From the Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030
Received for publication, October 29, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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Protein-disulfide isomerase (PDI)
catalyzes the formation and isomerization of disulfides during
oxidative protein folding. This process can be error-prone in its early
stages, and any incorrect disulfides that form must be rearranged to
their native configuration. When the second cysteine (CGHC) in the PDI
active site is mutated to Ser, the isomerase activity drops by
7-8-fold, and a covalent intermediate with the substrate accumulates.
This led to the proposal that the second active site cysteine provides
an escape mechanism, preventing PDI from becoming trapped with
substrates that isomerize slowly (Walker, K. W., and Gilbert, H. F. (1997) J. Biol. Chem. 272, 8845-8848). Escape also
reduces the substrate, and if it is invoked frequently, disulfide
isomerization will involve cycles of reduction and reoxidation in
preference to intramolecular isomerization of the PDI-bound substrate.
Using a gel-shift assay that adds a polyethylene glycol-conjugated
maleimide of 5 kDa for each sulfhydryl group, we find that PDI
reduction and oxidation are kinetically competent and essential for
isomerization. Oxidants inhibit isomerization and oxidize PDI when a
redox buffer is not present to maintain the PDI redox state. Reductants
also inhibit isomerization as they deplete oxidized PDI. These rapid
cycles of PDI oxidation and reduction suggest that PDI catalyzes
isomerization by trial and error, reducing disulfides and oxidizing
them in a different configuration. Disulfide reduction-reoxidation may
set up critical folding intermediates for intramolecular isomerization,
or it may serve as the only isomerization mechanism. In the absence of
a redox buffer, these steady-state reduction-oxidation cycles can
balance the redox state of PDI and support effective catalysis of
disulfide isomerization.
The folding of proteins destined for the secretory pathway occurs
in the endoplasmic reticulum where a quality control system ensures that secreted proteins are correctly folded (1), including the
correct formation of disulfide bonds. Disulfides that form early in the
folding process are often incorrect; cysteines can be mispaired (2), or
disulfides can be formed in the wrong temporal order, making it
difficult to oxidize buried cysteines (3). To rectify these errors, the
incorrect disulfides must be broken and new ones formed in a
different configuration.
The endoplasmic reticulum contains folding assistants that help
proteins achieve their correct disulfide arrangement. Protein-disulfide isomerase (PDI)1 is a
55-kDa protein of the endoplasmic reticulum which catalyzes disulfide formation (oxidase activity) as well as the rearrangement of
incorrect disulfide pairings (isomerase activity) (4), accelerating both processes without drastically altering the refolding pathway (1).
PDI catalyzes the chemical changes but does not appear to guide the
process or unfold misfolded substrates actively (5, 6). PDI has two
active sites, one near the amino terminus and the other near the
carboxyl terminus (7). Each active site contains two cysteines in the
sequence WCGHCK which mediate the catalytic activities. The oxidase
activity of the enzyme clearly requires the reaction of an oxidized PDI
active site with a reduced substrate to introduce a substrate
disulfide. Isomerization results in no net redox state change, but a
substrate disulfide must be broken to initiate rearrangement. The first
step of isomerization involves a reduced PDI active site attacking a
substrate disulfide (8). After the initial reaction, two different
mechanisms could result in substrate isomerization (Fig.
1). In the first mechanism, the
sulfhydryl of the substrate cysteine that was released from a disulfide
reacts intramolecularly with a different substrate disulfide, resulting
in a different disulfide configuration. The intramolecular
rearrangement concludes when a substrate cysteine displaces PDI from
the covalent complex, forming another disulfide in the substrate and
regenerating reduced PDI for another round of catalysis. In the second
model, reduced PDI could simply engage in cycles of reducing substrate
disulfides and reoxidizing them in a different orientation, much in the
same way that the ATP-dependent chaperonin, GroEL/ES
provides a folding substrate with numerous attempts at reaching the
native state through cycles of substrate binding and release (9).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Mechanism of PDI-catalyzed disulfide
isomerization. Two alternative pathways can accomplish disulfide
isomerization. The top (blue) pathway represents
an intramolecular rearrangement of the substrate while bound to PDI as
a disulfide. The bottom (tan) pathway represents
cycles of disulfide reduction and reoxidation in a different
orientation. GSH (red) inhibits the reduction-reoxidation
pathway by depleting oxidized PDI, which is required to reoxidize the
reduced substrate.
