCK represents a prime
target for ONOO
-induced damage (30). ONOO
is known to play a role in cells under conditions of oxidative stress,
as is the case in heart disease and after ischemia/reperfusion (31,
32), as well as in certain neurodegenerative diseases such as
amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's
disease (for review, see Ref. 33). All these pathologies are known to
show a compromised CK system (34-38). Brain-type CK was identified as
a specific target of protein oxidation in Alzheimer's disease (39).
Several reactions of ONOO
with amino acid side chains are
described in the literature, including nitration of tyrosine (40, 41)
and tryptophan (40, 42), as well as oxidation of methionine (43, 44)
and cysteine (45). Formation of nitrotyrosine serves as a marker for
ONOO
-induced damage in tissues (for review, see Ref.
46).
In the present study, we addressed the question of whether
ONOO
administered in vitro has differential
effects on enzymatic activity and octamer stability of the two human
MtCK isoenzymes. Divergent properties of human sMtCK and uMtCK have
already been reported (19, 20, 47) and may be relevant to the different
pathologies in heart and neuronal tissue, where an impaired CK system
is involved. For a molecular description of ONOO
-induced
MtCK damage, mass spectrometry was applied to identify those amino acid
residues that are modified by ONOO
.
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EXPERIMENTAL PROCEDURES |
Chemicals--
All enzymes and coenzymes were obtained from
Roche Molecular Biochemicals (Rotkreuz, Switzerland),
-cyano-4-hydroxycinnamic acid was from Aldrich (Buchs, Switzerland),
and 2,5-dihydroxybenzoic acid was from Fluka (Buchs, Switzerland).
Further chemicals were purchased from standard suppliers and were of
the highest purity commercially available. Human MtCK isoforms (19) and
chicken sMtCK were heterologously expressed according to a protocol
described previously (48). ONOO
was a generous gift from
Prof. W. Koppenol (Laboratorium für Anorganische Chemie, ETH
Zürich, Switzerland).
ONOO
Administration--
The concentration of
ONOO
stock solution, synthesized from the reaction of
gaseous NO with solid potassium superoxide (49), was determined
photometrically at 302 nm in 10 mM NaOH
(
mm = 1.67) before use. ONOO
,
in a volume of 20 ml of 10 mM NaOH, was added to 200 µl
of protein solution containing 0.5 mg/ml MtCK in 100 mM
Na3PO4, 150 mM NaCl, pH 7.2, to
reach final ONOO
concentrations between 1 and 1000 µM. To the control, 20 µl of NaOH but no
ONOO
was added, which led to a pH-shift in the protein
solution by <0.05 pH-units.
Determination of Octamer Content--
The oligomeric state of
MtCK was determined by gel permeation chromatography on a Superose 12 column (Pharmacia) at room temperature in running buffer (50 mM Na3PO4, 150 mM NaCl,
2 mM
-mercaptoethanol, 0.2 mM EDTA,
pH 7.0) at a flow rate of 0.8 ml/min. Peak areas in the elution profile
were quantified by graphical integration using Biocad Sprint HPLC
software (Applied Biosystems, Foster City, CA).
CK Activity Determination--
The specific CK activities were
assayed photometrically in the reverse reaction, using the
glucose-6-phosphate-dehydrogenase/hexokinase-coupled enzyme assay as
described previously (19) at room temperature (22 °C).
Tryptic Digest of MtCK--
ONOO
-treated MtCK, as
well as native MtCK, were dialyzed against 50 mM
NH4HCO3 and 20 mM methylamine, pH
8.0, before adding urea to a final concentration of 1 M.
The proteins were digested for 16 h at 37 °C by adding trypsin
in an enzyme-to-protein ratio of 1:100. The reaction was stopped by
freezing the samples.
Mass Spectrometric Analysis--
Mass spectra were performed on
a Voyager-DE Elite MALDI-TOF (Applied Biosystems) in positive
ion reflector mode, using either
-cyano-4-hydroxycinnamic acid or
2,5-dihydroxybenzoic acid in a 2:1 mixture of acetonitril, 0.1%
trifluoroacetetic acid in H2O. For sample preparation, 0.5 µl of the digested protein solution was mixed without further
preparation with 0.5 µl of the matrix solution directly on target and
the solution was air-dried. Postsource decay spectra were recorded on
an Axima-CFR MALDI-TOF (Kratos/Shimadzu, Manchester, UK) with a curved
field reflection.
