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INTRODUCTION |
Phosphoribulokinase
(PRK)1 (EC 2.7.1.19) and
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.13) form
multienzyme complexes. Several multienzyme complexes with different
compositions have been isolated from chloroplasts (1-21).
The PRK·GAPDH core complex is linked to photosynthesis, as these two
enzymes are part of the Benson-Calvin cycle and use ATP and NADPH
produced by the primary reactions of photosynthesis. PRK catalyzes the
ATP-dependent phosphorylation of ribulose 5-phosphate to
form ribulose 1,5-bisphosphate, the CO2 acceptor in
photosynthetic organisms, and GAPDH catalyzes the reversible reduction
and dephosphorylation of 1,3-bisphosphoglycerate (BPGA) to
glyceraldehyde 3-phosphate using NADPH.
We have purified a complex from the green alga Chlamydomonas
reinhardtii (10) that is made up of two dimeric PRK and two tetrameric GAPDH. The protein CP12 (20-22) was found recently to be
associated with this supramolecular edifice (23). The two enzymes, PRK
and GAPDH, may each be obtained in a free independent state. When they
are not associated with each other they form dimers (PRK) or tetramers
(GAPDH). CP12 is tightly associated with GAPDH (23). The complex can be
dissociated by harsh reduction and reversed by oxidizing conditions,
because the oxidized partners can spontaneously reform a complex
in vitro that is quite similar to the native state (10). In
only a few cases has it been possible to assemble particles from their
separate parts in vitro that resemble the native complexes
(24, 25). The association of these two enzymes also gives rise to new
regulatory properties. PRK and GAPDH within the complex are regulated
by NADP(H) rather than by NAD(H), whereas the independent stable
enzymes are not (26). Oxidized PRK may be active when associated with
GAPDH or when dissociated from the complex upon dilution (14). We have
also shown that the complex may exist under mild reducing conditions
(12) even if it is dissociated by severe reducing conditions (DTT
concentrations up to 20 mM). But it dissociates faster upon
dilution, as reduction weakens the complex.
Whereas plant enzymes are heterotetrameric
(A2B2), algal GAPDHs are homotetrameric and
made up of only A subunits (27, 28). The B subunit has a C-terminal
extension that contains two cysteine residues believed to be involved
in the regulation of the enzyme (29-31). Nonetheless, studies on crude
extracts of Chlamydomonas indicate that the algal enzyme
that lacks these 28 amino acid residues can be activated reductively by
light (32). We have studied the regulation of this enzyme upon
reduction or oxidation in its isolated state or within the
PRK·GAPDH·CP12 complex to see if this reductive light activation is
explained by the interaction of GAPDH with its protein partners. The
same study was performed on the other partner, PRK. In previous studies
(14) we have fully characterized the activity of isolated PRK and PRK
in the complex. PRK in the so-called oxidized complex was active as
mentioned above, but this result has been disputed (33), as the
cysteinyl sulfhydryls of PRK in the complex have never been directly
tested. We have therefore used mass spectrometry coupled with protein chemistry to analyze the cysteinyl sulfhydryl contents of PRK, GAPDH,
and CP12 in the complex, whatever its redox state.
Finally, we have used a biosensor to study the interaction between the
two enzymes depending on their redox states, as cellular redox
signaling contributes to the control of the Benson-Calvin cycle and
many other physiological processes (34). The qualitative and
quantitative aspects of these interactions have been analyzed.
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EXPERIMENTAL PROCEDURES |
Materials--
Oxidized glutathione, thioredoxin,
iodoacetamide, ATP, ribose 5-phosphate, 3-phosphoglyceric acid,
and phosphoribose isomerase were from Sigma, NADPH was from Calbiochem,
and other reagents were supplied by Roche Molecular Biochemicals.
