doi:10.1016/j.jmb.2011.03.024 J. Mol. Biol.

(2011) 409, 238249

Contents lists available at www.sciencedirect.com

Journal of Molecular Biology

j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b

Reexamination of the Role of Interplay between

Glutathione and Protein Disulfide Isomerase
Anna-Kaisa Lappi and Lloyd W. Ruddock
Department of Biochemistry, University of Oulu, PO Box 3000, Oulu 90014, Finland

Received 10 November 2010; Protein disulfide isomerase (PDI) has an essential role in the process of
received in revised form disulfide bond formation, where it catalyzes disulfide bond formation,
11 March 2011; reduction, and isomerization. It is thought that the major route for oxidizing
accepted 14 March 2011 dithiols in folding proteins to disulfides is via Ero1-mediated oxidation of
Available online PDI. Since the discovery of Ero1, the role of glutathione in disulfide bond
22 March 2011 formation has been downplayed. In this study, the role of glutathione in
disulfide bond formation was reexamined. Here we have studied in vitro the
Edited by C. R. Matthews kinetics of the glutathione-mediated oxidation and reduction of the catalytic
a domains of human PDI and yeast Pdi1p. The results obtained from
Keywords: stopped-flow and quenched-flow experiments show that the reactions of
disulfide bond formation; PDI and Pdi1p are faster and more complex than previously thought. Our
PDI; results suggest that the kinetics of oxidation of PDI and Pdi1p by oxidized
oxidation; glutathione are remarkably similar, whereas the kinetics of reduction by
reduction; reduced glutathione shows clear differences. The data generated here on the
stopped-flow kinetics rapid reactivity of PDI towards glutathione suggest that reevaluation is
required for several aspects of the field of catalyzed disulfide bond
formation, including the potential physiological role of glutathione.
2011 Elsevier Ltd. All rights reserved.

Introduction physiological relevance during protein folding. In

Protein folding in the endoplasmic reticulum (ER)
is often associated with the formation of structure-
stabilizing native disulfide bonds.1 The formation of
disulfide bonds is a complex process, and for many
secreted and outer membrane proteins, it is also the
rate-limiting step in their synthesis (for reviews, see
Freedman et al.,2 Ellgaard and Ruddock,3 and
Hatahet and Ruddock4). The pathways for native
disulfide bond formation in the ER are complex and
not fully understood. Dithiols in a folding protein
can be oxidized directly by molecular oxygen;
however, this reaction is probably too slow to have
*Corresponding author. E-mail address:
lloyd.ruddock@oulu.fi.
Abbreviations used: PDI, protein disulfide isomerase;
ER, endoplasmic reticulum; GSSG, oxidized glutathione;
GSH, reduced glutathione; EDTA,
ethylenediaminetetraacetic acid.
addition, dithiols can be oxidized by low-molecular-
weight biochemical compounds such as oxidized
glutathione (GSSG),5 by reactive oxygen species
(e.g., hydrogen peroxide),6,7 by dehydroascorbate,810
or by enzymes belonging to the thioredoxin super-
family [e.g., protein disulfide isomerase (PDI)].3,4,11
Currently, it is believed that the major route for
the oxidation of dithiols to disulfides is via the
oxidation of PDI by members of the Ero1 sulfhydryl
oxidase family, followed by the oxidation of
substrate dithiols by PDI.1217 However, this has
recently been questioned by mouse knockout
studies, which show relatively minor physiological
changes upon knockout of mouse Ero1 family
members.18 It is unclear whether this indicates that
Ero1-mediated oxidation is not the major route in
mammals or whether Ero1-mediated oxidation is
the major physiological route, but that alternative
routes for dithiol oxidation can composite for its
loss.
Since the discovery of Ero1, the role of GSSG as
a potential route in disulfide bond formation has

