Discrimination between Native and Non-native Disulfides by
Protein-disulfide Isomerase*
Ji
Zheng and
Hiram F.
Gilbert
From the Verna and Marrs McLean Department of Biochemistry, Baylor
College of Medicine, Houston, Texas 77030
Received for publication, December 19, 2000, and in revised form, February 1, 2001
 |
ABSTRACT |
The folding assistant and chaperone
protein-disulfide isomerase (PDI) catalyzes disulfide formation,
reduction, and isomerization of misfolded proteins. PDI substrates are
not restricted to misfolded proteins; PDI catalyzes the
dithiothreitol (DTT)-dependent reduction of native
ribonuclease A, microbial ribonuclease, and pancreatic trypsin
inhibitor, suggesting that an ongoing surveillance by PDI can test even
native disulfides for their ability to rearrange. The mechanism of
reduction is consistent with an equilibrium unfolding of the substrate,
attack by the nucleophilic cysteine of PDI followed by direct attack of
DTT on a covalent intermediate between PDI and the substrate. For
native proteins, the rate constants for PDI-catalyzed reduction
correlate very well with the rate constants for uncatalyzed reduction
by DTT. However, the rate is weakly correlated with disulfide
stability, surface exposure, or local disorder in the crystal. Compared
with native proteins, scrambled ribonuclease is a much better substrate
for PDI than predicted from its reactivity with DTT; however, partially
reduced bovine pancreatic trypsin inhibitor (des(14-38)) is
not. An extensively unfolded polypeptide may be required by PDI to
distinguish native from non-native disulfides.
 |
INTRODUCTION |
Protein folding is the search for a unique, biologically active
conformation among almost endless possibilities. The linear information
in the protein sequence is decoded to give rise to three-dimensional
structure in a way that is not completely understood. Although maximum
thermodynamic stability is certainly one property that identifies the
native states of many proteins, proteins like
-lytic protease are
biologically active in a metastable conformation that is kinetically
trapped from converting into a more stable structure (1).
In the cell, protein folding occurs in the presence of folding
catalysts and molecular chaperones. Structural features that mark the
biologically active conformation must be decoded by these folding
assistants so that they can discriminate native from misfolded conformations. This is particularly true in the endoplasmic reticulum, where an elaborate quality control system (2) ensures that misfolded
proteins are retained in the endoplasmic reticulum (or degraded),
whereas correctly folded proteins are allowed to exit. Protein-disulfide isomerase
(PDI)1 is a 55-kDa folding
assistant and chaperone of the eukaryotic endoplasmic reticulum (3-5).
It catalyzes thiol-disulfide exchange reactions, including disulfide
formation, disulfide reduction, and disulfide isomerization. The
mechanism of PDI-catalyzed disulfide isomerization involves cycles of
disulfide reduction and oxidation so that incorrect disulfides are
reduced and converted eventually, through a trial and error process, to
native disulfides that are resistant to further isomerization (6). A
similar cycle of trial and error unfolding/refolding operates in
ATP-dependent chaperones, such as GroEL/ES (7,
8).
For chaperone-mediated folding, the native conformation is resistant to
further structural rearrangement. This resistance of a structure to
further attempts at refolding could be due to thermodynamic or kinetic
stability, and it is likely that both are important. Chaperones
recognize the non-native state by its exposed hydrophobic surface that
is normally buried in the folded protein. They fail to interact with
the native state because the "recognition" sites are not accessible
(9). However, some chaperones can facilitate protein unfolding (10);
thus, there may be ongoing surveillance of the native state, or the
chaperone may trap the native protein when it spontaneously unfolds.
