(Received for publication, March 18, 1997, and in revised form, June 4, 1997)
From the Institut de Biotechnologie des Plantes, ERS 569 CNRS, Bâtiment 630, Université de Paris-Sud, 91405 Orsay Cedex, France
The chloroplastic NADP-malate dehydrogenase is activated by thiol/disulfide interchange with reduced thioredoxins. Previous experiments showed that four cysteines located in specific N- and carboxyl-terminal extensions were implicated in this process, leading to a model where no internal cysteine was involved in activation. In the present study, the role of the conserved four internal cysteines was investigated. Surprisingly, the mutation of cysteine 207 into alanine yielded a protein with accelerated activation time course, whereas the mutations of the three other internal cysteines into alanines yielded proteins with unchanged activation kinetics. These results suggested that cysteine 207 might be linked in a disulfide bridge with one of the four external cysteines, most probably with one of the two amino-terminal cysteines whose mutation similarly accelerates the activation rate. To investigate this possibility, mutant malate dehydrogenases (MDHs) where a single amino-terminal cysteine was mutated in combination with the mutation of both carboxyl-terminal cysteines were produced and purified. The C29S/C365A/C377A mutant MDH still needed activation by reduced thioredoxin, while the C24S/C365A/C377A mutant MDH exhibited a thioredoxin-insensitive spontaneous activity, leading to the hypothesis that a Cys24-Cys207 disulfide bridge might be formed during the activation process. Indeed, an NADP-MDH where the cysteines 29, 207, 365, and 377 are mutated yielded a permanently active enzyme very similar to the previously created permanently active C24S/C29S/C365A/C377A mutant. A two-step activation model involving a thioredoxin-mediated disulfide isomerization at the amino terminus is proposed.
NADP-dependent malate dehydrogenase (NADP-MDH)1 (EC 1.1.1.82) catalyzes the reduction of oxaloacetate into malate in higher plants. In C4 plants, such as sorghum or maize, it is located in the chloroplasts of mesophyll cells where it participates in the exportation of reducing equivalents needed for the photosynthetic fixation of atmospheric CO2 into organic molecules in bundle sheath cell chloroplasts (1). Among all the malate dehydrogenases studied so far, the NADP-dependent isoform exhibits a unique property. Whereas MDHs using NAD are permanently active, the NADP-dependent isoform is totally inactive in the dark and activated in the light (2). It is now clearly established that this activation is mediated via the photosynthetic electron transfer and the ferredoxin/thioredoxin system and corresponds to the reduction of disulfides present in the inactive form (3). By thiol derivatization before and after activation and site-directed mutagenesis, the disulfide bridges reduced by thioredoxins have been identified (4, 5). To reach full activity, two disulfide bridges must be reduced: an amino-terminal one (cysteines 24 and 29) and a carboxyl-terminal one (cysteines 365 and 377). When these four cysteines are mutated, the mutant protein is permanently active. The two regulatory disulfide bridges belong to two sequence extensions characteristic of the NADP-dependent isoform and absent from the NAD-dependent isoforms (6). It has been proposed that the carboxyl-terminal extension shielded the access to the active site in the oxidized form and moved upon reduction (7), whereas the reduction of the amino-terminal bridge triggered a slow conformational change shaping the active site in a high activity conformation (5).
The four regulatory cysteines have no catalytic role since their replacement by alanines does not impair the catalytic activity. Nevertheless, it has been shown that the NADP-MDH activity was sensitive to thiol reagents, such as iodoacetate or iodoacetamide. Moreover, the permanently active C24A/C29A/C365A/C377A mutant protein is sensitive to diamide, a reagent known to reoxidize vicinal thiols into disulfides (5). This observation suggested that an internal cysteine might be important for catalytic activity and also raised the question of the possible existence of an internal disulfide bridge. The NADP-MDH contains eight cysteines, at strictly conserved positions for all the NADP-isoforms; these conserved cysteines are not found in the NAD-dependent isoforms (6). Four of those cysteines, belonging to sequence extensions, have been shown to play a role in the activation process and are not involved in catalytic activity. Concerning the four internal cysteines (cysteines 175, 182, 207, and 328), Cys175, located at the active site, has been studied earlier and shown to have no catalytic function (8). In the present study, we examined the possible role of the three other internal cysteines in the activity of the enzyme. Quite unexpectedly, one of them, i.e. Cys207, appeared to have a role in the reductive activation of the enzyme.