BIAcore 2000 system, CM5 sensorchips, HBS-EP buffer (10 mM
Hepes, 150 mM NaCl, 3.4 mM EDTA, P20 surfactant
0.005% (v/v), pH 7.4), and the amine coupling kit
(N-hydroxysuccinimide,
N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide, ethanolamine hypochloride) were from BIAcore AB (Uppsala, Sweden).
Purification of GAPDH·CP12·PRK and
Isolated Forms from C. reinhardtii--
The complex GAPDH·CP12·PRK
from C. reinhardtii (WM3
) cells grown
mixotrophically was purified to apparent homogeneity as previously described (10). The isolated enzymes (GAPDH/CP12 and PRK) were obtained
by reducing the purified bi-enzyme complex with 20 mM DTT
for 1 h. This mixture was loaded onto a DEAE-trisacryl column equilibrated in buffer supplemented with 5 mM DTT (9, 23). The resulting purified enzymes were stored at
80 °C in 10%
aqueous glycerol.
Activity Measurements--
PRK and GAPDH activities were
determined using a Pye Unicam UV2 spectrophotometer (6, 23). The
enzymes were reduced by incubating them for 30 min with 20 mM dithiothreitol or 10 µM reduced
thioredoxin at 30 °C. GAPDH was oxidized with 5 mM
oxidized glutathione (5 mM cystine or 2 mM
diamide), or 10 µM oxidized thioredoxin at 4 °C, with
or without 5 mM BPGA or 5 mM NADP(H). Aliquots
were withdrawn at intervals and activity was measured using NADPH as
cofactor. PRK was also oxidized with the same oxidizing agents but
protection experiments were carried out using its substrates (5 mM ribose 5-phosphate or 5 mM ATP). All
experimental data were fitted to theoretical curves using Sigma plot
5.0. Protein concentrations were determined by the Bradford method
(35).
Native Electrophoresis and Immunoblotting
Experiments--
Native PAGE was performed on 4-15% minigels using
the Phastsystem apparatus from Amersham Biosciences. Proteins
separated by PAGE were transferred to nitrocellulose filters (0.45 µm, Schleicher and Schuell) by passive diffusion for 24 h. The
filters were then immunoblotted with a rabbit antiserum against spinach
PRK. Antibody binding was revealed using alkaline phosphatase (36).
Mass Spectrometry Analysis of Free Cysteine Residues--
The
free SH groups in PRK and GAPDH, both isolated or in the complex, were
quantified by alkylation of the free cysteine residues with
iodoacetamide prepared as in Ref. 37. The enzymes were incubated with
reducing or oxidizing agents or left untreated. They were alkylated
with 100 mM iodoacetamide for 1 h at room temperature
in the dark and analyzed by mass spectrometry MALDI-TOF (Voyager DE Pro
mass spectrometer from Applied Biosystems). PRK and GAPDH were analyzed
using sinapinic acid (3,5-dimethoxy-4-hydroxycinnaminic acid) as
matrix. Samples for analysis were desalted on C18 zip-tips (Millipore) and eluted in 50% acetonitrile, 0.1%
trifluoroacetic acid and 50% water, 0.1% trifluoroacetic acid.
Sensorchip Coupling--
PRK or GAPDH that had been treated with
reducing or oxidizing agents were coupled to the sensor chips using the
amine coupling kit and the automated immobilization application wizard
included in the BIAcore software to give a coupling level of around 100 resonance units. One flow cell of each CM5 sensor chip served as
negative control and was used to subtract the bulk refractive index.
Interaction Cycles--
We studied the interaction of GAPDH with
flow cells coupled to the various PRK in SPR experiments at 20 µl/min
in HBS running buffer plus 0.1 mM NAD. In each interaction
run, a given concentration was injected for 180 s to record the
association phase to the flow cells, after which buffer was injected
for 180 s to record the dissociation phase. Last, 10 µl of 10 mM glycine, pH 3, were injected to regenerate the flow
cells. The whole process was automated, and we performed multiple
interaction runs with different GAPDH concentrations. We checked that
there was no mass transfer. The sensorgrams were processed by
subtracting the corresponding reference flow cell sensorgram to remove
contributions because of base-line drift, bulk, and nonspecific
interactions. The reverse experiments were performed by immobilizing
GAPDH and injecting different concentrations of PRK.