With PDI, recognition of native versus non-native structure
might be based on any of the structural or chemical differences between
the two states. The native structure may be resistant to PDI attack
because its disulfides are inaccessible. PDI and related proteins are
the only molecular chaperones that interact covalently with their
substrates. One of the active site cysteines (the nucleophilic
cysteine) forms a mixed disulfide with a cysteine from the substrate
(6, 11, 12). Once a native disulfide is attacked by PDI, the disulfide
may reform and expel PDI much faster than it rearranges, implying
chemical stability of the protein disulfide. Finally, the native
protein may be subject to PDI attack, just like misfolded proteins, but
the thermodynamic stability of the native state ensures a low
equilibrium concentration of misfolded proteins.
To explore how (or if) PDI discriminates native from non-native
structures, we compared the reactivity of several native protein disulfides with dithiothreitol in the presence and absence of PDI. The
protein substrates we examined are all very well characterized structurally and thermodynamically, including knowledge about the
contribution of specific disulfides to the structure. The proteins
include bovine pancreatic ribonuclease A (RNase A) (13), bovine
pancreatic trypsin inhibitor (BPTI) (14), and microbial ribonuclease
(RNase Sa) (15). The proteins have different structures, disulfide
exposures, and thermodynamic stabilities. All are PDI substrates. The
rate constants for PDI-catalyzed reduction are directly correlated with
the rate constants for uncatalyzed reaction with DTT over a reactivity
range that spans six orders of magnitude. Scrambled RNase, with its
randomly formed disulfides, is considerably more reactive with PDI than
expected based on the reactivity of the native protein, implying that
PDI recognizes extensively unfolded proteins. This extensive
interaction is evidently not available for the transiently unfolded
structures that are formed from native proteins.
 |
MATERIALS AND METHODS |
Materials--
BPTI, RNase A, DTT, and iodoacetic acid were
obtained from Sigma. Wild-type PDI and mutants were expressed in
Escherichia coli strain BL 21 (DE3) obtained from Novagen
Inc. (Madison, WI). RNase Sa and its mutants, D17K Sa and E74K Sa, were
a generous gift from Dr. C. Nick Pace (Texas A & M University,
College Station, TX). PDI was purified to >90% homogeneity, as
determined by SDS-polyacrylamide gel electrophoresis, as described
previously (16). The concentration of PDI was determined by absorbance
at 280 nm using E0.1% of 0.94 mg/ml
1 cm
1 (17).
All absorbance measurements were performed using a Beckman DU-70
spectrophotometer. PDI was reduced in 10 mM DTT overnight at room temperature. The DTT was removed by centrifugal gel filtration using BioGel P-6 equilibrated in 10 mM Tris-HCl buffer, pH
7.5.
Preparation of Scrambled Ribonuclease--
Scrambled RNase
A (sRNase) was produced by a modification of the method of Hillson
et al. (18). Native RNase A (30 mg/ml) was incubated with
130 mM DTT (15-fold excess over RNase disulfides) in 50 mM Tris-HCl containing 9 M urea and 50 mM sarcosine, pH 8.6, for 18-20 h at room temperature and
followed by extensive dialysis against 0.1 M acetic acid in
the cold. The RNase A was then diluted into 50 volumes of 9 M urea, 50 mM Tris, and 50 mM sarcosine, pH 8.5. Oxygen gas was bubbled slowly through solution in
the dark for 2 days at room temperature. The solution was dialyzed extensively against 0.05 M NH4HCO3
and then lyophilized. The sRNase concentration was determined by
absorbance at 280 nm using
= 9800 M
1 cm
1
(19). Thiol content was analyzed using Ellman's assay (20) and was determined to be <4% of the RNase cysteine residues.