Restriction endonucleases, DNA modification enzymes, T4 DNA ligase, and T4 DNA polymerase were from Appligene. DEAE-Sephacel and Matrex Red A chromatographic supports were respectively from Pharmacia and Grace-Amicon. Chemicals (from Sigma, Boehringer, or Prolabo) were of analytical grade. Oligonucleotides were purchased from Eurogentec. Radiolabels were from Amersham Corp.
Escherichia coli strain XL1 blue (CLONTECH) was used to produce high yields of plasmids and M13 single-stranded DNA. All the other strains and vectors were the same as described in Ref. 9. E. coli strain RZ1032 and M13mp19 phage (Pharmacia Biotech Inc.) were used to produce deoxyuridine-substituted M13 single-stranded DNA for site-directed mutagenesis, pUC9 was used when needed for cloning strategy, and E. coli strain BL21 (DE3) (10) was used for the production of mutated NADP-MDHs encoded by recombinant pET-8c vectors. Bacteria were grown at 37 °C on Luria broth medium; ampicillin at 50 µg/ml was added when the bacteria carried plasmids conferring drug resistance.
Preparation of Mutated mdh cDNAsThe cDNAs encoding the NADP-MDHs bearing the single mutations C207A, C207S, C182A, and C328A were obtained by site-directed mutagenesis using the method of Kunkel (11). The single-stranded M13 template used was M13mdh, a vector in which the wild-type NADP-MDH cDNA is cloned (9). The mutagenic oligonucleotides are presented in Table I. A screening of the mutants was performed by sequencing single-stranded M13 DNA obtained from a few plaque isolates. The cDNA coding for the NADP-MDH with the double mutation C175A/C207A was obtained using the same method and the same template except that for the annealing reaction a mixture of both mutagenic oligonucleotides was used (see Table I).
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The cloning strategies used to combine the mutations are summarized in
Fig. 1. The cDNAs bearing the
mutations C24S/C365A/C377A, C29S/C365A/C377A, and C207A/C365A/C377A
were obtained by exchanging the NcoI-NheI
fragment of the C365A/C377A mdh cDNA cloned in pUC9 for
the corresponding fragment of, respectively, C24S mdh
cDNA, C29S mdh cDNA, and C207A mdh
cDNA. To obtain the cDNA with the double mutation C207A/C328A,
the NheI-BamHI fragment of the C207A/C365A/C377A mdh cDNA cloned in pUC9 was exchanged for the
corresponding fragment of the C328A mdh cDNA. Because
the NheI-BamHI encompasses cysteines 328, 365, and 377, this cloning strategy yields a cDNA containing only the
mutations C207A and C328A. The C29S/C207A/C365A/C377A cDNA was
obtained by exchanging the ApaI-ApaI fragment of
the C207A/C365A/C377A mdh cDNA cloned in pUC9 for the
corresponding fragment of the C29S/C365A/C377A mdh cDNA.
The NcoI-BamHI fragments of all the mutated MDH
cDNAs were transferred to a pET vector for production of the
modified proteins.
DNA Sequencing
The DNA sequencing was done using the dideoxy chain termination method (12) (T7 sequencing kit, Pharmacia). For every mutated cDNA, the sequence of the whole NcoI/BamHI fragment in the expression vector was checked prior to recombinant protein production.
Production and Purification of Recombinant NADP-MDHsThe mutated NADP-MDHs were produced using the pET/BL21 production system. The experimental procedures for the production in E. coli and purification of the recombinant proteins were essentially as described in Ref. 9. The main steps consisted of ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sephacel, and affinity chromatography on Matrex Red A. When needed, a hydrophobic interaction step on TSK Phenyl 5PW was added. The column was equilibrated with 1.8 M ammonium sulfate in 0.1 M potassium phosphate buffer, pH 7.2. The enzyme was eluted with a linear decreasing gradient of ammonium sulfate (1.8-0 M) in the same buffer. The NADP-MDH was then dialyzed against 20 mM potassium phosphate buffer, pH 7.2, 1 mM EDTA and concentrated.
Polyacrylamide Gel ElectrophoresisProteins were separated by vertical SDS-PAGE following the method of Schägger and von Jagow (13). They were visualized by Coomassie Brilliant Blue staining.