0022-2836/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
Glutathione and PDI
been downgraded from a major pathway to being
potentially physiologically irrelevant. Previous eval-
uations of the rates of reaction of PDI with
glutathione have suggested that the rates of reaction
may be sufficiently rapid to be physiologically
relevant. However, these studies have been per-
formed at a maximal concentration of 0.15 mM,19
which is probably not physiological. The total
concentration of glutathione in the ER is unknown,
but it is estimated to be around 9 mM.20
Here we reexamine the potential role of glutathi-
one in the catalytic cycle of PDI, studying both
oxidation and reduction of PDI by glutathione in
vitro. Our data suggest that at physiological concen-
trations of GSSG, the rate-limiting step for the
oxidation of the active site of PDI by GSSG is the
nucleophilic attack by the C-terminal active-site
cysteine on the reduced glutathione (GSH)PDI
mixed disulfide, and that the mixed disulfide is a
relatively long-lived species. The kinetics of reduc-
tion by GSH is more complex but rapid. These
results imply that the role of glutathione in the
catalytic cycle of PDI in vivo should not be down-
played. In addition, the comparison of the stopped-
flow kinetics of the catalytic a domains of human
PDI and yeast Pdi1p shows that these enzymes are
not equivalent, and that interpretations of the data
observed from protein folding studies in yeast
should be carefully considered before generalizing
them into mammalian systems.
Results
Oxidation of the active site of the PDI a domain
by GSSG
To date, there have been no reported studies on
the kinetics of glutathione-mediated oxidation or
reduction of the active sites of PDI family members
at or near physiological concentrations of glutathi-
one. Previous studies have implied that the reactions
may be rapid, with a reported second-order rate
constant of 200 M 1 s 1 for the reduction of the
active site of PDI by GSH.19 With physiological
glutathione concentrations of up to 10 mM being
reported,20 this implies a pseudo-first-order rate
constant of ca 1.4 s 1 (i.e., a subsecond half-time for
the reaction). To measure such rapid kinetics, we
initially chose to perform fluorescence-based
stopped-flow measurements. Previously, we have
reported studies on the kinetics of oxidation of the
catalytic a domain of human PDI by peroxide with
rate constants of up to 23 s 1 21 by using a W128F
mutant. This mutation in the a domain does not
significantly change the catalytic ability of the
protein22 and results in the protein having only a
single tryptophan (W52). W52 is juxtaposed to the
239
active site and acts as a useful and direct spectro-
scopic marker of the redox state of the active site.22
When GSSG was added to the reduced PDI a
domain W128F, a time-dependent decrease in
fluorescence was observed (Fig. 1a). The average
decrease in fluorescence was 49 1%, consistent
with the difference in fluorescence of the dithiol
and disulfide states of the active site22 and with
the 48 1% change observed by stopped-flow
measurements for the peroxide-mediated oxida-
tion of the active site.21 However, while peroxide-
mediated oxidation fits to a pseudo-first-order
single-exponential process when peroxide is in
excess, 21 GSSG-dependent oxidation of the a
domain of PDI shows nonrandom residuals when
fitted to a single-exponential process (Fig. 1a, inset).
Hence, with GSSG-dependent oxidation, multiple
kinetic steps are observable. This is consistent with
the expected reaction pathway, as GSSG-dependent
oxidation of PDI is expected to be a two-step
process. In the first step, reduced PDI will form a
mixed disulfide with glutathione by a second-order
process. Under the conditions used, GSSG is in
large excess over reduced PDI; hence, the first step
would be expected to be a pseudo-first-order
process. In the second step, intramolecular nucleo-
philic attack by the C-terminal active-site cysteine
on the mixed disulfide results in the formation of
the oxidized active site. Since this is an intramolec-
ular reaction, it will be a first-order process. Hence,
the expected kinetic scheme would be a sequential
set of two first-order-type reactions, with the rate of
reaction being dependent on the GSSG concentra-
tion for the first step and being independent of the
GSSG concentration for the second step. Such
kinetics can be examined by fitting to a double-
exponential process to obtain the rate constants, or
they can be fitted to sequential kinetics if the rate
constants and the (fluorescence) parameters of the
three species are desired. While the decrease in
fluorescence of PDI upon GSSG-mediated oxidation
does not fit to a single-exponential function (Fig. 1a,
inset), over the concentration range 120 mM GSSG,
all of the data fit to a double-exponential or
sequential process with random residuals. We
have no evidence of any systematic deviation
from this fit, and since it was the simplest kinetic
scheme that gave random residuals, this scheme
was used in all further analyses.
When fitted to this scheme, one derived rate
constant was independent of GSSG concentration,
and one was dependent (Fig. 1b), as expected. Much
of the fluorescence change is associated with the
GSSG-dependent rate constant, consistent with the
formation of a spatially adjacent disulfide bond
quenching W52 tryptophan fluorescence. At low
GSSG concentrations, the two rate constants became
similar, making kinetic analysis difficult. In addi-
tion, the longer times for reaction made accurate
240 Glutathione and PDI