Native Protein Reduction--
Reduction and unfolding reactions
were carried out in the presence of 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.2 M KCl, and various concentrations of DTT and PDI. All of the solutions except the protein
stocks were purged of oxygen by bubbling with argon for 10 min. At
various times after the reactions were initiated by addition of
protein, aliquots were quenched with a solution containing 0.25 M iodoacetate (adjusted to pH 8.0 with KOH). After 15 min at room temperature, the solutions were placed on ice. For BPTI, RNase
A, and sRNase reduction, the sample was analyzed on a native polyacrylamide gel (27%) using the low-pH discontinuous system of
Reisfield et al. (21). Electrophoretic separation was at constant voltage (130-160 V) at 4 °C for 1-2 h. Staining was
performed with 0.1% (w/v) Coomassie Brilliant Blue in 10% (w/v)
trichloroacetic acid and 10% (w/v) sulfosalicylic acid overnight. The
gels were destained by diffusion into 7.5% acetic acid and 5%
methanol. RNase Sa reduction was analyzed in the same way, except that
the oxidized and reduced proteins were separated by SDS-polyacrylamide gel electrophoresis (12%). The concentration of various species was
determined by densitometry of Coomassie Brilliant Blue-stained gels.
Determination of Rate Constants--
The rate constants for the
reduction of native proteins were determined by using an exponential
function to fit the time course for the disappearance of native
protein. With some proteins and at some of the lower DTT and PDI
concentrations, the rate of reduction was sufficiently slow that the
initial rate of disappearance of the native protein was used to
estimate the rate constant. Under these conditions, the initial
concentration of DTT was assumed to equal the added concentration of
DTT, avoiding the complications of air oxidation that occurs with
prolonged incubations. The rate constants observed by initial rates
agreed with those observed at higher DTT concentrations, where the
reduction of native protein was observed to go to completion. Rates in
the presence of PDI were corrected for any spontaneous reaction with
DTT observed in the absence of PDI. The PDI-catalyzed reaction was
generally more than 1.5-2-fold higher than that in the absence of
PDI.
 |
RESULTS |
The contribution of disulfides to protein stability depends on the
redox environment (22). Although denaturants are often added to
facilitate the reduction of native proteins, reduction of native
proteins can be observed at high concentrations of good reducing agents
such as DTT (23). If PDI can interact with native proteins and
facilitate the reduction of native disulfides, the presence of PDI
should increase the rate of reduction when sufficiently high
concentrations of DTT are present to drive the equilibrium toward the
reduced protein.
Three disulfide-containing proteins were examined for PDI-catalyzed
reduction: RNase A, BPTI, and RNase Sa. These were chosen because they
have all been well characterized with respect to the contribution of
specific disulfide(s) to stability. The reduction of native proteins by
DTT at pH 7.5 (10 mM Tris HCl, 0.2 M KCl, and 1 mM EDTA) can be observed by native or SDS-polyacrylamide gel electrophoresis after quenching the reaction with a high
concentration of iodoacetate (0.25 M) to prevent any
further thiol-disulfide exchange and to introduce additional negative
charges into the protein (24). The relative concentrations of reduced
and native proteins at various times after initiating reduction were
determined by densitometry of the gels.
PDI catalyzes the DTT-dependent reduction of all the native
proteins examined (Table I). A
representative example is shown in Fig.
1, where 3.5 µM PDI
accelerates the conversion of native RNase A to the reduced protein
approximately 5-fold. During the reduction of native RNase A (four
disulfides) (Fig. 1), no specific, partially reduced intermediates are
observed by gel electrophoresis, although high pressure liquid
chromatography shows that reduction proceeds by preferential reduction
of the 65-72 and 40-95 disulfides (25). Consequently, only the first
step of the reduction (N
R) was observed. Both the band corresponding
to the native protein and the broad band(s) of the fully reduced
protein were used to determine rate constants for conversion of the
native protein to the collection of reduced proteins.
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Table I
Properties of native and non-native disulfides and the rate constants
for protein reduction by DTT in the presence and absence of PDI
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Fig. 1.
Reduction of native RNase by 10 mM DTT catalyzed by PDI. The reaction was performed at
pH 7.5 (10 mM Tris-HCl, 0.2 M KCl, 1 mM EDTA, 37 °C). At the times indicated, an aliquot was
quenched with 0.25 M iodoacetate (pH 8.0) and analyzed by
native polyacrylamide gel electrophoresis (see "Materials and
Methods"). A, reduction in absence of PDI; B,
reduction in the presence of 3.5 µM PDI.