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RESULTS |
Inactivation of MtCK by Peroxynitrite--
The sensitivity of
human sMtCK and uMtCK toward ONOO
was first compared with
respect to enzymatic activity. Activity of both human MtCK isoenzymes,
as determined immediately after ONOO
administration,
decreased in a dose-dependent fashion at already very low ONOO
concentrations (Fig.
1). The IC50 was determined
by graphical estimation to be about 8 µM. This
inactivation was not reversed by subsequent addition of 20 mM dithiothreitol, which reduces disulfides and reverses
S-nitrosylation (22).

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Fig. 1.
Dose response of peroxynitrite-induced
inactivation of human MtCK isoenzymes. Enzymatic activity of human
uMtCK (filled squares), human sMtCK (open
squares), and chicken sMtCK C278G mutant (filled
triangles) is expressed in percentage of control activity to allow
direct comparison of inactivation patterns. Note that in absolute
terms, mutant C278G has a residual CK activity of ~3 to 5% compared
with wild-type enzyme. IC50 values were determined by
graphical estimation. Data are given as mean value of four independent
experiments; error bars are within the boundaries of
symbols. Note that both human MtCK isoenzymes show very similar
inactivation kinetics by peroxynitrite, whereas the C278G active site
cysteine mutant is resistant toward this reagent.
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Target of Peroxynitrite in the MtCK Active Site--
Cysteine 278 is a highly conserved residue in the active site of CK, which is known
to be very reactive and essential for substrate binding and synergism
in all guanidino kinases (18, 50). To test whether MtCK inactivation
was caused by oxidation of this residue, a glycine replacement mutant
(C278G) of chicken sMtCK (50) was treated with ONOO
in
the same manner. Chicken sMtCK is almost identical to human sMtCK (97%
sequence identity) and shows exactly the same dose-response curve for
ONOO
-inactivation. The residual enzymatic activity of the
C278G mutant, which is about 3-5% compared with wild type depending
on the assay conditions, was completely unaffected by
ONOO
(Fig. 1).
Octamer Dissociation of MtCK by Peroxynitrite--
In parallel
experiments, sMtCK and uMtCK solutions with an initial octamer content
of more than 80% were treated with increasing concentrations of
ONOO
and kept overnight at room temperature to reach a
stable octamer-dimer equilibrium. Subsequent gel permeation
chromatography was performed to determine the oligomeric state of the
isoenzymes as a function of peroxynitrite concentration (Fig.
2). Although ONOO
administration destabilized both MtCK isoenzymes and favored their
dissociation into dimers, uMtCK was significantly more stable than
sMtCK. Whereas for the latter, a concentration of only about 240 µM ONOO
was sufficient to dissociate 50%
of the initial octamer concentration into dimers (C50
value), this concentration was about 790 µM for the
brain-type uMtCK (Fig. 2).

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Fig. 2.
Dose response of peroxynitrite-induced
octamer dissociation of human MtCK isoenzymes. Octamer content of
human uMtCK (filled squares) and sMtCK (open
squares) as measured by integration of elution peaks from high
performance liquid chromatography gel filtration chromatography.
C50 values were determined by graphical estimation. Data
are given as mean ± S.E. of three independent experiments. Note
that in terms of octamer destabilization by ONOO , human
sMtCK is much more susceptible than human uMtCK.
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MALDI-TOF Analysis of Peroxynitrite-modified MtCK Residues--
To
further identify the MtCK residues modified by ONOO
,
human uMtCK, human sMtCK, and chicken sMtCK were treated with
increasing concentrations of ONOO
as before and, after
exhaustive digestion by trypsin, subjected to mass spectrometric
analysis by MALDI-TOF. Chemically modified peptides of
ONOO
-treated MtCK were identified by their higher
molecular mass compared with peptides derived from untreated control
protein. Fig. 3 summarizes all peptides
that could be unambiguously identified by mass spectrometry in the
native, as well as the ONOO
-treated human isoenzymes
(highlighted in gray in Fig. 3A), aligned with
the amino acid sequences. Some theoretically predicted fragments that
could harbor putative ONOO
-sensitive residues (Fig.