Protein SequencingThe amino-terminal sequence of the purified proteins was verified with an Applied Biosystems model 476A automated sequencer equipped with an on-line phenylthiohydantoin-derivative analyzer.
Molecular Mass Determination by Size Exclusion ChromatographyGel filtration experiments were performed using a TSK 3000 SW column (300 × 7.5 mm). After loading the protein, elution was achieved with a 30 mM Tris-HCl, pH 7.9 buffer, containing 0.15 M NaCl. Polypeptides with known molecular masses were used as standards.
Activation and Enzyme Activity AssaysThe enzymes were activated at 25 °C, in 100 mM Tris-HCl buffer, pH 7.9, by 20 µM E. coli thioredoxin, reduced either chemically with 10 mM DTT or photosynthetically in a reconstituted light activation system (14). Recombinant E. coli thioredoxin was purified as in Ref. 15. NADP-MDH activity was measured on aliquots, at 30 °C, by following the decrease in absorbance at 340 nm, in a standard assay mixture (1 ml) containing 100 mM Tris-HCl, pH 7.9, 780 µM oxaloacetate, and 140 µM NADPH. The Km values for NADPH and oxaloacetate were determined on preactivated enzymes, unless otherwise indicated, by varying the concentrations of each substrate, the other being kept constant. S1/2 for thioredoxin were determined by measuring the initial rates of NADP-MDH activation in the presence of different concentrations of thioredoxin.
DEPC TreatmentThe sensitivity of the C207A and C207A/C365A/C377A mutant MDHs to inhibition by DEPC was tested by adding the reagent at a 1 mM concentration either before or after activation of the enzymes in the same conditions as described in Refs. 7 and 16. Substrate protection experiments were performed on the C207A/C365A/C377A mutant by adding 1 mM NADPH before the DEPC treatment. After the treatment, the enzyme was extensively dialyzed and submitted to activation by DTT-reduced thioredoxin.
As mentioned in the introduction, the NADP-MDH mutated
on Cys175 had been studied earlier (7). To get a complete
picture of the roles of cysteines in NADP-MDH, the three other internal
cysteines, e.g. Cys182, Cys207, and
Cys328, were individually mutated into alanines. After
production in E. coli and purification to homogeneity, the
three mutant proteins were found to be still active after activation by
reduced thioredoxins. It thus appeared clearly that none of the
internal cysteines was necessary for the catalytic process. No
spontaneous activity could be measured prior to activation. Activation
kinetics were followed by measuring NADP-MDH activity as a function of
time after addition of thioredoxins reduced either chemically by DTT
(Fig. 2) or photosynthetically in a
reconstituted light activation system (data not shown). The activation
time courses of the C182A and C328A mutant proteins, as well as the
half-saturation concentrations for thioredoxins (S1/2), were similar to those of the wild-type
enzyme and of the C175A mutant studied previously (7). In contrast, the
C207A mutant protein exhibited an accelerated activation time course and a 2 to 3 times lower S1/2 for thioredoxin (Table
II).
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The kinetic parameters of the mutant proteins were also determined after full activation (see Table II). No significant differences for the Km OAA could be detected between the proteins mutated on cysteine 182, 207, or 328 and the WT enzyme. The Km NADPH of the C328A NADP-MDH was close to that of the WT enzyme. The Km NADPH of the C182A and C207A mutants was about 4 times higher than that of the WT enzyme. When kcat values of the different mutants were compared, it appeared that the replacement of cysteine 182 or 207 did not impair but increased the catalytic activity (respectively about 5- and 2-fold), whereas the replacement of Cys328 by alanine resulted in a decrease of the catalytic activity (by about 2 times). Thus the presence of a cysteine at position 328 seemed to be favorable for the catalytic activity even though it was not directly implicated in the catalytic process. The higher Km NADPH of the C182A NADP-MDH suggested a possible role of Cys182 in the coenzyme binding, in accordance with its predicted position, close to the nicotinamide ring of the cofactor (5). The overall effects of these individual mutations on the catalytic efficiencies of the mutated proteins (kcat/Km) were rather limited, increased kcat being compensated by increased Km for NADPH. In contrast, the acceleration of the activation rate upon replacement of Cys207 by an alanine was rather unexpected and suggested that this cysteine could be involved in the activation process.