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PDI also catalyzes the reduction of native BPTI (28). BPTI has three
disulfides, and the fastest step is the reduction of the 14-38
disulfide to produce des(14-38) BPTI (29). At a DTT concentration of 5 mM (Fig. 2), the first step
occurs with a half-time of 0.5-1 min. PDI at a concentration of 0.5-2
µM increases the rate of this step ~1.5-2-fold. This
is followed by a slow intramolecular rearrangement or direct reduction
of the remaining two disulfides (28). In the absence of PDI, the
partially reduced intermediate is at this reduced very slowly
DTT concentration, consistent with the results reported by Creighton
et al. (28). However, PDI increases the reduction of this
intermediate to fully reduced protein, even at low concentrations of
DTT (0.5 mM) (Fig. 2).

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Fig. 2.
Reduction of BPTI by 5 mM DTT
catalyzed by PDI at pH 7.5 (10 mM Tris-HCl, 0.2 M KCl, 1 mM EDTA, 25 °C). At the times
indicated, an aliquot was quenched with 0.25 M iodoacetate
(pH 8.0) and analyzed by native polyacrylamide gel electrophoresis (see
"Materials and Methods"). N represents the mobility of
native BPTI, II represents species with two disulfides (one
disulfide reduced), and R represents the fully reduced
molecule. A, reduction in absence of PDI; B,
reduction in the presence of 2 µM PDI.
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In the presence or absence of PDI, the disappearance of the native
protein is first order and can be described by an exponential function.
For all of the proteins examined (RNase, BPTI, and RNase Sa), the
native protein can be fully reduced in the presence of PDI at high DTT
and PDI concentrations. Fig. 3 shows the
rate constant for RNase A reduction as a function of the DTT
concentration. For all of the proteins examined, including RNase A, the
rate constant for reduction is proportional to DTT concentration. Thus, the rate-limiting step involves a molecule of DTT, even when the reaction is catalyzed by PDI. The rate constants for native protein reduction are also linearly dependent on the PDI concentration (representative data are shown in Fig.
4), with no evidence of saturation up to
a PDI concentration of 3.5 µM. The rate constants for
catalyzed and uncatalyzed reduction are summarized in Table I. For the
reduction by DTT, the rate constant is second order overall (units of
M
1
min
1) and first order in native protein and
DTT concentration. With PDI-dependent reduction, the rate
of reduction is third order overall (units of
M
2
min
1) and first order in native protein, PDI,
and DTT.

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Fig. 3.
Dependence of the rate constant for the
reduction of native RNase on the DTT concentration at pH 7.5 (10 mM Tris-HCl, 0.2 M KCl, 1 mM EDTA,
37 °C). , no PDI; , 3.5 µM PDI. The rate
constants, which were determined from least squares fitting, are shown
in Table I.
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Fig. 4.
Dependence of the rate constant for the RNase
reduction on the PDI concentration at pH 7.5 (10 mM
Tris-HCl, 0.2 M KCl, 1 mM EDTA, 37 °C).
The DTT concentration was 10 mM.
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The first-order dependence of the PDI-catalyzed reduction on the
concentration of DTT, substrate protein, and PDI suggests a simple
mechanism where the rate-limiting step involves direct reduction of a
disulfide formed by attack of PDI on the native protein. Consistent
with the covalent participation of PDI in the reaction, a PDI mutant
with no active site cysteines is inactive (Fig.
5). PDI is a multidomain protein with two
independent active sites, one near the amino terminus of the protein
and another near the carboxyl terminus (6). Each active site has two
cysteines in the sequence CGHC. At either active
site, the first cysteine (CGHC) is exposed to solvent and
reacts with substrate. The second active site cysteine
(CGHC) is buried and restricted to react with only the other
active site cysteine. If a substrate-PDI mixed disulfide intermediate
is reduced directly by DTT, the second active site cysteine
(CGHC) nearer the carboxyl terminus should be dispensable
for catalysis. This prediction is confirmed experimentally. Both the
wild-type PDI (CGHC ...