To account for the observation that PDI active sites lacking the carboxyl cysteine (CGHS) are deficient in catalyzing disulfide isomerization unless supported by a glutathione redox buffer, Walker and Gilbert (10) proposed the scanning and escape model for PDI-assisted disulfide isomerization of scrambled ribonuclease. In this model, the first (more amino-terminal) active site cysteine of PDI initiates a disulfide rearrangement by attacking a reactive disulfide within the substrate and forming a covalent complex (scanning). The resolution of this complex depends on the relative rates of an intramolecular isomerization pathway versus the escape of PDI from the covalent complex using the second active site cysteine (Fig. 1). By this model, escape serves as an internal clock providing some time for an intramolecular substrate isomerization to occur but limiting the time PDI spends tied up in rearranging complexes. However, the escape pathway releases the substrate from the covalent complex forming a reduced RNase and oxidized PDI. To complete the substrate isomerization, oxidized PDI must then locate and reoxidize the reduced sRNase molecule in an alternate configuration to generate the rearranged substrate and recycle PDI to the reduced state to initiate further rounds of substrate reduction and oxidation.
The scanning and escape model predicts that both oxidized and reduced
PDI are necessary for the efficient catalysis of isomerization, even
when there is no net disulfide reduction or oxidation. We have
monitored the redox state of PDI during steady-state disulfide isomerization and found that both reduced and oxidized PDI are essential to observe net isomerization. Rapid reduction-reoxidation cycles accompany the isomerization of sRNase. They are kinetically competent to account for substrate isomerization, and intramolecular substrate isomerization is a relatively rare event during the PDI-assisted isomerization of sRNase A. These reduction-reoxidation cycles can also balance the redox state of PDI in a nonequilibrium steady state because of the relatively slow reaction between PDI and a
glutathione redox buffer.
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EXPERIMENTAL PROCEDURES |
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Materials--
Ribonuclease A, glutathione disulfide (GSSG),
glutathione (GSH), cCMP, DTT, 5,5'-dithiobis(2-nitrobenzoic acid),
glutathione reductase, and NADPH were obtained from Sigma.
Mal-PEG was obtained from Shearwater Corporation (Huntsville,
AL). PDI was purified as described (11). The concentration was
determined by absorbance at 280 nm using an E0.1% of 0.94 (mg/ml)1 cm
1 (12). Mutation of the two
noncatalytic cysteines of PDI was performed using the
QuikChange® Multi Site-directed Mutagenesis kit purchased
from Stratagene. The mutations were verified by dideoxy sequencing. The
production and isolation of other mutants have been described
previously (13). Scrambled ribonuclease was prepared by the method of
Hillson et al. (14) with minor changes.
Assay of RNase Refolding--
PDI activity was measured by
observing the activity caused by the formation of native RNase using
cCMP as a substrate (12, 15). The cCMP at an initial concentration of
4.5 mM was monitored continuously at 296 nm using a
of 0.19 mM
1 cm
1. The active
RNase concentration at any time was calculated from the first
derivative of the absorbance versus the time curve. The
assay was performed at pH 8.0, 25.0 °C in 0.1 M Tris-HCl
containing various components of a glutathione redox buffer and PDI as
described in the figure legends. The reaction was initiated by the
addition of sRNase.
In experiments where it was necessary to exclude GSSG, the rate of refolding of sRNase was measured in the presence of 0.75 unit/ml glutathione reductase and 0.27 mM NADPH. Assays were performed in 100 mM Tris-HCl, 2 mM EDTA, pH 8.0.