Fig. 1. The kinetics of oxidation of the reduced active-site CGHC motif of the W128F mutant of the catalytic a domain of
human PDI. (a) Representative trace of the kinetics of oxidation of the PDI a domain W128F at 10 M in the presence of
5 mM, 10 mM, and 20 mM GSSG (from right to left), as observed by changes in fluorescence. Inset: The residuals to fitting
to a single-exponential function (nonrandom) and a sequential function (random) for the 10 mM GSSG reaction. (b) Linear
dependence of the pseudo-first-order rate constant for the first step of oxidation on the concentration of GSSG and the
concentration independence of the second step. At all concentrations of GSSG examined, the kinetics showed random
residuals to a double-exponential function. The data are shown as mean standard deviation (n = 67). The second rate
constant is independent of GSSG concentration over the range 530 mM. (c) Kinetics of oxidation of the PDI a domain
W128F at 40 M in the presence of 10 mM GSSG, as observed by quenched-flow measurement and mass spectrometry.
The reduced species is shown as filled circles, and the oxidized species is shown as open circles. For clarity, only time
points up to 4 s are shown. The lines of best fit are to a single-exponential function. (d) Detection of the PDIglutathione
mixed-disulfide form in quenched-flow experiments may be unsuccessful due to a competition reaction between
intermolecular nucleophilic attack by the C-terminal active-site cysteine on the quenching reagent and an intramolecular
nucleophilic attack. The inaccessibility of the C-terminal active-site cysteine favors reaction 2 over reaction 1.

correction for photobleaching effects problematic.
However, it is clear that an extrapolation of the
GSSG-dependent rate constant does not pass
through the origin (i.e., the extrapolated rate
constant for [GSSG] = 0 is not zero). The same effect
has been observed for some other PDI family
catalytic domains and mutants tested (data not
shown) for GSSG-dependent oxidation, but not for
peroxide-mediated oxidation.21 Hence, at low con-
centrations of GSSG, a more complex kinetic scheme
appears to be operating.
With this complication excluded, the derived
second-order rate constant for the formation of the
PDIglutathione mixed disulfide from the gradient
of the GSSG dependence was 191 3 M 1 s 1, while
the average first-order rate constant for the loss of
the mixed disulfide over the range 520 mM GSSG
was 0.23 0.03 s 1 . These data imply that at
physiological concentrations of GSSG, the rate-
limiting step for the oxidation of the active site of
PDI by GSSG is the nucleophilic attack by the
C-terminal active-site cysteine on the GSHPDI
mixed disulfide, and that the mixed disulfide is
a relatively long-lived species.
To confirm that the rate constants derived from the
stopped-flow experiments represent the predicted
steps in the reaction, we undertook quenched-flow
experiments. Here reduced PDI was added to GSSG,
and at set time intervals, a large molar excess of a
quenching reagent was added. Since the reaction
being studied is based on thioldisulfide exchange,
the quencher primarily used was a thiol-alkylating
agent N-ethyl maleimide. Analysis of the products
by electrospray mass spectrometry allows the
Glutathione and PDI 241

