(Received for publication, July 31, 1995; and in revised form, October 16, 1995)
From the
Nitric oxide (NO)-related activity has been associated with an
NAD-dependent modification of the glycolytic enzyme,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). However, the
mechanism by which NO effects covalent attachment of nucleotide and its
role in regulation of enzyme activity are controversial. Recent studies
have shown that S-nitrosylation of GAPDH (Cys
)
initiates subsequent modification by the pyridinium cofactor. Here we
show that NADH rather than NAD
is the preferred
substrate. Transnitrosation from active site S-nitrosothiol to
the reduced nicotinamide ring system appears to facilitate protein
thiolate attack on the enzyme-bound cofactor. This results in
attachment of the intact NADH molecule. Moreover, we find that S-nitrosylation of GAPDH is responsible for reversible enzyme
inhibition, whereas attachment of NADH accounts for irreversible enzyme
inactivation. S-Nitrosylation may serve to protect GAPDH from
oxidant inactivation in settings of cytokine overproduction and to
regulate glycolysis. NADH attachment is more likely to be a
pathophysiological event associated with inhibition of gluconeogenesis.
The versatility of NO as a biological messenger reflects its
participation in rich additive and redox chemistry. Pathways of NO
oxidation involve reactions with O,
O
, and transition metals, which support
the formation of surrogates retaining NO-like
bioactivity(1, 2) . This is exemplified in the case of S-nitrosothiols that are formed in vivo and serve as
NO-group donors(3, 4, 5) . In particular,
NO
donation (heterolytic decomposition) appears to be
the predominant mechanism of RSNO (
)metabolism in many
biological systems (6, 7, 8) . Examples of
NO
-related activities include allosteric modulation of
the N-methyl-D-aspartate receptor involved in
neuroprotection(9) , the antimicrobial effects of
RSNO(10) , the inhibition of many sulfhydryl-containing
enzymes(11, 12) , the activation of p21
and tissue plasminogen activator, and the down-regulation of
transcriptional activators(13, 14, 15) .
NO-related signal transduction can be broadly classified as
cGMP-dependent or mediated through redox signaling events (1) .
The latter is, perhaps, best exemplified in the regulation of protein
function by S-nitrosylation(16) . In the case of
enzymes that contain critical thiols at their active site, covalent
attachment of the NO group leads uniformly to functional attenuation.
Examples of enzymes in this category include cathepsin B, aldolase,
-glutamylcysteinyl synthetase, aldehyde dehydrogenase, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (see (1) and
references therein). Studies on the potential regulation of GAPDH have
received particular attention in view of evidence that NO-related
activity (nitric oxide synthase activity or NO donors) stimulates an
NAD
-dependent posttranslational modification of active
site thiol in association with the loss of protein function (3, 17, 18) . The demonstration of such a
modification in cells has led to the proposal that NO induces an
ADP-ribosylation reaction reminiscent of that catalyzed by bacterial
and mammalian enzymes (19, 20, 21) . In this
reaction, the ADP-ribose moiety of NAD
is transferred
to acceptor amino acids with the release of nicotinamide. Studies by
Pancholi and Fischetti (22) may provide the strongest evidence
in favor of a true thioglycosidic linkage induced by NO. More recently,
however, this mechanism has been challenged by the demonstration that
both the ribose and the nicotinamide moieties of NAD
are incorporated by GAPDH(23) . This activity implies
linkage of the intact molecule to the active site of the enzyme.
Furthermore, inhibition of enzyme activity has seemed to correlate
better with the extent of S-nitrosylation than the attachment
of
P-nucleotide, the latter representing only a small
fraction of the total protein(23) .
We recently probed the
mechanism of GAPDH modification using several NO donors. Our studies
revealed that NO transfer to active site thiol is
requisite for subsequent modification by
[
P]NAD
(3) . These data,
however, raise a fundamental paradox, as the pathway by which S-nitrosylation facilitates covalent modification by
NAD
is not readily apparent. We reasoned, therefore,
that NADH rather than NAD
is involved in this
reaction, since reduction of nicotinamide would make it susceptible to
activation via nitrosative attack. Here we show that 1) S-nitrosylation promotes covalent attachment of reduced
nicotinamide; 2) covalent modification by
[
P]NADH occurs (largely) via a thionicotinic
linkage; 3) S-nitrosylation of GAPDH accounts for reversible
enzyme inhibition, and 4) covalent modification by NADH is responsible
for irreversible protein inactivation.
