Discrimination between Native and Non-native Disulfides by Protein-disulfide Isomerase*

Ji Zheng and Hiram F. GilbertDagger

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 epsilon  = 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Nright-arrowR) 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.

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.

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; open circle , 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.

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. black-square, no PDI; , wild-type PDI; , mutant PDI with one active site cysteine (SGHS ... CGHS); open circle , mutant PDI with no active site cysteines (SGHS ... SGHS). The concentration of both wild-type and mutant enzymes was 2.0 µM.

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
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ABSTRACT
INTRODUCTION
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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
k<SUB><UP>DTT</UP></SUB>=K<SUB><UP>un</UP></SUB>×k<SUB><UP>ex,DTT</UP></SUB> (Eq. 1)
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.


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Fig. 6.   Mechanism for PDI catalysis of DTT-dependent reduction of native proteins.

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.

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 beta -lactamase that are buried in a folded structure (6). In this case, the beta -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 beta -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.

Dagger 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

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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