Partitioning between Reduction and Isomerization--
At higher
concentrations of GSH, the background oxidation of NADPH caused
absorbance changes at 296 nm which interfered with continuously
monitoring the cCMP concentration over the time course of native RNase
formation. Consequently, we monitored the partitioning between sRNase
reduction and isomerization to native RNase. The rate of reduction of
27 µM sRNase was determined at various concentrations of
GSH ranging from 0 to 7 mM. All assays were done in the
presence of 0.75 unit/ml glutathione reductase and 0.27 mM
NADPH to reduce any GSSG present. The PDI concentration was as
indicated in the figure legends. Assays were conducted in a buffer of
90 mM Tris-HCl, 1.8 mM EDTA, pH 8.0, at
25 °C. The rates of reduction were determined from the slope of
absorbance at 340 nm versus time using an extinction coefficient of 6.23 mM1
cm
1.
To measure the partitioning between sRNase reduction and refolding, the amount of native RNase was determined by measuring its enzymatic activity after an overnight incubation of sRNase with PDI, various concentrations of GSH, and 0.75 unit/ml of glutathione reductase and 0.27 mM NADPH. After overnight incubation at room temperature, cCMP was added to a concentration of 4.5 mM, and the initial rate of hydrolysis was measured. A small background rate of NADPH oxidation caused by GSH oxidation by air was subtracted from the observed rate.
Preparation of Reduced and Oxidized PDI-- Reduced PDI was prepared by incubating PDI with 0.5 mM DTT overnight at room temperature. The DTT was removed by centrifugal gel filtration over Sephadex G-50 coarse from Amersham Biosciences. Oxidized PDI was prepared by incubating PDI with 10 mM GSSG overnight at room temperature. The GSSG was removed by centrifugal gel filtration over Sephadex G-50 coarse from Amersham Biosciences. Controls showed no significant carryover of DTT or GSSG during gel filtration.
Gel-shift Assays of the PDI Redox State--
To capture the
redox state of PDI during steady-state turnover or in equilibrium with
a glutathione redox buffer, the reaction mixture was quenched at the
appropriate time with an equal volume of ice-cold 40% trichloroacetic
acid to precipitate the proteins and stop further
thiol/disulfide exchange. After a 1-h incubation on ice, the
precipitated proteins were isolated by centrifugation, and the pellets
were washed twice with acetone to remove the trichloroacetic acid. The
pellet was dissolved in 30 µl of nonreducing 2× SDS sample buffer
and split into two equal portions. One portion immediately received 5 mM Mal-PEG (final concentration), and the other received an
equivalent amount of buffer only. This provides an internal recovery
control for each sample. Controls in which the trichloroacetic acid
pellet was dissolved directly into Mal-PEG showed that the short time
before the addition of Mal-PEG to the split samples did not result in
any redox changes. The unbound Mal-PEG in the sample buffer resulted in
distortion of the protein migration so it was removed by dialysis (1 h
at room temperature) of the split samples against sample buffer using
10,000 MWCO microdialysis devices from Pierce. SDS-PAGE was performed
on a nonreducing 4-20% Tris-HCl polyacrylamide gel. Protein bands
were visualized by staining with Coomassie Blue, or the proteins were
transferred to nitrocellulose and detected using a monoclonal anti-PDI
antibody (Stress-Gen) or a polyclonal anti-PDI antibody.
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RESULTS |
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Substrate Inhibition-- One distinction between an intramolecular mechanism of disulfide rearrangement and a mechanism involving cycles of substrate reduction and reoxidation lies in the consequences of releasing a reduced substrate and forming a molecule of oxidized PDI. If reductive escape happens frequently, increasing the concentration of an oxidized substrate such as sRNase would tend to oxidize more and more of the PDI. As the concentration of sRNase rises, it should also become more difficult for this oxidized PDI to locate an appropriately reduced substrate to complete the catalytic cycle. A large excess of fully oxidized substrate should compete with the much lower concentration of partially reduced substrate for binding to oxidized PDI. Consequently, substrate inhibition at high concentrations of sRNase would be expected if reductive escape is a frequent phenomenon, but simple saturation behavior would be characteristic of an exclusively intramolecular mechanism.