Theory--
The analyte in the injected sample interacts with
ligand immobilized on the sensor surface to give the association phase, which continues until a steady state is reached and association and
dissociation rates are in equilibrium. The analyte begins to dissociate
as soon as injection is stopped and replaced by buffer. The association
and dissociation phases can be used to derive kinetic constants.
Assuming pseudo-first order kinetics,
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(Eq. 1)
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the rate of complex formation during analyte injection is given
by,
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(Eq. 2)
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where dR/dt is the rate of change of the
SPR signal, ka is the association rate constant,
kd the dissociation rate constant, C is
the analyte concentration, Rmax the maximum analyte binding capacity in response units (RU), and
Rt is the signal at time t.
The rate of dissociation can be expressed as the following.
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(Eq. 3)
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At equilibrium, association and dissociation rates are equal and
the equilibrium-dissociating constant (Kd) is given by the following.
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(Eq. 4)
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Global fittings of the exponential curves (sensorgrams) giving
both the ka and kd values were
performed using Biaevaluation 3.1.
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RESULTS |
Titration of Sulfhydryl Groups within the PRK·GAPDH· CP12
Complex--
PRK in the complex was alkylated using iodoacetamide. The
Chlamydomonas PRK monomer contained five cysteine residues
(SH) but only three molecules of iodoacetamide bound to each PRK
monomer within the oxidized complex (mass increment of 168.34 Da). This increase indicated that there was one disulfide bridge and three free
thiol groups (SH) per monomer. The PRK released by dissociating the
complex, the so-called metastable PRK, also had one disulfide bridge.
All five cysteine residues in the PRK monomer were free when the
complex was reduced with 20 mM DTT for 30 min, as shown by
the mass increment of 274.36 Da (Fig.
1).

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Fig. 1.
MALDI-TOF mass spectra of native oxidized
complex (A), oxidized complex treated with
iodoacetamide (B), and reduced complex treated with
iodoacetamide (C). The concentration of the
bi-enzyme complex in all experiments was 1.3 µM.
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GAPDH and CP12 in the oxidized complex were also alkylated
simultaneously. The four cysteine residues per subunit of GAPDH (mass
increment of 222.7 Da, indicating the binding of four molecules of
iodoacetamide) were all free, but no molecules of iodoacetamide were
bound to CP12. As CP12 contains four cysteine residues, this indicated
that CP12 has two disulfide bridges when it is present in the oxidized
complex. Two populations of CP12 were found after the complex had been
reduced with 20 mM DTT for 1 h. The CP12 in one was
fully reduced (with 4 SH per monomer titrated with iodoacetamide),
whereas the CP12 in the other was half-reduced (with 2 SH per monomer
titrated with iodoacetamide). The fully reduced CP12 was only obtained
after drastic dilution of the complex (100-fold) followed by reduction
with 20 mM DTT for 1 h.
GAPDH Activity in the Reduced Complex--
The enzyme activity of
PRK in the oxidized complex was greatly enhanced by reduction of its
disulfide bridge with DTT. No direct effect of the reducing agent could
be detected as the GAPDH in the so-called oxidized complex already had
4 SH groups per subunit. Nonetheless, there was a reproducible 2- or
3-fold activation of the NADPH-dependent activity of GAPDH
after reduction of the complex with DTT or reduced thioredoxins (from
40 to 100 units/mg). The NADH-dependent activity of
the complex was also measured as chloroplast GAPDH is specific for both
NADH and NADPH. This activity decreased from 40 to 17 units/mg, so that
the ratio of NADPH/NADH activities increased from 1 to 6 upon
reduction. This ratio was constant at 6 for isolated GAPDH (26) (Fig.