For preparation of
[nicotinamide-C]NADH, 20 µl (1 nCi)
[nicotinamide-
C]NAD
(41 mCi/mmol) was incubated with 2 mM MnCl
,
10 mM isocitrate (pH 7.5), 22 units of isocitrate
dehydrogenase (NAD
-specific, 31 units/mg), and 100
mM Hepes (pH 7.5) in a total volume of 100 µl at 37 °C
for 45 min. Separation of
C-labeled
NAD
/NADH was performed as described above.
Figure 1:
Modification of GAPDH by
[P]NAD
and
[
P]NADH. Modification of GAPDH was performed
with 10 µM reduced or oxidized
P-labeled
nucleotide cofactor (each 200,000 cpm/assay), SNP, and DTT. Detection
of radioactivity was made by PhosphorImager analysis. Experimental
details are described under ``Experimental Procedures.''
Results are representative of three similar
assays.
Importantly, NO
donors of several different molecular classes were found to induce
[P]NADH-dependent GAPDH labeling. However, the
time course of enzyme modification varied among compounds (Fig. 2). For example, SNP-induced labeling was detected at 2.5
min and reached saturation by 5 min, whereas SIN-1 modification
occurred at a much slower rate. Radiolabeling was noted after 10 min
and required 40 min to achieve levels comparable with those with SNP.
Nonspecific labeling was observed with NADH but required reducing
conditions, i.e. DTT, and much longer reaction times (i.e. labeling was not detectable during the first 20-30 min).
Figure 2:
Time-dependent modification of GAPDH by
NADH in the presence of SNP and SIN-1. Modification of GAPDH was
carried out as described under ``Experimental Procedures''
with 10 µM [P]NADH (120,000
cpm/assay). Detection was performed using a PhosphorImager. Data are
representative of three similar
experiments.
The pH optimum for nucleotide incorporation was 7.5 for NADH and
above 8.5 for NAD, in agreement with involvement of
enzyme (active site) thiolate (Fig. 3). In aggregate, these data
are compatible with reports that S-nitrosylation of GAPDH is
rate-limiting, since SNP is a better nitrosating agent than SIN-1. We
speculate that the higher pH optimum for NAD
may
reflect its more efficient reduction by reduced thiol at alkine pH (26) . Nonspecific labeling by reduced nucleotide is also more
prevalent under alkine conditions.
Figure 3:
pH-dependent modification of GAPDH by
NAD and NADH. Modification of GAPDH in the presence of
10 µM [
P]NAD
(120,000 cpm/assay) and 10 µM [
P]NADH (120,000 cpm/assay) was determined
under the following assay conditions: 100 mM Mes, pH 6.5; 100
mM Hepes, pH 7.5; 100 mM Tris, pH 8.5. SNP and DTT
were incubated in each reaction mixture for 20 min. For experimental
details see Fig. 1. One of three representative experiments is
shown.
The amount of radioactivity
incorporated into GAPDH (based on equivalent amounts of cold and
radioactive labeled nucleotide) was much higher with NADH than
NAD. Under optimal labeling conditions (10 µM NADH, 2.5 mM DTT, 20 min, n = 16), SIN-1
(200 µM) stimulation led to 1.14 ± 0.37 mol of
NADH/mol of GAPDH. Similar rates of incorporation were achieved using
200 µM SNP (1.05 ± 0.15 mol of NADH/mol of GAPDH,
mean ± S.D., n = 8; 2.5 mM DTT for 10
min). These results demonstrate the modification of approximately one
GAPDH subunit/molecule holoenzyme (GAPDH consists of four identical
39-kDa polypeptide chains), and are to be contrasted with relatively
trivial modification by NAD
((23) , Fig. 3and Fig. 4).
Figure 4:
Modification of GAPDH by
[nicotinamide-C]NAD
and [nicotinamide-
C]NADH. GAPDH
labeling was performed for 20 min with SIN-1, DTT, 10 µM [nicotinamide-
C]NAD
(40,000 cpm/assay) or 10 µM [nicotinamide-
C]NADH (40,000
cpm/assay). Further details are outlined under ``Experimental
Procedures.'' Similar results were obtained in two separate
assays.
Experiments performed with
-NAD
further support the notion that binding
involves the intact pyridine cofactor. Upon cleavage of the
-nicotinamide bond in
-NAD
by pertussis
toxin, the fluorescence of the molecule increased, much as described
previously for NADase(25) . In comparison, modification of
GAPDH using
-NAD
did not result in a change in
fluorescence (Fig. 5), even though the substitution for
NAD
led to a comparable degree of GAPDH inhibition.