3A, bold capital letters in white
areas) were not covered by the mass spectrometric analysis. However, we
did not pursue their identification, because they are neither located
in the active site or at the dimer/dimer interface nor have any other
known functions for the enzyme been attributed to them. From the MtCK
peptides recovered by MALDI-TOF, only three were modified by
ONOO
treatment (Fig. 3A, highlighted in
black with residues marked by arrowheads): (i)
peptide Gly-263-Arg-271 containing two tryptophans, Trp-264 and
Trp-268, (ii) peptide Leu-272-Arg-287 containing the active site
Cys-278, and (iii) peptide Ser-340-Arg-360, including the C-terminal
Cys-358. The cysteine residues in the latter two fragments were
modified by single and double oxidation. Thus, combined with results
described above for the C278G mutant, modification of Cys-278 is
sufficient to explain the loss of enzymatic MtCK activity upon
peroxynitrite treatment.

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Fig. 3.
Peptides identified by MALDI-TOF in untreated
and peroxynitrite-treated human sMtCK and uMtCK. A,
amino acid sequence alignment of human uMtCK and human sMtCK (15).
Peptides recovered from both native unmodified and
ONOO -treated protein are highlighted in gray;
those peptides found to be modified by ONOO are
highlighted in black. Residues potentially susceptible to
ONOO -induced damage are bold, and the modified
residues identified by MALDI are marked by arrowheads.
B, molecular structure of a dimer of human uMtCK (16),
showing the spatial localization of ONOO -modified
residues (prepared with Rasmol 2.6 (61)). The dimer/dimer-interface
that would connect to neighboring dimers in the octameric structure is
situated at the lower, convex side of the banana-shaped dimer. Trp-264
(red), Met-267 (blue) and to minor degree Trp-268
(red) are located at the dimer/dimer interface, Cys-278
(yellow) identifies the active site, and Cys-358
(yellow) is near the octamer face that binds to
mitochondrial membranes. Sequences covered by MALDI-TOF analysis are in
white. Note also the far-reaching N termini of uMtCK that
are mainly responsible for higher octamer stability of uMtCK by
providing additional polar interactions between the dimers (16,
20).
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The modified peptide Gly-263-Arg-271 is located at the dimer/dimer
interfaces of the octameric MtCK isoenzymes, as revealed by the x-ray
structures of sMtCK (6) and uMtCK (16). All mass shifts occurring with
this peptide (Fig. 4) can be explained by
reactions of ONOO
with methionine and tryptophan. The
difference of 16 Da would point to oxidation of methionine Met-267,
whereas further mass shifts are caused by nitration of Trp-264 and/or
Trp-268. The mass shift of 45 Da corresponds to nitration of a
tryptophan residue, whereas the mass shift of 29 Da can be explained by
the loss of one oxygen from the nitro group. This loss of oxygen can
occur as a laser-induced photochemical decomposition, as has been shown for the similar aromatic side chain nitro-tyrosine (51). Finally, the
mass shift of 61 Da can be explained by the nitration of one tryptophan
plus the additional oxidation of methionine (Fig. 4). Because peptide
Gly-263-Arg-271 contains only Trp-264 and Trp-268, but no tyrosine,
this modification-pattern unambiguously identifies tryptophan
nitration. All these latter modifications are at or near the
dimer/dimer interface and show the same concentration dependence as
octamer dissociation (compare Figs. 2 and 4). This strongly indicated
that these modifications are involved in octamer dissociation. In fact,
Trp-264 in particular has been identified by site-directed mutagenesis
to be crucial for octamer stability (52). To identify which of the two
tryptophans in the peptide Gly-263-Arg-271 was modified, we applied
postsource decay MALDI-TOF with human sMtCK. When mass selection was
set to pass the unmodified parent ion at a measured
m/z of 1255 (calculated 1254.4), all b- and
y-fragments could be detected, leaving no doubt about the identity of
this peptide. However, postsource decay analysis of the
nitration-modified parent ion did not reveal a single modified tryptophan. By contrast, we detected a b-ion (b4) that is a
unique fragment to the peptide modified at Trp-264 but also a y-ion
(y6) that is unique to the peptide modified at Trp-268,
suggesting that both Trp-264 and Trp-268 are partially modified.