To strengthen this hypothesis, this residue was also replaced by a serine. The C207S mutant protein exhibited the same kinetic parameters as the C207A mutant protein, that is a Km OAA close to that of the wild-type enzyme, and a Km NADPH about 3 times higher (Table II). Concerning the activation process, the C207S mutant protein exhibited an accelerated activation time course and a lower S1/2 for thioredoxin, i.e. characteristics similar to those of the C207A mutant (Table II and Fig. 2) and to those obtained previously for the amino-terminal mutants mutated on either or both of the most amino-terminal cysteines (9).
Based upon these experiments, it could be concluded that cysteines 182, 207, and 328, like the previously studied Cys175, have no direct catalytic role in NADPH-MDH, but that Cys207 seemed to be implicated in the activation process.
Involvement of Cysteine 207 in the Activation by ThioredoxinsIn previous studies (5, 7), we have shown that the removal of the carboxyl-terminal disulfide of NADP-MDH by mutation of cysteines 365 and/or 377 opened the access to the active site but did not accelerate the slow activation rate of the enzyme which was linked to the presence of the amino-terminal disulfide. In contrast, mutation of amino-terminal cysteines (Cys24 and/or Cys29) accelerated the activation rate (9) and loosened the interaction between subunits (17) but did not give access to the active site. The kinetic effects of mutation of Cys207 into alanine or serine described above strongly suggested that this residue was involved in the activation process but did not identify the step in which this cysteine was implicated.
The role of the carboxyl-terminal bridge had been suggested first by
the existence of a slight spontaneous activity in the mutants where the
carboxyl-terminal disulfide had been removed. Supporting evidence for
this role came from experiments using DEPC, a histidine-derivatizing
reagent, which has been shown to constitute a good probe of the
accessibility of the active site (16): it inhibits the enzyme activity
only when the carboxyl-terminal disulfide bridge is open by reduction
or suppressed by site-directed mutagenesis. To investigate a possible
role of Cys207 in the accessibility of the active site, the
effect of DEPC was tested before and after the activation of the C207A
mutant MDH. The activated mutant was inhibited by DEPC to the same
extent as the activated WT enzyme (data not shown), but no inhibition occurred when the reagent was applied prior to activation,
i.e. the mutant enzyme could be fully activated by reduced
thioredoxin after removal of DEPC (Fig.
3). In contrast, when the C207A mutation was combined with the double mutation C365A/C377A which eliminates the
carboxyl-terminal disulfide, the unactivated enzyme was inhibited by
DEPC and could not be activated further (Fig. 3). As in the case of the
double carboxyl-terminal mutant, NADPH provided a protection against
inhibition (data not shown). Clearly, the mutation of
Cys207 alone did not give access to the active site.
Interestingly, the combined triple mutant exhibited a higher
spontaneous activity than the corresponding double carboxyl-terminal
mutant: about 20% of the activity of the activated enzyme (Fig. 3)
versus about 5% for the latter (5). Thus, a
Km for NADPH and a Km for
oxaloacetate could be measured for the non-activated triple mutant
enzyme (Table III). The
Km for oxaloacetate of this enzyme prior to
activation was found to be 220 µM, a value falling
between the Km of the fully activated
C207A/C365A/C377A triple mutant or WT enzymes (approximately 38 µM, Tables II and III) and the Km of
the non-activated C365A/C377A NADP-MDH (1100 µM, Ref. 5).
In other words, in the enzyme mutated on Cys207, the
conformation of the oxaloacetate binding site was intermediary between
the low affinity conformation of the unactivated wild-type enzyme and
the high affinity conformation of the fully activated wild-type enzyme.
This was an additional indication that Cys207 is implicated
in the activation process. More specifically, this result suggested
that this cysteine is involved in the conformational change of the
active site proposed to occur upon the reduction of the amino-terminal
bridge. Another feature suggesting that the Cys207 mutation
exhibited characteristics similar to those of the amino-terminal Cys
mutations rather than the carboxyl-terminal Cys mutations is the
sensitivity of the activation of this mutant to inhibition by NADP
(data not shown), which is a a well known property of the WT enzyme and
amino-terminal mutants and which is suppressed by mutation of the
carboxyl-terminal cysteines (5). The question can be raised of whether,
as is the case for the amino-terminal cysteine mutations, the mutation
of Cys207 loosened the interaction between subunits. To
answer this question, size exclusion chromatography experiments were
run on the unactivated C207A mutant under varying ionic strength
conditions. However, the oxidized protein behaved much like the WT
protein, i.e. could not be dissociated into monomers under
these conditions (data not shown).