For example, modification of GAPDH (10 µg) by 200 µM SIN-1 (2.5 mM DTT) in the presence of either 10
µM NAD
or 10 µM
-NAD
resulted in a 55 ± 0.5% versus 54 ± 3.5% decrease in enzyme activity relative
to controls after 40 min, respectively.
Figure 5:
Pertussis toxin and GAPDH catalyzed
reactions using -NAD
. A, pertussis
toxin-catalyzed ADP-ribosylation was carried out as described under
``Experimental Procedures'' using 10 µM
-NAD
at 37 °C for 40 min. B,
GAPDH modification was performed as described above, using 10
µM
-NAD
. Changes in fluorescence
were recorded as difference spectra. Experimental details are described
under ``Experimental
Procedures.''
We further reasoned that
nitrosation of reduced nicotinamide was required for ring activation.
This would then facilitate protein thiolate attack on the nucleotide by
increasing its electrophilicity. To test the validity of this
mechanism, we first examined the effects of
nitrosonium-tetrafluoroborate (BFNO), a strong
NO
donor (Fig. 6). In the presence of DTT,
BF
NO potently induced GAPDH labeling by
[
P]NADH. Moreover, the
NO
-donor nitronium-tetrafluoroborate
(BF
NO
), which on theoretical grounds should be
equally capable of nicotinamide activation(3) , resulted in
comparable degrees of GAPDH modification (Fig. 6). With both
agents, maximal labeling was achieved at concentrations of 50
µM under reducing conditions.
Figure 6:
NADH-dependent covalent modification of
GAPDH induced by BFNO and BF
NO
.
Modification of GAPDH was performed with 10 µM [
P]NADH (150,000 cpm/assay), DTT,
BF
NO, and BF
NO
. Details are given
under ``Experimental Procedures.'' This figure is
representative of three similar
experiments.
Cleavage experiments with
HgCl were then performed in order to confirm that the NADH
linkage involved protein thiol groups (i.e. Cys
of GAPDH). Specifically, the enzyme preparation was treated with
HgCl
after covalent modification had been induced with
SIN-1. HgCl
(5 mM) was found to displace the
greater part of the [
P]NADH radiolabel (Fig. 7).
Figure 7:
HgCl cleavage of ADP-ribose
following pertussis toxin treatment and of NADH following NO
stimulation. Human platelet microsomes (80 µg/assay) were incubated
with 10 µM [
P]NAD
(0.5 µCi/assay) and pertussis toxin as outlined under
``Experimental Procedures.'' Labeling of GAPDH (10
µg/assay) was performed with 200 µM SIN-1, 2.5 mM DTT, and 10 µM [
P]NADH
(200,000 cpm/assay) for 20 min. For HgCl
cleavage
experiments, protein pellets were resuspended in 100 mM Hepes
buffer (pH 7.5) and incubated for 90 min with HgCl
.
Following protein precipitation, the remaining radioactivity was
measured as described under ``Experimental Procedures.'' Data
are representative of three experiments.
Importantly, radioactivity incorporated by
incubating GAPDH with 10 µM NADH and 2.5 mM DTT, i.e. nonspecific labeling, could be readily discriminated from
active site modifications due to its resistance toward Hg treatment. 5 mM HgCl
or 10 mM DTT
was unable to remove any such incorporated radioactivity.
NO-independent radioactivity, however, was partially cleaved (around
40% decrease) by treatment with 5 mM NH
OH for 90
min. For comparison, the effects of HgCl
were examined on
human platelet membranes that had been ADP-ribosylated by treatment
with pertussis toxin. As previously shown, HgCl
(0.5
mM) removed all the radiolabel. Furthermore, over 90% of the
radioactivity remained bound to GAPDH following treatment with
hydroxylamine.
To further identify the cysteine residue involved by
NO, a tryptic digestion of radiolabeled GAPDH was performed, attachment
followed by sequence analysis of the single fragment that contained
radioactivity. Amino acid sequencing identified the peptide IVSNAS,
after which analysis resisted further cycling. The sequence matches
identically with the predicted tryptic digestion fragment containing
the active site Cys residue of rabbit muscle GAPDH. Thus,
cleavage experiments combined with tryptic digestion strongly suggest
that the modification of GAPDH occurs at Cys
.
In general, GAPDH
inhibition appeared to correlate well with the extent of nucleotide
incorporation. In particular, enzyme inhibition induced by SIN-1 was
greatest with NADH. NAD was significantly less
effective and equal in activity to
-nicotinamide mononucleotide.