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Fig. 4.
MALDI mass spectra of peptide
Gly-263-Arg-271 treated with increasing peroxynitrite
concentrations. The molecular masses of species arising from this
fragment at m/z 1255 due to modification by
peroxynitrite are indicated at the top, and the difference
from the unmodified fragment is given. The unmodified peak from each
spectrum was standardized to a relative intensity of 1.0 to show the
increases of additional peaks relative to the unmodified species.
ONOO concentrations are given to the right.
Note that molecular mass shifts of 45 and 29 Da identify
nitro-tryptophan, and the shift of 16 Da indicates methionine oxidation
(see text and Ref. 51).
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DISCUSSION |
In this study, we show that both human mitochondrial CK isoenzymes
are very sensitive to ONOO
-induced damage but differ in
part in the dose-dependence of the deleterious modifications. The
concentration for half-maximal inactivation was 8 µM
ONOO
for both sMtCK and uMtCK, whereas concentration for
half-maximal octamer dissociation was only 240 µM
ONOO
for sMtCK but was 790 µM for uMtCK. We
could also identify the MtCK residues involved in ONOO
damage, namely active site Cys-278, as well as Met-267, Trp-264, and/or
Trp-268 at the dimer/dimer interface.
Half maximal inactivation of human MtCK isoenzymes occurred at very low
ONOO
concentrations (i.e. 8 µM),
which corresponds to a ONOO
:MtCK monomer-ratio of 0.7:1.
This is in line with earlier results on chicken sMtCK, showing
inactivation with an IC50 of 35 µM (30); the
slightly decreased IC50 value in our study could be
attributable to species-specific differences or to the stronger
buffering capacity used. Cytosolic muscle CK was reported to be
inactivated with an IC50 of 2.5 µM
ONOO
(26). However, their protein concentrations were
much lower (0.4 µg/ml, compared with the 0.5 mg/ml used here),
resulting in a significantly higher ONOO
:protein ratio of
about 50:1 at the IC50 value. It is well known that
ONOO
added in a single bolus can decompose very quickly
in water without reacting with the highly diluted protein. Our work has
taken into account the high MtCK concentration in the mitochondrial
intermembrane space and the limited stability of MtCK octamers below a
concentration of 0.5 mg/ml (20).
In earlier studies, it was speculated that modifications of the highly
reactive active site cysteine (Cys-278 in MtCK, Cys-283 in cytosolic
CK) cause enzymatic inactivation (26, 30). Here, we show unambiguously
the modification of this residue and its role in inactivation, using
MALDI-MS analysis of ONOO
-modified wild-type enzyme and
activity measurements of a C278G replacement mutant. As shown
previously, site-directed mutagenesis of Cys-278 leads to significant
but not complete inactivation of the enzyme and, most importantly, to a
complete loss of substrate synergism (50). This residue is located in
the active site of MtCK (6), and its homologue in the transition state
structure of the closely related arginine kinase is in direct contact
with the reactive guanidino group of the arginine substrate (18). Mutagenesis studies and the CK x-ray structures identified another catalytically important residue in the active site of MtCK that can
potentially be modified by peroxynitrite, the active site Trp-223 (6,
52). However, peptide Gly-211-Arg-231 containing this tryptophan
showed no mass shift after ONOO
exposure, demonstrating
that Trp-223 is not modified by ONOO
, similar to
inactivation by radiation (21). Because the active site is highly
conserved throughout all CK isoenzymes and even the whole group of
guanidino kinases (17, 18), it can be tacitly assumed that the active
site cysteine is involved as the prime target of ONOO
in
all these kinases. In addition, this homology also explains why there
is no difference between the two human MtCK isoenzymes with regard to
inactivation. Oxidation at the C-terminal cysteine Cys-358 may lead to
reduced membrane binding of MtCK, as we have observed after
anthracycline-induced oxidation of human MtCK in vitro (47).
Anthracyclines are efficient cancer chemotherapeutics, but they also
induce oxidative damage of proteins such as MtCK that may cause their
well known cardiotoxic side-effects.