When the isomerization of sRNase (four disulfides) is initiated by the
addition of reduced PDI, isomerization proceeds rapidly when the
concentration of sRNase is low (8 µM). Under these
conditions, a glutathione redox buffer has little effect on the
isomerization rate (Fig. 2), suggesting
that there is no large imbalance in the PDI redox state. However, when
the substrate concentration is increased to 32 µM, the
initial velocity of native RNase formation actually falls 7-fold (Fig.
2), even though the Km for this substrate is
high (40 µM), and the rate would have been expected to
increase by almost 4-fold. If the excess substrate is preventing the
oxidized PDI that is formed from locating a reduced substrate, adding
more oxidized PDI should increase the rate of RNase oxidation and help restore the isomerization activity by increasing the rate of substrate oxidation and by generating more reduced PDI in the process. Adding oxidized PDI to the assay, along with reduced PDI, does increase the
isomerase activity, but the increase is still significantly less
than that produced by adding a redox buffer (Fig. 2).
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PDI Redox State-- The depletion of reduced PDI during the early stages of the reaction could also compromise initiating new rounds of substrate reduction. To visualize the redox state of PDI during steady-state turnover, a gel-shift assay was developed to detect the number of available sulfhydryl groups present on PDI under turnover conditions. The basis of the assay is the modification of the available PDI sulfhydryl groups by a PEG-conjugated maleimide (Mal-PEG) with an average molecular weight of 5,000 (16). Because disulfides are not maleimide-reactive, only reduced PDI molecules show a shift. PDI normally has two cysteines outside the active site (in the b' domain). They do not contribute to catalysis (17), but their presence creates ambiguity in the site(s) of modification. Consequently, the initial experiments were performed with a mutant PDI (internal mutant, ImPDI) in which the two internal, nonactive site cysteines are mutated to serines. This restricts the location of the modification by Mal-PEG to the active site cysteines. The mutation of the internal cysteines to serine has no effect on the catalytic activity compared with wild-type PDI (data not shown).
The ability of Mal-PEG to trap the cysteines of reduced PDI and capture
the PDI redox state accurately was examined by incubating ImPDI with
glutathione redox buffers designed to set the active site redox state
at various extents of reduction. It is essential to quench redox
changes rapidly and to remove small molecule thiols before trapping the
free cysteines with Mal-PEG. Consequently, after equilibration, the PDI
was precipitated with cold trichloroacetic acid, washed extensively
with acetone to remove traces of trichloroacetic acid, and the
precipitates were dissolved under denaturing conditions in the presence
of Mal-PEG. If Mal-PEG is added directly to the isomerization reaction
mixture without trichloroacetic acid precipitation, the competing
reactions of PDI disulfide reduction by the GSH and the modification of
the PDI thiols with Mal-PEG shift the PDI redox state toward more
reduced PDI (data not shown). As the redox buffer is varied, individual
bands corresponding to zero, one, two, three, and four Mal-PEG shifts
are observed (Fig. 3). The most
predominant bands are at zero, two, and four SH groups. Bands visible
at one and three modified cysteines are weaker because of the
difficulty of modifying both cysteines within a single active site
(CGHC) with this bulky reagent. Prolonging the reaction time causes
these bands to disappear but at the risk of reaction at residues other
than cysteine. A molecular mass shift of ~15 kDa/Mal-PEG
addition is observed (Fig. 3), consistent with the addition of a bulky
PEG molecule that is highly solvated and interacts weakly with SDS. The
presence of two active sites means that a shift of one or two SH groups
identifies a molecule where one of the two active sites is oxidized,
whereas a shift of three or four SH groups indicates a completely
reduced PDI.