identification of three potential species: the oxidized
protein (Mr = 14,301), the glutathione mixed disulfide
with the C-terminal active-site cysteine alkylated
(Mr = 14,733), and the reduced alkylated protein
(Mr = 14,553). For all experiments, only the oxidized
and reduced alkylated proteins were observed.
Separate experiments indicated that this was inde-
pendent of the quenching reagent or the conditions
used (see Materials and Methods for other method-
ologies tested). The kinetics of conversion of the
reduced protein to the oxidized protein deduced by
quenched-flow measurement for 10 mM GSSG gave
a pseudo-first-order rate constant of 2.6 0.3 s 1
(Fig. 1c), consistent with the results obtained for the
GSSG-dependent pseudo-first-order rate constant
determined by a stopped-flow measurement of
2.5 0.1 s 1 under the same conditions. While the
glutathioneprotein mixed disulfide was not ob-
served in this reaction, there are good reasons that
it might not be connected with the accessibility of
the C-terminal cysteine and the relative reactions
rates of intramolecular versus intermolecular nucle-
ophilic attack (Fig. 1d). Hence, while the quenched-
flow data are not able to confirm the reaction
scheme, the data are consistent with the stopped-
flow derived data.
Reduction of the active site of the PDI a domain
by GSH
With the use of the same W128F mutant, it is
possible to study the reduction of the active-site
disulfide by GSH using stopped-flow measurements.
When GSH was added to the oxidized PDI a
domain W128F, a time-dependent increase in fluo-
rescence was observed (Fig. 2a), with an average
increase in fluorescence equivalent to that observed
as a decrease during oxidation. GSH-dependent
reduction of oxidized PDI is expected to be a two-
step process. The first step is the nucleophilic attack
of GSH on the active-site disulfide to form a mixed
disulfide, while the second step is the nucleophilic
attack of another molecule of GSH on the mixed
disulfide to yield the reduced active site and GSSG.
Both of these reactions are second order; however,
GSH is in large excess over oxidized PDI in the
reaction, and hence both will be expected to be
pseudo-first-order reactions, with the rate constant
being proportional to the concentration of GSH. An
alternative reaction mechanism would be via the
formation of a trimolecular complex of two GSH and
one protein molecule. Here the reaction would be
third order, but again since GSH is in large excess

Fig. 2. The kinetics of reduction of the oxidized active-site CGHC motif of the W128F mutant of the catalytic a domain
of human PDI. (a) Representative trace of the kinetics of reduction of the PDI a domain W128F at 10 M in the presence of
5 mM, 10 mM, and 20 mM GSH (from right to left), as observed by changes in fluorescence. Inset: The residuals to fitting
to a single-exponential function for the 10 mM GSH reaction. (b) Dependence of the pseudo-first-order rate constant for
the reduction on the concentration of GSH. At all concentrations of GSH examined, the kinetics showed random residuals
to a single-exponential function. The data are shown as mean standard deviation (n = 78). The line of best fit is to the
reaction scheme shown in (c). (c) The kinetic partitioning of the mixed disulfide into products or back to the reactants
depends on the concentration of GSH.
242 Glutathione and PDI

over oxidized PDI, the reaction would be observed