CGHC) and a mutant PDI with a single nucleophilic
cysteine at the more carboxyl-terminal active site (SGHS ...
CGHS) are effective catalysts of BPTI reduction (Fig. 5).
Only one active site cysteine is required to catalyze
DTT-dependent reduction, showing that the intramolecular
displacement of the substrate cysteine by the second active site
cysteine is slow compared with the attack of DTT.

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Fig. 5.
Reduction of native BPTI by DTT in the
presence of wild-type and mutant PDI. Reduction of the 14-38
disulfide was measured at pH 7.5 (10 mM Tris-HCl, 0.2 M KCl, 1 mM EDTA, 25 °C) in the presence or
absence of 5 mM DTT. , no PDI; , wild-type PDI; ,
mutant PDI with one active site cysteine (SGHS ...
CGHS); , mutant PDI with no active site cysteines
(SGHS ... SGHS). The concentration of both wild-type and mutant
enzymes was 2.0 µM.
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Ribonuclease Sa is a small (96 amino acids) extracellular ribonuclease
isolated from growth media of Streptomyces aureofaciens BM-K
(30). It has one disulfide between residues 7 and 96 that contributes
4.6 kcal/mol to the overall stability of the protein (6.4 kcal/mol)
(15). As with other native proteins, RNase Sa is reduced catalytically
by PDI. To observe the effect of thermodynamic stability on reduction
in the absence of large sequence variation, mutants of RNase Sa, D17K
and E74K, were also examined. The mutants are 1.1 kcal/mol less or more
stable than RNase Sa, respectively. The PDI-catalyzed reduction of
wild-type and mutant Sa was measured using nonreducing
SDS-polyacrylamide gel electrophoresis and quantifying the intensity of
the native protein band after staining, similar to the methods used
with RNaseA and BPTI. The least stable Sa mutant, D17K, is reduced
faster than wild-type RNase Sa, and the more stable mutant, E74K, is
reduced more slowly than wild-type RNase Sa (Table I).
To compare PDI-catalyzed reduction of native disulfides to its
reduction of a random, unstructured disulfide, the reduction of
scrambled RNase was measured. As expected, the reduction of sRNase was
much faster than that of native proteins (Table I). The rate
constant for sRNase reduction catalyzed by PDI is about 106
faster than that seen for reduction of native RNase A. When
statistically corrected for the number of reactive disulfides (four
disulfides), the PDI-catalyzed reduction of sRNase is ~100-fold
faster than the PDI-catalyzed reduction of the native, 14-38 disulfide
of BPTI.
 |
DISCUSSION |
Mechanism of Reduction--
Whether the substrate is a native
protein or one that is misfolded, PDI can clearly involve it in
thiol-disulfide exchange and catalyze its reduction by DTT. The absence
of PDI-catalyzed reduction in a mutant with no active site cysteines
(Fig. 5) suggests that PDI participates covalently in
DTT-dependent reduction, as in the catalysis of substrate
oxidation and isomerization. The first-order dependence of the rate of
reduction on the substrate, DTT, and PDI concentrations is consistent
with the mechanism for disulfide reduction shown in Fig.
6. According to this model, the protein
spontaneously unfolds to expose a disulfide, followed by attack of PDI
and rate-limiting attack of DTT on a PDI-substrate mixed disulfide.