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The two independent active sites have similar redox potentials and are
expected to titrate together. When the active sites are half-oxidized,
a statistical distribution of redox states would generate 25%
unshifted PDI (both sites oxidized), 25% fully shifted PDI (both sites
reduced), and 50% shifted by two modifications (one site oxidized, one
site reduced). From integrating the band intensities and calculating
the fraction of the active sites that were reduced, the average redox
potential
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(Eq. 1) |
Using this gel-shift assay, the redox state of PDI was examined during
sRNase isomerization. Reduced ImPDI was incubated for a short time (1 min) with high or low concentrations of sRNase in the presence or
absence of a glutathione redox buffer (Fig. 4A). As predicted, reduced PDI
was oxidized more extensively by 40 µM sRNase in the
absence of a redox buffer, but the presence of a redox buffer helps
maintain a higher concentration of reduced PDI without eliminating the
oxidized PDI. In the absence of a glutathione redox buffer, changing
the substrate concentration from 10 to 40 µM shifts the
PDI redox state from 48 ± 4% reduced (n = 4) to
28 ± 4% reduced (n = 4). A glutathione redox
buffer (1 mM GSH, 0.2 mM GSSG) also increases
the amount of PDI in its reduced form (74 ± 4%,
n = 4), suggesting that depletion of the amount of
reduced PDI contributes to the inhibition by high concentrations of the
sRNase substrate. Control experiments without PDI verify that sRNase
does not shift significantly under these conditions so that sRNase
multimers are distinguishable from PDI (Fig. 4A). Western
blots to visualize only the PDI (data not shown) confirm that the
indicated bands contain PDI.
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The Mal-PEG assay was also performed during isomerization catalyzed by wild-type PDI (Fig. 4B) with comparable results. Oxidized PDI shifts ~30 kDa upon the addition of Mal-PEG-MAL, suggesting that the two internal, nonactive site cysteines are still reduced (Fig. 4B). PDI in the completely reduced control shifts the equivalent of six Mal-PEG additions, as expected. As with ImPDI, the active site cysteines become more oxidized as the sRNase concentration increases.
Role of the Redox Buffer--
For a reduction-reoxidation
mechanism of isomerization, both oxidized and reduced PDI would have to
participate in the reaction. By contrast, an intramolecular
isomerization would only require reduced PDI. If oxidized PDI is
necessary to complete a redox cycle during isomerization, one might
expect that eliminating the oxidant, GSSG, from the redox buffer would
inhibit isomerization. To eliminate even small amounts of GSSG, the
GSSG was rapidly recycled to GSH using NADPH and glutathione reductase.
Under these conditions, GSH by itself can relieve the substrate
inhibition (Fig. 5), without a
requirement for GSSG.
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Although GSH with an NADPH trap will completely reduce PDI in the
absence of substrate, low concentrations of GSH (< 0.5 mM) are not sufficient to deplete the steady-state
concentration of oxidized PDI in the presence of high concentrations of
sRNase (Fig. 6). Thus, the reduction of
PDI by GSH must be slower than the formation of oxidized PDI through
substrate reduction. At even higher GSH concentrations (>0.5
mM) a substantial fraction of the PDI becomes reduced.
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A technical limitation of the isomerization assay precludes continuously monitoring the RNase-catalyzed hydrolysis of cCMP at GSH concentrations >0.5 mM. The background oxidation of NADPH as the reduction of the sRNase substrate produces absorbance changes at 296 nm which are too large to permit an accurate assessment of the isomerization rate during a continuous assay. Although sRNase is reduced by GSH and NADPH, native RNase is sufficiently stable that once formed, it is resistant to reduction by the highest GSH used (data not shown). The partitioning between reduction and isomerization will depend on the ratio of the velocities of the two alternative pathways. By measuring the velocity of sRNase reduction from the absorbance change caused by NADPH oxidation (Fig. 6A) and the amount of native RNase that is formed when the reaction is complete (Fig. 6B), we can determine how higher concentrations of GSH influence the isomerization of sRNase (Fig. 6C). At low GSH concentrations, all of the sRNase that can fold to the native structure does so (typically 50-70% of the sRNase present, varying among sRNase preparations). As the GSH concentration increases, the sRNase partitions more and more completely toward reduction so that less and less of the sRNase is converted to the native molecule. The sRNase partitions equally between reduction and isomerization when the GSH concentration is 1.2 mM (Fig. 6A).
The rate of reduction (Fig. 6A) increases linearly with
increasing GSH concentrations in the presence and absence of PDI. Because the isomerization pathway has to at least keep up with the
increasing reduction rate at low GSH (where the extent of isomerization
is constant and large) the rate of isomerization must increase as well.