The octamer can be considered the physiological form of MtCK. Impaired
octamer stability leads to reduced creatine stimulated mitochondrial
respiration (14). We show here that ONOO
also
destabilizes the octameric structure of MtCK, albeit at higher
concentrations than necessary for inactivation and by affecting human
sMtCK much more than human uMtCK. The higher octamer stability of uMtCK
could be caused by reduced reactivity toward ONOO
or an
intrinsically higher octamer stability compared with sMtCK. However,
the latter mechanism is strongly supported by different observations. A
very similar difference in octamer stability between human uMtCK and
sMtCK was observed in vitro after incubation with transition
state analogue complex (20), as well as after treatment with
anthracyclines (47). Finally, an exact comparison of the x-ray
structures (6, 16) and a direct biophysical approach (20) revealed that
the mainly hydrophobic dimer/dimer interfaces of uMtCK are larger than
those of sMtCK and contain additional polar interactions that preserve
the octameric state of uMtCK. Peroxynitrite not only destabilizes the
octameric structure, at much lower concentrations it already prevents
formation of octamers from dimers as shown in vitro with
chicken sMtCK (53). Thus, inactivation and dimerization of MtCK
in vivo eventually occur simultaneously.
According to our mass spectrometric results, destabilization of
octameric MtCK by ONOO
is caused by the chemical
modification of peptide Gly-263-Arg-271, which is part of the
dimer/dimer interface and responsible for hydrophobic stabilization of
the MtCK octamer (6, 16). The modifications are clustered and involve
oxidation of Met-267 and nitration of Trp-268 and/or Trp-264. Trp-264
is a key residue in the hydrophobic dimer/dimer interaction patch, as
revealed by x-ray crystallography (6) and replacement mutants leading to octamer destabilization (52), but modifications rendering the nearby
residues such as Trp-268 more polar may also contribute to
destabilization. Both tyrosine residues present in the dimer/dimer interface, Tyr-15 and Tyr-34 (6), were not modified by
ONOO
at least in sMtCK, because the peptides containing
these residues (Leu-8-Arg-19 and Lys-20-Lys-36, respectively) did not
show any mass shift upon ONOO
treatment.
The CK system plays an important physiological role for cellular
energetics in health and disease (for review, see Ref. 54). A
compromised CK system, caused, among others, by oxygen radical damage
to the enzyme, has been implicated in heart disease (34), as well as in
many neurodegenerative diseases such as Huntington's disease (35),
amyotrophic lateral sclerosis (36), and Alzheimer's disease (for
reviews, see Refs. 33 and 37). In brains of persons with Alzheimer's
disease, CK activity decreased, whereas mRNA levels did not change
significantly (55, 56) and cytosolic brain-type CK was oxidatively
modified (39, 57). Besides the integrity of the active site, the
octameric state of MtCK seems also to be crucial for efficient
vectorial channeling of high-energy phosphates from mitochondria to the
cytosol (4, 14). In addition, octameric MtCK has been shown to
stabilize mitochondria from going into permeability transition (58).
Thus, a decrease in MtCK octamer content caused by ONOO
could interrupt the creatine/PCr-shuttle and also lead to early events
of apoptosis (59). In animal models of short- and long-term ischemia-reperfusion damage, as well as diseased human heart, a
significant decrease of the octamer/dimer ratio was observed (53). Some
of the main pathological symptoms of these cardiomyopathies, but also
of many neurodegenerative diseases, are caused by reactive oxygen and
nitrogen species and involve mitochondria (33, 60). Therefore, it is
highly probable that ONOO
-induced modifications at the
dimer/dimer interfaces of MtCK octamers, which we have observed
in vitro, also occur in vivo. The fact that uMtCK
is significantly more stable than sMtCK could explain why a decreased
octamer/dimer ratio seems to be an early event in heart disease,
whereas it may occur in brain and neuronal tissue only at a more
advanced stage of neurodegeneration.
In summary, this study shows that ONOO
has severe effects
on enzyme activity, as well as on the stability of human MtCK octamers, with sMtCK being more susceptible than uMtCK in the latter case. Among
the modified amino acid side chains, we could identify some key
residues, such as active site Cys-278 and dimer/dimer interface Trp-264. In vivo, molecular damage of MtCK would lead to an
interruption of the creatine/PCr-shuttle and therefore to a lower
cellular energy state, with all its far-reaching consequences.