2).

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Fig. 2.
Activation/inhibition kinetics of the GAPDH
in the bi-enzyme complex in the presence of DTT. The bi-enzyme
complex (300 nM) was incubated with 10 mM DTT
and the NADPH- ( ) or the NADH- ( ) dependent activities were
measured. The ratio between the NADPH- and NADH-dependent
activities is shown in the inset. The bi-enzyme complex
concentration in the assay cuvette was 0.6 nM.
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GAPDH was no longer activated by reducing agent if the complex had
first been dissociated by dilution. On the other hand, in
vitro reconstitution of this complex (9, 10) using dissociated PRK
and GAPDH restored the capacity of GAPDH to become activated (data not shown).
Treatment of the Complex and Isolated Enzymes with
Oxidants--
We also treated the reduced complex with oxidant as the
activities of PRK and GAPDH within the complex were modulated by
reducing agents. The GAPDH activity decreased by one-half as soon as
the reducing agent was removed, whereas the PRK activity remained constant and stable throughout the experiment. The GAPDH activity was
lost as soon as the PRK-GAPDH interactions were weakened. This indeed
happened when the reducing agent was removed by dilution on a desalting
column. The oxidant GSSG had no further effect on the GAPDH or PRK
activities of the complex. However, GAPDH activity was slightly reduced
(20%) by a smaller oxidant like cystine, whereas the activity of the
PRK in the complex was drastically decreased. The loss of PRK activity
was reversible as it was restored by adding DTT, but that of GAPDH was not.
We used native electrophoresis followed by immunoblotting
with anti-PRK antibody (Fig. 3) to
examine the difference between the effect of cystine and GSSG on PRK
activity within the complex. The cystine appeared to be responsible for
the dissociation of the complex and thus led to oxidation of
dissociated PRK, whereas GSSG treatment left the complex intact. Both 5 mM ATP and, to a lesser extent, 5 mM ribulose
5-phosphate protected PRK from oxidation by cystine (data not
shown).

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Fig. 3.
Immunoblotting following native PAGE of the
bi-enzyme complex. The bi-enzyme complex (control, lane
3, 700 ng; treated with GSSG, lane 1, 660 ng; or with
cystine, lane 2, 660 ng) was electrophoresed under native
conditions, then immunoblotting experiments were carried out using
antibodies against phosphoribulokinase. Lane 4 pertains to
isolated free phosphoribulokinase (6 µg) treated with oxidized
glutathione. C stands for the complex and R for
the dissociated phosphoribulokinase.
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Isolated GAPDH and PRK were also treated with oxidant. All PRK activity
was lost by incubation with GSSG or cystine. DTT restored almost full
activity (85%), as expected. Treatment of isolated GAPDH with 5 mM GSSG resulted in a 50% decrease in activity (Fig. 4) but adding DTT (up to 25 mM) did not restore any activity. Reduction also failed to
restore the activity of GAPDH incubated with 5 mM cystine. This irreversible decrease in GAPDH activity was not linked to simple denaturation of the enzyme, as the
fluorescence spectrum underwent no red shift (data not shown).
Interestingly, the substrate BPGA prevented the loss of activity
because of oxidation, but NADP(H) did not (Fig. 4). Oxidized
thioredoxin had no effect on isolated GAPDH activity, unlike GSSG,
cystine, or diamide.

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Fig. 4.
Kinetics of oxidation of isolated GAPDH.
The NADPH-dependent activity of GAPDH incubated with 5 mM GSSG ( ) or 5 mM GSSG plus 6 mM BPGA ( ) was followed over time. The final
concentration of GAPDH in the assay cuvette was 5 nM.
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The activity of GAPDH within the complex was less severely inhibited by
incubation with cystine than was that of the isolated enzyme, although
cystine disrupted the complex. This could be because of these two forms
of GAPDH having different conformations, as indicated elsewhere (26).