When the isomerization pathway lags behind and more of the sRNase is
reduced, the velocity of isomerization must either stay constant or
drop as the GSH concentration increases further. Relative to the
velocity of sRNase reduction, the velocity of the competing
isomerization reaction will be given by
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(Eq. 2) |
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DISCUSSION |
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Two mechanisms can be envisioned for the PDI-catalyzed disulfide isomerization of sRNase (Fig. 1). In the intramolecular rearrangement mechanism, a free thiol is generated in the misoxidized substrate by attack of the more amino-terminal thiolate of the PDI active site. The free thiol of the substrate would then be required to attack another disulfide of the substrate, leading eventually to rearrangement and release of the substrate by reforming a different disulfide and expelling PDI. There is precedence for such a rearrangement in the uncatalyzed, intramolecular rearrangement of folding intermediates of bovine pancreatic trypsin inhibitor (20).
The alternative is that disulfide isomerization results from cycles of reduction and oxidation, simply trying again and again until isomerization leads to a product that is resistant to further rearrangement (10). Walker and Gilbert (10) suggested that the loss in activity resulting from mutation of the second (more carboxyl-terminal) cysteine in either active site could be most easily accommodated by a reduction-reoxidation mechanism because this cysteine does not participate in the intramolecular isomerization pathway. The timing of the intramolecular clock provided by the second active site cysteine will govern the partitioning between intramolecular isomerization and reduction-reoxidation pathways. If intramolecular substrate isomerization takes too long, the second active site cysteine will initiate escape and reduce the substrate. Using equilibrium and rate constants for the equilibration of the PDI active site with glutathione, Darby and Creighton (18) estimated the half-time for the intramolecular expulsion of glutathione to be ~50 ms, although the rate constant could be different for protein substrates. Whether or not reductive escape is an important feature of the mechanism depends on how frequently it occurs during substrate turnover and whether or not redox cycling of the substrate and catalyst are essential during catalysis of isomerization.
Substrate reduction and oxidation by PDI are both kinetically competent to participate in a reduction-reoxidation mechanism of isomerization. When reduced PDI and sRNase (40 µM) are mixed, the redox state changes are finished within 1 min (kobs for reducing sRNase is >8 µM substrate reduced/min/µM PDI) (Fig. 6A), and at high substrate, most of the PDI is quickly converted to its oxidized form (Fig. 4A). The oxidation of reduced substrate by PDI is also fast. Lyles and Gilbert (15) found that a catalytic amount of PDI will convert a reduced RNase substrate to an oxidized RNase within 1 min after 0.1 mM GSSG is added. This places a lower limit of >26 µM substrate formed/min/µM PDI on the rate constant for oxidation of a reduced substrate by PDI. Because the turnover number for sRNase isomerization is 1 µM native RNase formed/min/µM PDI, both substrate oxidation and substrate reduction are sufficiently fast that they would be catalytically competent to participate in an isomerization mechanism composed of reduction-reoxidation cycles.
A reduction-reoxidation mechanism also predicts that high concentrations of an oxidized substrate should inhibit the isomerization reaction by competing with the low concentration of reduced substrate for binding to oxidized PDI. The accumulation of oxidized PDI resulting from a rapid reaction of reduced PDI with the oxidized substrate is verified by a gel-shift assay designed to monitor the redox state of PDI during substrate turnover under inhibiting conditions (Fig. 4). The substrate inhibition is decreased by either dropping the concentration of the substrate (Fig. 4), adding oxidized PDI (Fig. 2), or by making more reduced PDI available by adding GSH (Figs. 5 and 6). These diverse effects on the rate of isomerization are more easily accounted for by a reduction-reoxidation mechanism than by intramolecular isomerization (Fig. 1).
Increasing concentrations of GSH (in the presence of an NADPH/glutathione trap to remove GSSG), initially stimulate isomerization as they convert some of the PDI active site back into the reduced state (Fig. 6), but at higher concentrations significant inhibition is observed concomitant with the loss of oxidized PDI during steady-state turnover. A simple intramolecular isomerization mechanism would predict that there should be no requirement for oxidized PDI and that increasing the extent of PDI reduction should only stimulate isomerization, not inhibit it.