to be a pseudo-first-order reaction, with the rate
constant being proportional to the concentration of
GSH squared.
Analysis of the stopped-flow data showed that the
data fitted to a single-exponential process (Fig. 2a)
with random residuals at all GSH concentrations,
suggesting that only a single pseudo-first-order rate
constant could be observed. However, a plot of the
derived rate constants versus GSH concentration
(Fig. 2b) showed that they were proportional neither
to the concentration of GSH nor to the concentration
of GSH squared. Hence, the kinetics of reduction by
GSH is complex. The initial mixed disulfide formed
from the reaction of GSH with oxidized PDI
undergoes kinetic partitioning. Either the mixed
disulfide can react with another molecule of GSH
to generate the reduced protein and GSSG, or the
C-terminal active-site cysteine can initiate an intra-
molecular nucleophilic attack to regenerate the
starting material (Fig. 2c). This kinetic partitioning
clearly depends on the concentration of GSH. Fitting
the data obtained for the GSH dependence of the
rate to this reaction scheme gave a good fit and
allowed us to obtain values for the three rate
constants. Since this was the simplest kinetic scheme
that was consistent with the data obtained, this
scheme was used in all further analyses. With the
use of this reaction scheme, the first nucleophilic
attack by GSH on the oxidized a domain gave a
second-order rate constant of 11,200 M 1 s 1. The
second nucleophilic attack by GSH on the PDIGSH
mixed disulfide gave a second-order rate constant of
4600 M 1 s 1, while the reverse reaction initiated by
the C-terminal active-site cysteine gave a first-order
rate constant of 430 s 1.
Oxidation of the active site of the Pdi1p a
domain by GSSG
While humans have ca 20 PDI family members,
the yeast Saccharomyces cerevisiae has only 5 PDI
family members, the archetypal member of which
is Pdi1p. Generally, human PDI and yeast Pdi1p
are considered to be homologous proteins; often
during comparison of in vitro and in vivo results,
no significant differences between the two are
considered (see Hatahet and Ruddock4 for a review).
However, the two proteins are only 29% identical,
and few direct comparative studies have been
undertaken. We therefore wanted to study whether
the a domain of yeast Pdi1p showed similar reaction
kinetics as the a domain of human PDI.
The a domain of yeast Pdi1p contains only a single
tryptophan residue, W60, which is juxtaposed to the
active site in a position analogous to that of W52 in
human PDI and hence can play the same role as a
spectroscopic marker for the redox state of the active
site. When GSSG was added to the reduced Pdi1p a
domain, we observed a time-dependent decrease in
fluorescence similar to that observed for the reduced
a domain of human PDI. In addition, as per the
human protein, the kinetic traces gave nonrandom
residuals when fitted to a single-exponential process
and a good fit with random residuals when fitted to
a sequential two-step process. One of the kinetic
steps was dependent on the concentration of GSSG,
and the other was independent (Fig. 3a), again
similar to the results obtained on the human protein.
The derived second-order rate constant for the
formation of the Pdi1pglutathione mixed disulfide
was 163 5 M 1 s 1, while the first-order rate
constant for the loss of the mixed disulfide was