We therefore compared the kinetic parameters of the two forms to test
this possibility. Kinetic experiments were carried out with a
saturating concentration of NADPH and the concentration of BPGA was
varied (Fig. 5).

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Fig. 5.
Steady-state kinetics of GAPDH within the
complex. The bi-enzyme complex was placed in the reaction mixture
containing 0.25 mM NADPH with BPGA concentrations of 0-1.8
mM and the appearance of product was monitored. The
experimental points were fitted to a hyperbola. The bi-enzyme complex
concentration in the assay cuvette was 0.65 nM.
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The form released from the complex displayed Michaelis-Menten kinetics
unlike the isolated form, which displayed positive co-operativity (23).
The data were fitted to a hyperbola to estimate the catalytic constant
(kcat) and the Km for BPGA.
The kcat was 650 ± 10 s
1 and
the Km was 262 ± 11 µM. These
parameters were compared with those of the isolated enzyme (Table
I).
Interaction Analysis of PRK and GAPDH/CP12 Using
SPR--
We then studied the interaction of PRK with GAPDH using
biosensor technology to determine whether the redox state of PRK altered complex formation.
GAPDH and PRK can both be covalently coupled to the CM5 sensorchip, but
the enzymes were each immobilized under different conditions. Amine
coupling is based on favorable electrostatic attraction between
negative charges of the dextran carboxylic groups and positive charges
on the immobilized ligand. Thus, PRK (pI 5.68) was covalently bound
using HBS-EP buffer containing 10 mM acetate, pH 4.8 (immobilization buffer A), whereas GAPDH (pI 8.6) was covalently bound
using HBS-EP buffer, pH 7.4. We studied the stability of the kinase
under these conditions by preincubating the enzyme in immobilization
buffer A for up to 40 min before measuring its activity. No activity
was lost during the time of the Biosensor experiment.
Reduced PRK was immobilized and measurements at different GAPDH
concentrations were performed (Fig. 6).
These curves were fitted to a model equivalent to the Langmuir model
for adsorption to a surface (Equation 1). The average apparent
equilibrium dissociation constant was 62 ± 10 nM.

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Fig. 6.
Plasmon resonance monitoring of GAPDH
interaction with PRK surface. Net sensorgrams (after subtracting
the bulk refractive index) were obtained with immobilized reduced PRK
using 0.14 µM (1), 0.29 µM
(2), 0.44 µM (3), and 0.58 µM (4) reduced GAPDH as analyte. The beginning
of the association phase and the beginning of the dissociation phase
were marked with a and d, respectively. The
experimental data were analyzed using global fitting assuming a 1:1
interaction with the Biaevaluation 3.1.
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We also studied the interaction of oxidized PRK with different amounts
of GAPDH. GAPDH from rabbit muscle was also injected onto oxidized PRK
to check specificity. The responses at equilibrium (Req) for Chlamydomonas and rabbit
muscle GAPDH for their interactions with immobilized oxidized PRK are
reported as a function of their concentrations (Fig.
7). The data were fitted to the following hyperbola function.
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(Eq. 5)
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The average apparent equilibrium dissociation constant was 14 ± 1.6 nM for algal GAPDH and 628 ± 1.3 nM for rabbit muscle GAPDH.

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Fig. 7.
Specificity of the interaction of C. reinhardtii GAPDH with PRK. The amplitude of the
plateau SPR signal (Req) was plotted against the
concentration of C. reinhardtii GAPDH ( ) or rabbit muscle
GAPDH ( ). The experimental data were fitted to Equation 5 in the
main text.
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Experiments were also carried out at 25 and 37 °C and at pH 7 or 8 to determine whether temperature or pH affected the interaction between
GAPDH and PRK. No difference was observed. The same dissociation constants were obtained whether or not GAPDH had been treated with GSSG
or DTT.