A reduction-reoxidation cycle also requires that the PDI active site
disulfide be able to survive long enough to reoxidize the reduced
substrate. In the presence of GSH and NADPH, redox equilibrium would
predict complete reduction of the PDI active site. However, under
steady-state conditions, the rapid reduction of the substrate by PDI
and the relatively slow reaction of the PDI active site with GSH permit
a significant steady-state accumulation of oxidized PDI, sufficient to
support the reoxidation of the reduced substrate. The rate constant for
the reduction of the PDI active site by 1 mM GSH is 12 min1 (22), suggesting that the survival of the oxidized
PDI active site during steady-state isomerization is caused by the
relatively slow reduction of PDI by GSH and the rapid oxidation of
protein substrates. At higher GSH, the rate of active site reduction by GSH becomes competitive with protein substrate oxidation, and the rate
of isomerization falls.
The observation of rapid cycles of reduction and reoxidation does not exclude the occurrence of intramolecular isomerization reactions during the formation of native RNase. In fact, the reduction-reoxidation cycles may serve only to set up the formation of a critical intermediate during sRNase refolding which isomerizes through an intramolecular pathway. Shin et al. (23) have recently elaborated specific pathways for PDI-dependent conversion of an ensemble of RNase species with three disulfides into native RNase. Some of these critical interconversions on the pathway to the native structure may occur by intramolecular isomerizations; however, our data would suggest that if their conversion occurs by an intramolecular mechanism, their formation must be dependent on reduction-oxidation cycles.
The disulfide isomerization of sRNase is accompanied by numerous reduction-reoxidation cycles, but the isomerization of other substrates may be dominated by intramolecular rearrangements. As a result there may be no "universal" mechanism for catalysis of disulfide isomerization. PDI may simply provide multiple mechanisms that can be used differently by different substrates. The contribution of intramolecular isomerization of the substrate compared with cycles of reduction-reoxidation will be governed by how fast the intramolecular rearrangement of the substrate occurs compared with the clock that is set by the PDI second active site cysteine. This path will invoke escape (reduction-reoxidation) if the intramolecular rearrangement it too slow. In addition, preferential covalent or noncovalent interactions between PDI and various sites on individual substrates might also influence this partitioning by affecting differentially the rates of the various ways in which the PDI-substrate complex is resolved.
The occurrence of cycles of reduction and oxidation may also have
implications toward the mechanism of disulfide formation in
vivo. Previously we reported that the PDI redox state could be
maintained in a steady state that was effective for catalysis of
disulfide isomerization by using a sulfhydryl oxidase to catalyze oxidation of a reduced RNase substrate and GSH to maintain PDI in a
reduced state (24). The GSH was required to offset the oxidation of the
PDI active site by either the sulfhydryl oxidase or through oxidation
of the reduced substrate. Thus, the PDI redox state can be maintained
by a nonequilibrium, steady-state balance between the kinetics of
transfer of oxidizing and reducing equivalents to the substrate. Frand
and Kaiser (25) along with Pollard et al. (26) have found
that PDI is oxidized in the yeast endoplasmic reticulum by an oxidase,
Ero1p. Interestingly, a mutant allele of this gene is suppressed by a
mutation in the GSH1 gene (21), responsible for the
biosynthesis of glutathione, suggesting that GSH may be used to balance
the oxidase activity of Ero1p.
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ACKNOWLEDGEMENT |
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We thank Anton Solovyov for providing the construct expressing a mutant PDI with the nonactive site cysteines mutated to serines.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants R01-GM-40379 (to H. F. G. and M. S.) and T32-GM-08280 (to B. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-5880; Fax: 713-796-9438; E-mail:hgilbert@bcm.tmc.edu.
Published, JBC Papers in Press, December 15, 2002, DOI 10.1074/jbc.M211036200
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ABBREVIATIONS |
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The abbreviations used are: PDI, protein-disulfide isomerase; DTT, dithiothreitol; ImPDI, internal mutant PDI; Mal-PEG, a conjugate of polymeric polyethylene glycol and maleimide; RNase A, bovine pancreatic ribonuclease; sRNase, scrambled RNase.
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