Fig. 3. The kinetics of oxidation and reduction of the active-site CGHC motif of the catalytic a domain of yeast Pdi1p.
(a) Linear dependence of the pseudo-first-order rate constant for the first step of oxidation on the concentration of GSSG
and the concentration independence of the second step. At all concentrations of GSSG examined, the kinetics showed
random residuals to a double-exponential function. The data are shown as mean standard deviation (n = 67). The
second rate constant was independent of GSSG concentration over the range 520 mM. (b) Dependence of the pseudo-
first-order rate constant for the reduction on the concentration of GSH. At all GSH concentrations examined, the kinetics
showed random residuals to a single-exponential function. The data are shown as mean standard deviation (n = 78).
The line of best fit is to the kinetic partitioning reaction scheme shown in Fig. 2c.
Glutathione and PDI
0.13 0.01 s 1. These results suggest that while the
mixed disulfide is a more stable species for the yeast
Pdi1p a domain than for the human protein, the
kinetics of oxidation of the two proteins by GSSG are
remarkably similar.
In contrast to these results, very significant
differences in kinetics were observed for the
reduction of the two proteins (compare Figs. 2b
and 3b). When GSH was added to the oxidized yeast
Pdi1p a domain, we observed a time-dependent
increase in fluorescence, which, as per the human
PDI a domain, fitted to a single-exponential function
with random residuals and showed a complex
dependence on the concentration of GSH (Fig. 3b).
Fitting the data obtained for the GSH dependence of
the rate to the same kinetic partitioning reaction
scheme used for human PDI, we obtained values for
the three rate constants. The first nucleophilic attack
by GSH on the oxidized yeast Pdi1p a domain gave
a second-order rate constant of 720 M 1 s 1. The
second nucleophilic attack by GSH on the Pdi1p
GSH mixed disulfide gave a second-order rate
constant of 170 M 1 s 1, while the reverse reaction
initiated by the C-terminal active-site cysteine gave
a first-order rate constant of 6.4 s 1. These rate
constants are between 15 times and 68 times slower
than the corresponding rate constants for the human
protein.
The reactivity of the N-terminal cysteine is
influenced by the C-terminal cysteine
The data for the GSSG-mediated oxidation of both
the human PDI a domain and the yeast Pdi1p a
domain indicate that the rate-limiting step is the
intramolecular nucleophilic attack by the C-terminal
active-site cysteine on the mixed-disulfide species.
This suggests that the mixed disulfide is a relatively
long-lived species with a half-time of around 3 s and
5 s, respectively. To date, it has been assumed that
the reactive species during the oxidation of a peptide
substrate is the oxidized species (i.e., intramolecular
disulfide form of PDI). However, there is no reason
to suppose that the PDIglutathione mixed disulfide
could not also be a target for nucleophilic attack by a
thiolate on a peptide or protein substrate (Fig. 4a). If
it can, then C56 mutants of PDI would also be
expected to be active in catalyzing the GSSG-
mediated oxidation of a peptide substrate.
For in vitro and in vivo studies on the mechanisms
of action of PDI family members, the C-terminal
cysteine is often mutated to a serine, since serine is
similar to cysteine in terms of size and polarity.
However, serine is also a strong -sheet and -helix
breaker, and so the C-terminal cysteine has also been
mutated to alanine in some studies. To examine both
possibilities, we made the C56S and C56A mutants
of the a domain of human PDI in the W128F
background, along with the C56S mutant in the
243
wild-type a domain. As further control, the C53M
mutant was made, with the methionine retaining the
sulfur atom at the N-terminal active-site cysteine
position but not being able to do a similar
nucleophilic attack, and hence the mutant was
expected to have no activity. Using a standard
peptide oxidation assay23 but with GSH omitted, we
determined the activity of the C56A/W128F mutant
at equivalent concentrations to have around 1%
of the activity of the wild-type enzyme (Table 1).
This low activity was not due to the W128F
mutation, as this mutation had N90% of the activity
of the wild-type enzyme. While the C56A/W128F
mutant had low activity, it did retain some activity,
while the C53M mutant did not. To further examine
the residual activity, we increased the enzyme
concentrations of the C56 mutant enzymes 10-fold.
It should be noted under these conditions that the
substrate peptide is only in small excess over the
enzyme concentration, and that due to the nature of
the assay, the usable concentration range of the
peptide substrate in the assay is narrow. Analysis of
the C56S/W128F mutant activity under these con-
ditions showed that it had 25% of the activity of the
wild-type enzyme, implying that on a per-molar
basis, it had around 2.5% of the wild-type
enzyme activity. In contrast to this, the C56A/
W128F mutant showed more complex kinetics.
Instead of a change in fluorescence that could be
fitted to a single-exponential process, a clear
double-exponential process was observed (Fig.
4b). The relative change in fluorescence suggested
that the slower of the two processes was linked to
the formation of the oxidized peptide. This would
imply an activity for this mutant that is 1.2% of
that of the wild-type enzyme on a per-molar basis.
The faster rate constant was 4.9 1.1 10 2 s 1.
Since the enzyme concentration is high in these
assays, we thought that this rate constant might
represent the formation of the glutathioneenzyme
mixed disulfide.
To examine this in more detail, we undertook a
mass-spectrometry-based quenched-flow analysis
of the reaction of the C56 mutants with GSSG. In
these reactions, two products were observed: the
initial reduced species (containing a single reactive
thiol) and the mixed disulfide with glutathione.
Quantification of these two species allowed the
calculation of the first-order rate constants. These
were 1.24 0.23 s 1 for C56A/W128F (Fig. 4c),
0.18 0.01 s 1 for C56S/W128F (Fig. 4d), and
0.21 0.01 s 1 for C56S (Fig. 4e). These are signifi-
cantly lower than the equivalent values obtained
for the wild-type protein by stopped-flow measure-
ment (2.5 s 1) and quenched-flow measurement
(2.6 s 1). Since the concentration of glutathione used
was 10 mM, these convert into second-order rate
constants of 124 M 1 s 1, 18 M 1 s 1, and 21 M 1 s 1
for the C56A/W128F, C56S/W128F, and C56S
244 Glutathione and PDI