This model predicts that the second active site cysteine of PDI does
not participate in DTT-dependent reduction, presumably
because the intermolecular attack by DTT is faster than attack of the
second active site cysteine to form a PDI active site disulfide, a
prediction that is confirmed experimentally (Fig. 5). According to this
mechanism, uncatalyzed protein disulfide reduction can be separated
into two linked steps, as proposed by Li et al. (25). In the
first step (Kun), spontaneous unfolding of some
portion of the native structure exposes the protein disulfide to
attack. As suggested by Li et al. (25), the energetics of spontaneous unfolding can be approximated by assuming that the exposed
disulfide reacts with DTT like a typical, exposed cysteine disulfide
(kex,DTT = 1205 M
1
min
1). The observed rate constant for DTT
reduction is then
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(Eq. 1)
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The free energy corresponding to unfolding
(Kun) is due to destroying noncovalent
interactions in the native state that must be broken to convert the
protein disulfide to one that is as reactive as an exposed disulfide in
scrambled RNase.
Using the same approximation, Goldenberg (31) estimates that the 5-55
and 30-51 disulfides of BPTI in the des(14-38) BPTI intermediate
require extensive unfolding of the core of the molecule. Li et
al. (25), on the other hand, find that native RNase A reduction
((65-72) or (40-95) disulfides) requires only partial unfolding,
presumably local unfolding in the vicinity of the disulfide. When
several different substrate disulfides are considered, it is clear that
the extent to which the free energy stabilizing the surrounding protein
structure opposes reduction varies considerably from protein to protein
(Table I).
Structural Basis for Reactivity--
The native proteins we chose
to study have been characterized extremely well with respect to
structure, stability, and the contribution of the individual disulfides
to stability. Of the four disulfides of native RNase, reduction of
65-72 and 40-95 occur the fastest and by parallel pathways (25). With
BPTI, the 14-38 disulfide is the most easily reduced. With RNase Sa, there is only one disulfide bond. The common characteristic of these
disulfides is that they are all at least partially exposed to solvent
(Table I). Totally inaccessible disulfides are less reactive. However,
for the partially exposed disulfides in this study, there is only a
weak correlation between exposure and reactivity with DTT or PDI.
Native RNase is the least exposed and least reactive; however,
the exposures of the reactive disulfides of RNase Sa and BPTI are
similar, but BPTI is almost 100-fold more reactive.
The disulfides of these three proteins contribute differently to the
stability of the structure toward equilibrium denaturation. The
difference in the free energy of stabilization for the wild-type protein and mutants in which the cysteine has been replaced by alanine
are given in Table I. If unfolding of the structure that is stabilized
by the disulfides occurs in the transition state for reduction, then
stability would correlate directly to reactivity. However, this is not
the case for the proteins in this study. The 14-38 disulfide
contributes as much to the stability of BPTI as the 7-96 disulfide of
RNase Sa; however, the 14-38 disulfide of BPTI is 74-fold more
reactive with DTT. Apparently, the interactions that are stabilized by
a given disulfide must be destroyed to differing extents as the
disulfide is reduced. Kinetic flexibility of the structure surrounding
the disulfide might also affect the rate of disulfide reduction;
however, for these proteins, the disulfides are all located in
reasonably rigid portions of the molecule, and there appears to be no
correlation of the rate of reduction with the crystallographic
temperature factor of the structure surrounding the disulfide. As with
other factors that govern protein stability and kinetic reactivity, the
reactivity of disulfides with PDI and DTT reflects a delicate and
complex interplay between exposure and stability, possibly including
long-range interactions that stabilize the structure.
PDI Discrimination between Native and Misfolded
Substrates--
Native disulfides are clearly not immune from
PDI-initiated attempts at isomerization or reduction. Surveillance by
PDI is evidently an ongoing process for both native and non-native
disulfides. Substrate reduction, oxidation, and isomerization are all
initiated by the attack of a PDI active site cysteine on a substrate
sulfur; hence, the recognition events are likely to be similar for all PDI-catalyzed reactions.