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In a protein, the role of a cysteine residue can be related either to its hydrogen bonding capacity or to its hydrophobicity or to its ability to form a disulfide bridge. As the hydrophobic properties of cysteine are shared by alanine, and its hydrogen-bonding properties are similar to those of a serine, the identical results obtained with the mutations C207A and C207S are strongly in favor of the hypothesis that Cys207 reacts with another cysteine to form a disulfide bridge. In this case, a partner cysteine should be found, the mutation of which should yield the same modifications in the activation kinetics as the mutation of Cys207. None of the mutations of the other internal cysteines (cysteines 175, 182, and 328) altered the activation kinetics. Furthermore, combined mutations of each of these cysteines with Cys207 yielded proteins showing properties identical to those of the single Cys207 mutant (data not shown). Thus the formation of a disulfide bridge between any of these cysteines and Cys207 can be ruled out. Among the four regulatory cysteines located in extensions, single or double mutation of Cys365 and Cys377 did not yield accelerated activation time courses (5). Furthermore, as described above, the single mutation of Cys207 did not open the access to the active site and did not relieve the inhibition of activation by NADP. Thus it is highly unlikely that the partner cysteine of Cys207 would be one of the carboxyl-terminal cysteines. In contrast, the effects of mutation of Cys207 shared characteristics common with the mutation of the amino-terminal cysteines 24 and/or 29: accelerated activation time course and decreased S1/2 for thioredoxin (9). This suggested that the partner cysteine of Cys207 in a disulfide bridge might be one of the two amino-terminal cysteines.
From previous studies, it had been concluded that the activation of
NADP-MDH required the reduction of only two disulfides per monomer
(5). This conclusion was based on the observation that both single or
double amino-terminal mutations similarly accelerated the activation
kinetics and that the combined mutation of the two most amino-terminal
cysteines with either or both of the two most carboxyl-terminal
cysteines yielded permanently active proteins. However no attempt to
differentiate between possible different roles of Cys24 and
Cys29 in proteins where the carboxyl-terminal bridge had
been removed by mutation had been made. To fill this gap, proteins in
which a single mutation of one of the two amino-terminal cysteines was combined with the mutation of the two carboxyl-terminal cysteines were
produced. The biochemical properties of the C24S/C365A/C377A NADP-MDH
resembled those of the previously created C24S/C29S/C365A/C377A quadruple mutant. It was permanently active, and its activity could be
followed throughout the purification procedure without preactivation
(data not shown). However, as it was also rather unstable and
susceptible to oxidation, maximal activity of the pure protein could be
obtained only if 1 mM DTT were included in the reaction
medium (Fig. 4). In contrast, the
C29S/C365A/C377A NADP-MDH still required activation by reduced
thioredoxin to reach full activity (Fig. 4). Its activation rate was
very similar to the activation rate of the C207A/C365A/C377A mutant,
but its spontaneous activity was very similar to that of the
C365A/C377A NADP-MDH, i.e. approximately 5% of the activity
of the fully activated protein. The Km OAA of the
unactivated enzyme (477 µM) was higher than that of the
fully activated enzyme (66 µM, Table III) but somewhat
lower than that of the non-activated C365A/C377A NADP-MDH (1100 µM, Ref. 5). Clearly, this mutant shared some
similarities with the C207A/C365A/C377A mutant but also exhibited some
notable differences. In particular, despite the fact that the
thioredoxin-dependent activation of both mutants proceeded
at similar rates, the C207A/C365A/C377A mutant could be fully activated
either by DTT or by mercaptoethanol alone, although much more slowly,
whereas the C29S/C365A/C377A mutant was strictly dependent on reduced
thioredoxin for activation (Fig. 4). The thioredoxin dependence of the
activation can be unambiguously established by using a reconstituted
light activation system in which thioredoxins are reduced by the
photosynthetic electron transfer. In this system both C207A/C365A/C377A
and C29S/C365A/C377A triple mutants were activated at higher rates than
was the WT protein, without the typical lag in activation of the latter
(Fig. 5). In contrast, when the
C24S/C365A/C377A mutant was assayed in this system after having been
inactivated by dilution, its activity could not be restored unless DTT
was added either to the activation medium or to the reaction cuvette
(Fig. 5).