A range of PRK concentrations were tested with immobilized GAPDH to
obtain an average apparent equilibrium dissociation constant. The same
apparent equilibrium dissociation constants were obtained, as when PRK
was immobilized. Only when PRK was reduced did we find a higher
apparent equilibrium dissociation constant (62 ± 10 nM).
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DISCUSSION |
Many enzymes belonging to the Benson-Calvin cycle are regulated by
dark-light transitions via thioredoxins in vivo (38) (or
dithiothreitol in vitro). In particular, thioredoxin (39, 40) reduces the disulfide between Cys55 and
Cys16 of inactive oxidized spinach PRK (41, 42).
We have shown (14) that oxidized PRK from C. reinhardtii may
have some activity when it is associated with GAPDH, contrary to common
belief, but no direct evidence of the regulatory disulfide bridge
between Cys16 and Cys55 present in oxidized
PRK, was given. We have now used alkylation of the so-called oxidized
complex to show that there is one disulfide bridge in the PRK monomer.
The activity of PRK is greatly increased when it is reduced, indicating
that the Cys16-Cys55 bond is the target.
Another troubling aspect of the activity of oxidized PRK concerns the
P-loop. This contains residues that interact with the
- and
-phosphoryls of Mg2+-ATP (43). This loop should a
priori be free to move for efficient catalysis. Some data for
bacterial PRK (33, 44) suggest that the
Cys16-Cys55 disulfide bridge found only in the
eukaryotic enzymes could immobilize the P-loop, thereby preventing
catalytic turnover. However, it is worthwhile to pinpoint that there
are insertions and deletions in the plant and algal enzymes, such as an
insertion of 15 residues after the P-loop of the eukaryotic enzyme that
may modify its mobility even in the presence of the
Cys16-Cys55 disulfide bridge. These sulfhydryl
groups are also not essential for catalysis, as a
Cys16-Cys55 double mutant of spinach PRK still
has some activity (42, 45). The conformational constraint imposed by
this bond is more likely to be responsible for the decreased PRK
activity than the formation of the disulfide bridge itself (45). Thus,
although the bacterial PRK structure is a useful model, it may not be
wise to extrapolate data obtained with it to eukaryotic PRKs,
especially the eukaryotic enzyme in a multienzyme complex. Our results
indicate that the conformational constraint near the P-loop could be
attenuated when the PRK is within the complex, allowing oxidized PRK to
be active. The interaction of PRK with GAPDH modifies the conformation of this enzyme (46) and hence its kinetic properties (12, 14).
There is now considerable published evidence from NMR studies that
supramolecular edifices (protein-protein or DNA-protein complexes)
appear to be flexible (47, 48). The entropic cost of the decrease in
conformational freedom must be offset, to some extent, by preserving
the flexibility of other regions. These reports support the assertion
made above by the authors, but only structural data will clarify this
point. Our direct evaluation of the redox status of the regulatory
cysteine residues now clearly shows that oxidized PRK may be
catalytically active. This activity is, however, lower than the
activity of the released metastable form, as the PRK-GAPDH association
hampers the overall flexibility of these enzymes. The released PRK with
decreased conformational constraint around the P-loop, because of a
memory effect, and with greater overall flexibility is thus a better
catalyst than the enzyme in the complex.
Whereas each PRK monomer in the oxidized complex has one disulfide
bridge, all the cysteinyl sulfhydryl groups of GAPDH are free and CP12
has two disulfide bridges. We found five cysteinyl sulfhydryl groups
per PRK subunit by alkylation after reduction of the oxidized complex.
The GAPDH content of thiol groups obviously does not change, but two or
four thiol groups become titrated per CP12 monomer. Reduction of the
PRK disulfide bridge is followed by an increase in enzyme activity, as
mentioned above.
Chlamydomonas GAPDH is an A4 homotetramer; the regulatory
cysteine residues are thus absent (28, 31). Nonetheless, incubation of
a crude extract with DTT increased its enzyme activity 3-fold (32).