Some disulfides are simply more reactive with PDI than others,
and this difference is reflected both in the rate of PDI-catalyzed reduction and in the uncatalyzed reduction by DTT. In fact, reactivity with PDI correlates faithfully with the reactivity toward DTT over 6 orders of magnitude (Fig. 7). For native
proteins, the a plot of log(kDTT) against
log(kPDI) is linear, with a slope of 0.8 ± 0.03. The slope is determined by a series of different proteins with
greatly different reactivity. If only the three RNase Sa variants are
considered, the slope is 0.94 ± 0.3, very close to 1, suggesting
that the factors that dictate reactivity with DTT influence the
PDI-catalyzed reaction to the same extent. For these native protein
disulfides, PDI recognizes features of the native structure but with no
more discrimination than DTT. The determinants of native disulfide
reactivity with PDI are essentially those features of the disulfide
that govern its reactivity with DTT. Although BPTI is almost as
reactive with PDI and DTT as an exposed disulfide, the thermodynamic
stability of the structure of the native arrangement of disulfides is
the most stable one, preventing the accumulation of alternative
disulfide arrangements (27).

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Fig. 7.
Correlation between the uncatalyzed and
PDI-catalyzed reduction of protein disulfides by DTT. The least
squares line of a plot of log(kPDI) against
log(kDTT) has a slope of 0.8 ± 0.03 and an
intercept of 5.9 ± 0.3. Rate constants are taken from Table I.
The reactivity of sRNase with PDI is ~60× faster than expected from
its reactivity with DTT alone.
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The strong correlation between the rates of catalyzed and uncatalyzed
reduction of native disulfides shows that the factors that influence
reactivity with PDI and DTT are very similar. This is consistent with a
previous study on the formation of a disulfide from two cysteines of
-lactamase that are buried in a folded structure (6). In this case,
the
-lactamase has to unfold spontaneously before the thiols can be
oxidized by PDI. Compared with the disulfide of glutathione, PDI is
500-fold better in promoting the oxidation of folded, reduced
-lactamase. This difference in rate can be accounted for by the
higher chemical reactivity of the active site disulfide of PDI compared
with glutathione disulfide. Based on this precedent (6), it
seems unlikely that PDI facilitates the reduction of native disulfides
by interacting with the native structure and inducing it to unfold.
Based on the behavior of native proteins, the rate constant for
PDI-catalyzed reduction of the denatured protein sRNase is about
60-fold faster than expected from the rate of its reduction by DTT
(Fig. 7). Although PDI does not recognize differences between various
native proteins any better than DTT, it does detect unfolded substrates
better than DTT, presumably through interaction with an extensively
unfolded polypeptide. Klappa et al. (32) suggest that larger
protein substrates (like sRNase) interact with multiple peptide/protein
binding sites distributed on the PDI molecule. This more extensive
interaction would allow PDI to kinetically detect disulfides in the
context of extensively unfolded polypeptide and specifically reduce
them. Interestingly, reduction of the des(14-38) BPTI species falls on
the line established by native proteins, suggesting that this species
is not extensively unfolded (from the perspective of PDI) in the
absence of the 14-38 disulfide. Although native protein disulfides are
substrates for PDI-dependent reduction, extensively
unfolded substrates are preferred (Fig. 7).
 |
ACKNOWLEDGEMENTS |
We thank Dr. C. N. Pace and K. L. Shaw of the Department of Medical Biochemistry and Genetics, Texas A & M University for their generous gift of RNase Sa and its mutants.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM40379.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.
To whom correspondence should be addressed: Dept. of Biochemistry,
Baylor College of Medicine, Houston, TX 77030. Tel.: 713-798-5880; Fax:
713-796-9438; E-mail: hgilbert@bcm.tmc.edu.
Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M011444200
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ABBREVIATIONS |
The abbreviations used are:
PDI, protein-disulfide isomerase;
DTT, dithiothreitol;
RNase A, bovine
pancreatic ribonuclease;
RNase Sa, microbial ribonuclease;
sRNase, scrambled RNase A;
BPTI, bovine pancreatic trypsin inhibitor.
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