Summarizing the results obtained with the three combined triple mutations, it can be concluded that whereas in the C24S/C365A/C377A mutant there was no thioredoxin-reducible disulfide bridge left, a thioredoxin-reducible disulfide was still present in both the C29S/C365A/C377A and the C207A/C365A/C377A mutants. This observation suggested that Cys24 could form a disulfide bridge with Cys29, as shown previously, or alternately with Cys207. If this interpretation is valid, a quadruple mutant combining the mutation of Cys29 and Cys207 should yield a permanently active protein. This was indeed the case (Fig. 5). The C29S/C207A/C365A/C377A NADP-MDH was fully active, and its activity could not be enhanced by reduced thioredoxin. Its kinetic properties were very similar in all respects to those of the previously created C24S/C29S/C365A/C377A quadruple mutant (5) and, more generally, to those of the activated WT enzyme (Table III).
The present work where the roles of Cys182,
Cys207, and Cys328 of NADP-MDH were examined
allows us to draw a general picture of the roles of the eight strictly
conserved cysteines of NADP-MDH. It rules out the direct involvement of
any cysteine in the catalytic process. It confirms the previously
proposed hypothesis that the four cysteines belonging to sequence
extensions participate in the activation of the enzyme. Among the
internal cysteines, Cys182 and Cys328, as well
as the previously studied Cys175 (8) clearly have no role
in activation. Their substitution by site-directed mutagenesis did not
modify the activation kinetics. In contrast, mutation of
Cys207 into either Ala or Ser yielded mutants with
accelerated activation rates. This suggested that the role of
Cys207 was neither related to its hydrophobicity nor to its
hydrogen-bonding capacity but specifically to its ability to form
disulfide bridges. This conclusion was rather unexpected, as none of
the three other internal cysteines induced similar modifications upon
mutation, and hence none of them could be a partner of
Cys207. On the other hand, the four external cysteines had
already been shown to be paired: Cys24 with
Cys29 and Cys365 with Cys377. A
closer analysis of the properties of the Cys207 single
mutant proteins showed that they shared a number of characteristics with the proteins mutated on the Cys of the amino-terminal extension. Their activation rates were accelerated, and their half-saturation concentrations for thioredoxin were lowered to the same extent (9). In
contrast, they did not show any of the characteristics of the enzymes
mutated at the two most carboxyl-terminal cysteines, i.e.
slight spontaneous activity, slow activation kinetics, accessibility of
the active site histidine to DEPC in the unactivated form, and lack of
inhibition of activation by NADP (5). In an attempt to identify
unequivocally, among the three cysteines yielding similar functional
consequences upon mutation, the cysteine pair implicated in activation,
cumulated mutations of each of these cysteines were performed on
enzymes where the carboxyl-terminal bridge was removed by mutagenesis.
The functional consequences of these mutations are summarized in Fig.
6. When a permanently active,
thioredoxin-insensitive enzyme was obtained, it could be concluded that
no reducible disulfide remained. If the mutant enzyme was still
activable by reduced thioredoxin, the position of the remaining
disulfide could be identified. From Fig. 6 it is clear that a
thioredoxin-reducible disulfide can be formed between Cys24
and Cys29 (the previously identified amino-terminal
regulatory disulfide) and also between Cys24 and
Cys207. The question of the physiological relevance of
these alternate disulfides is of obvious importance. The mere fact that
both of them were much more efficiently reduced by thioredoxin than by DTT suggests that both belong to a physiological process. The presence
of the Cys24-Cys29 disulfide was first
established on the basis of chemical derivatization and amino acid
sequencing. In the unactivated enzyme, both amino-terminal cysteines
were unavailable to derivatization and hence appeared as gaps in amino
acid sequencing. Both became available (and visible in the primary
sequence) after full activation of the enzyme (4). Based on this
evidence, the amino-terminal disulfide initially present in the
oxidized enzyme can be identified as the
Cys24-Cys29 disulfide. The mutant protein
where only this disulfide remained was activated much more efficiently
by reduced thioredoxin than by DTT alone. It could be fully activated
by DTT, although much more slowly. The mutant protein where only the
Cys24-Cys207 bridge remained was also
activated very efficiently by reduced thioredoxin but could not be
activated by DTT alone under standard low ionic strength conditions.