Nicotinamide and nicotinamide N-propanesulfonic acid also
exhibited inhibitory effects, although they were the least active
compounds. The time- and concentration-dependent effects of NADH are
detailed in Table 2. Importantly, radiolabel incorporation
induced by S-nitrosylation of Cys
correlated
well with loss of enzyme activity, whereas NO-independent modification
of GAPDH by NADH did not (data not shown). Inhibition of GAPDH clearly
increased with time and with higher concentrations of the nucleotide.
Experiments were then performed to delineate the role of S-nitrosylation vis à vis covalent
NADH attachment in the inhibition of GAPDH (Fig. 8). GAPDH was
incubated with BFNO for 5 min followed by the addition of
either DTT or NADH. GAPDH catalysis (Fig. 8, upper
panel) was then monitored over the ensuing 30 min, and enzyme
activity was correlated with the amount of incorporated radioactivity (Fig. 8, lower panel).
Figure 8:
Action of BFNO and NADH on
GAPDH enzyme activity. In the upper panel GAPDH was incubated
with 100 µM nitrosonium tetrafluoroborate
(BF
NO) for 5 min, followed by the addition of 10 µM NADH or 10 mM DTT. Incubations were then continued for
the times indicated. Samples treated with NADH/DTT (but not
BF
NO) served as controls. In the lower panel GAPDH
was preincubated with 100 µM BF
NO, followed by
the addition of 10 µM NADH (150,000 cpm/assay).
Incorporation of radioactivity was carried out as described above. Data
are representative of four similar
experiments.
Following the addition of
BFNO, we observed an initial drop in enzyme activity by 40%
compared with untreated controls. Over the next 5 min, enzyme activity
partially recovered spontaneously. Thereafter, either 10 mM DTT or 10 µM NADH were added. With the addition of
DTT, GAPDH activity was restored to control values (90 ± 2.5%).
In contrast, NADH caused a time-dependent irreversible inhibition of
the enzyme. By 35 min, only 30% of the initial enzyme activity remained (Fig. 8, upper panel). Using
[
P]NADH, we confirmed that the extent of
radiolabeling incorporated by GAPDH paralleled the degree of inhibition
following the addition of nitrosating agent (Fig. 8, lower
panel). We conclude 1) that S-nitrosylation is responsible for
early, reversible enzyme inhibition and 2) that S-nitrosylation promotes subsequent irreversible attachment of
NADH to active site thiol.
Nitric oxide has been associated with a
mono-ADP-ribosylation-like reaction in which the pyridinium nucleotide
undergoes covalent attachment to the active site thiol of GAPDH.
However, the mechanism of this reaction is poorly understood, and its
contribution to changes in protein function is controversial. We
recently demonstrated that GAPDH modification is stimulated by S-nitrosylation, i.e. NO transfer
chemistry rather than reaction of nitric oxide(3) . In
particular, S-nitrosylation of the enzyme active site thiol
(Cys
) was found to initiate subsequent covalent
modification by NAD
. In order to rationalize this
finding, we reasoned that NAD
must first be reduced
under in vitro assay conditions(1, 3) .
Transnitrosation from active site RSNO to the nicotinamide ring could
then facilitate protein thiolate attack on the nucleotide(1) .
Here we show that NO-related activity does indeed depend on the
presence of reduced nicotinamide and that NADH rather than
NAD
is the preferred reaction substrate. Specifically,
labeling with NADH is much more efficient than with
NAD
, occurs more rapidly, and correlates better with
changes in enzyme activity.
Figure 9: Suggested reaction sequence of NO- induced NADH attachment to GAPDH. Modification of GAPDH follows the upper pathway leading to a thionicotinic linkage. For details see ``Discussion.''
This proposed
mechanism is supported by three findings: 1) that several reduced
nicotinamide derivatives can substitute for NADH; 2) that treatment
with HgCl liberates the reduced nucleotide from active site
thiol; and 3) that binding and inhibition by
-NAD
occurs without cleavage of the
-glycosidic bond. The
involvement of reduced nicotinamide might also explain reports of a
NO-associated ADP-ribosylation reaction involving a true thioglycosidic
linkage. In this scenario, activation of nucleotide (via
transnitrosation), engenders thiolate attack at ribose C-1` by making
nicotinamide a better leaving group. This reaction (Fig. 9, lower pathway) may be more viable in other proteins where
structural constraints place the RSNO in closer proximity to the sugar
moiety than the nicotinamide ring.