Somewhat surprisingly, we saw the same increase in
NADPH-dependent activity for GAPDH when it was part of the
complex, but not for the isolated enzyme. We therefore proposed (49)
that this modulation had a physiological role, as the NADPH
concentrations are increased in the light. Our present results also
show that reducing the complex leads to a decrease in the use of NADH.
As titration of the thiol groups in GAPDH in the complex reveals 4 SH
groups whatever the redox state of the complex, the modulation of GAPDH
activity is not linked to disulfide reduction. Our present finding also
indicates that these effects are linked to heterologous interactions
within the complex as there is no longer an effect once these
interactions are weakened or broken. This supports the idea mentioned
above that new properties may emerge as a consequence of conformation
change within multienzyme complexes. It also shows that the regulation
of an enzyme like PRK may modulate the regulation of another (GAPDH)
via a "domino-like" effect.
We have tested the effect of oxidizing agents on PRK and GAPDH
activities, as oxidized thioredoxin causes the oxidation of the
light-reduced enzymes. Treatment with oxidants caused the activity of
reduced PRK isolated or released from the complex (e.g.
using cystine) to decrease. This effect was reversed by reduction, as
expected. The situation is quite different for GAPDH, as the loss of
activity by the isolated enzyme that follows incubation with oxidized
glutathione is very likely because of a modification of the active site
cysteine (Cys156), as it is well documented for other
GAPDHs (50). This is also supported by protection experiments using
BPGA and by the fact that oxidized thioredoxins have no effect on GAPDH
activity. The inactivation is not reversed by a reducing agent. It has
been suggested that the oxidation of glycolytic enzymes may introduce an element of strain resulting from the formation of a disulfide bond,
which then causes an irreversible conformational change, perhaps with
displacement of an essential residue from the active site. GAPDH is not
significantly inactivated by oxidized glutathione even after
dissociation of the complex, like PRK. This may be because the
cysteinyl sulfhydryl in this molecule is less exposed than is that in
the isolated GAPDH. We checked this by measuring the enzyme kinetics of
the isolated GAPDH and of the released form from the complex. The
differences observed with the pseudo-affinity constants for BPGA
clearly support the assertion made above. Thus, GAPDH retains the
conformation it had within the complex (imprinting) even after the
physical separation of PRK and GAPDH, so that the sulhydryl group of
the Cys156 is poorly accessible to oxidized glutathione.
This corroborates the imprinting effect previously reported (12, 13,
26).
Finally, the complex we have purified is a suitable model with which to
study protein-protein interactions, but most of the information on it
stems from in vitro reconstitution experiments. This report,
for the first time, describes the use of Biosensor technology to
determine the dynamics of these interactions between PRK and the
GAPDH·CP12 subcomplex under different redox states. The dissociation
constants between the PRK and GAPDH·CP12 subcomplex are rather low
(nM range), with the lowest value (14 ± 1.6 nM) being obtained with oxidized PRK. The probable
conformational change caused by the chemical modification of GAPDH by
oxidation has no influence on the binding of GAPDH to PRK and
vice versa. The SPR results indicate that these enzymes (PRK
and GAPDH/CP12) may reconstitute the complex under reducing conditions
with a low dissociation constant (62 ± 10 nM) even if
complex formation is sensitive to the redox state of PRK. Our results
also suggest that this complex may form at pH 7, under oxidizing
conditions (dark conditions) or at pH 8, under mild reducing conditions
(light conditions).
To conclude, thiol/disulfide exchange influences the state of
activation of chloroplast PRK, in its isolated form and when it is part
of a complex. On the other hand, the modulation of PRK activity
influences the state of activation of GAPDH via protein-protein interactions, at least in the alga C. reinhardtii. A new
mode of light regulation could emerge that involves a "domino
effect" and this could well apply to enzymes that are not direct
targets of light.