Similarly, under the same conditions, the WT protein could not be
activated by DTT alone, the only difference between the mutant and WT
proteins being the slower activation rate of the WT enzyme. These
observations strongly suggest that the
Cys24-Cys207 bridge is a thioredoxin-reducible
physiologically important disulfide bridge formed during the activation
process and that the faster activation of the mutant protein containing
this single disulfide results from the fact that one activation step
had been suppressed by mutation, namely, the reduction of the
amino-terminal Cys24-Cys29 disulfide. The slow
activation rate linked to the reduction of the amino-terminal disulfide
bridge has been previously ascribed to a slow conformational change
following reduction, the disulfide reduction per se being
fast. The present mutagenesis experiments suggest that this
conformational change is linked to the isomerization of amino-terminal
disulfides followed by a second reduction step. It has been shown
previously that the elimination of the amino-terminal disulfide by
site-directed mutagenesis (9) or by proteolysis (18, 19) weakened the
interaction between subunits. No such effect was observed upon mutation
of Cys207. On the other hand, mutation of
Cys207 or, to a lesser extent, Cys29 combined
with the mutation of the two carboxyl-terminal cysteines led to a
decrease in the Km OAA of the unactivated proteins (Table III), suggesting that these residues (and especially
Cys207) are involved in the conformational change of the
active site toward a higher efficiency.
In an attempt to visualize the succession of events following the reduction of the Cys24-Cys29 disulfide bridge, we propose that this reduction would be followed by the formation and further reduction of a Cys24-Cys207 disulfide bridge, the formation of this second disulfide being favored by the loosening of the interaction between subunits. This proposal is supported by structural data. The core part of NADP-MDH has been modeled using the coordinates of the crystal structure of NAD-MDH (5, 20). In the model, Cys207 appears to be located at the subunit interface, in a half-buried position. A change in the interaction between subunits can bring it into a more exposed position. In this regard, it should be mentioned that Cys207 was one of the cysteines Reng et al. (21) proposed to be implicated in the activation process of pea NADP-MDH and that an amino-terminally truncated pea MDH is able to form mixed disulfides with glutathione (22).
The updated functional model we propose for NADP-MDH activation (Fig.
7) accomodates all the experimental data
gathered from site-directed mutagenesis experiments. It is also
consistent with the existence of the "preregulatory" disulfide
bridge proposed by Hatch and Agostino (23). These authors observed that
a pretreatment of NADP-MDH with mercaptoethanol did not activate the
enzyme but accelerated its further activation by reduced thioredoxin.
Such a result can be expected if mercaptoethanol reduced the
Cys24-Cys29 bridge but was unable to reduce
either the Cys24-Cys207 bridge formed
subsequently (Fig. 5) or the Cys365-Cys377
bridge (5, 9).
In the light of the present results, the reductive activation of NADP-MDH by thioredoxin can be viewed as a protein unfolding pathway, the activation resulting not only from reduction of disulfides but also from their transient isomerization leading to a conformational change of the active site. Until now, isomerization of disulfides was shown to be an obligate step of the protein folding pathway (24), but recently it has been demonstrated that isomerization could also occur upon an unfolding triggered by disulfide reduction (25). Correct protein folding is considered to be catalyzed by protein-disulfide isomerase (26), which is a strong oxidant. However, thioredoxin was also shown to possess a disulfide isomerase activity (27). The present work suggests that thioredoxin could also be involved in the isomerization of disulfides in conjunction with its disulfide reducing function. The case of NADP-MDH is the best documented among the light-activated chloroplastic enzymes in this respect. However, it is not the only known example. Recent mutagenesis experiments on the thioredoxin-regulated chloroplastic fructose-1,6-bisphosphatase showed that three cysteines were involved in the reductive activation of the enzyme (28). Thus the light regulation of some chloroplastic enzymes could be viewed as a general regulation process in which thioredoxin, in addition to its disulfide reducing activity, fulfills a disulfide isomerase function, thus ensuring a correct folding/unfolding of the light-regulated enzymes.
We thank Dr. J-P. Jacquot for providing E. coli thioredoxin, P. Decottignies and J-P. Jacquot for helpful discussions and critical reading of the manuscript, Dr. D. Knaff for help in revising the manuscript, Prof. P. Gadal for interest and support, and graduate students A. Lagorce and C. Reculé for some Km determinations.