Fig. 4. Reactivity of the N-terminal active-site cysteine. (a) Reaction scheme for the oxidation of a peptide substrate. The
upper panel shows the current dogma in the field with four covalent reactions and with nucleophilic attack by the peptide
cysteine on the oxidized site of PDI. The lower panel shows a plausible alternative reaction scheme requiring only three
covalent reactions and with nucleophilic attack by the peptide cysteine on the PDIglutathione mixed disulfide. For
simplicity, the thiol/thiolate state of the reactants, along with proton transfer reactions and the formation of Michaelis
Menten complexes, is not shown. (b) Representative trace for the kinetics of oxidation of a fluorescent decapeptide with
GSSG using 2 M PDI a domain C53A/W128F in the reaction buffer [0.1 M disodium phosphate, 0.1 M boric acid, 0.1 M
citric acid, and 1 mM EDTA (pH 7)] and 3.2 M substrate peptide. The line of best fit is to a sequential process. Inset: The
residuals to fitting to a sequential process. (ce) Quenched-flow analysis of the formation of the PDIglutathione mixed
disulfide from 40 M protein and 10 mM GSSG, as determined by mass spectrometry. The lines of best fit are to a single-
exponential process. (c) C56A/W128F. (d) C56S/W128F. (e) C56S.
Glutathione and PDI
Table 1. Relative rates of oxidation of a fluorescent
decapeptide using GSSG in a reaction buffer [0.1 M
disodium phosphate, 0.1 M boric acid, 0.1 M citric
acid, and 1 mM EDTA (pH 7.0)]; [peptide] = 3.2 M and
[GSSG] = 0.5 mM
Concentration
Construct (M) k (s 1)
Noncatalyzed 5.6 0.2 10 4
a domain 0.2 1.17 0.17 10 2
a domain W128F 0.2 1.09 0.01 10 2
a domain C53M 2.0 5.4 0.2 10 4
a domain C56A/W128F 0.2 6.5 0.4 10 4
a domain C56A/W128F 2.0 4.9 1.1 10 2
2.0 0.4 10 3
a domain C56S/W128F 2.0 3.3 0.3 10 3
Relative activity is calculated by setting the wild-type protein to
100% and the noncatalyzed rate to 0% and by correcting for the
concentration of enzyme used. NA is non-applicable. The first rate
constant from the C56A/W128F mutant at 2.0 mM probably
represents the reaction of the active site with GSSG (see the text).
mutants, respectively. A second-order rate constant
of 124 M 1 s 1 for the C56A/W128F mutant
would give an apparent pseudo-first-order rate
constant of 6 10 2 s 1, with the 0.5 mM GSSG
used in the peptide oxidation assay. This value
(6.2 1.2 10 2 s 1) is comparable with the fast rate
constant observed for this mutant in the peptide
oxidation assay (4.9 1.1 10 2 s 1), suggesting that
this rate does indeed represent the formation of the
glutathione mixed disulfide. From the quenched-
flow studies, the expected pseudo-first-order rate
constant for the C56S/W128F mutant reacting with
GSSG under peptide oxidation assay conditions
would be 9 10 3 s 1, and the similarity of this
rate constant with the observed rate of peptide
oxidation may be the reason that we were unable to
separate the two rate constants.
Discussion
Disulfide bond formation in the ER is complex,
with multiple possible routes for oxidative protein
folding. The widely accepted major route for dithiol
oxidation to a disulfide is Ero1 oxidation of PDI,
followed by PDI oxidation of substrate proteins.
Since the discovery of Ero1, the potential role for
GSSG as a physiologically relevant route for
disulfide bond formation in vivo has been down-
played. Indeed, the role of glutathione in the
formation of native disulfide bonds is now often
regarded either as being solely in the reduction of
incorrect disulfide bonds or as a competitor for the
formation of protein disulfides.20,24,25 However, this
viewpoint overlooks key facts from decades of in
vitro studies. Firstly, the reported GSSG/GSH ratio
present in the ER26,27 gives a redox potential that is
245
optimal for native disulfide bond formation in vitro.
Secondly, previous studies on the rates of reaction of
glutathione with PDI have shown the processes to
be rapid (e.g., Darby and Creighton19). In addition, a
recent elegant study has shown that after treatment
Relative with DTT, the redox potential of the ER is rapidly
activity restored to this optimal level without overshoot.28 It
(%) is difficult to envisage how this occurs or why it is
0 necessary without assuming a role for GSSG/GSH
100 in native disulfide bond formation.
93
0
Using stopped-flow and quenched-flow kinetics,
1 we show here that the reactions of the a domains of
NA PDI and Pdi1p are both more rapid and more
1.3 complex than previously thought. The concentra-
2.5 tions of GSSG and GSH in the ER are not known and
may vary between species, between cell types, or
even for one cell type under different physiological
conditions. However, the total mammalian cellular
glutathione concentration is probably around
10 mM (counting GSSG as two molecules of
glutathione). Thiscombined with the reported
ratio of 3:1 26 in microsomes isolated from the
mammalian ER and ignoring the thorny issue of
glutathioneprotein mixed disulfides27 and ques-
tions relating to how accurate this ratio is in vivo
gives putative concentrations of GSH and GSSG in
the ER of 6 mM and 2 mM, respectively. While these
are open to question, they can be used to give us
some estimates of how fast glutathionePDI ex-
change reactions may occur in vivo. Based on the
calculated rate constants we have obtained, this
would give us rate constants of 0.38 s 1 and 0.23 s 1
for the two steps of oxidation. The rate of reduction
is more complex, but at 6 mM GSH, the net rate of
reduction of oxidized PDI to form GSSG would be
3.5 s 1. While there are caveats on the in vivo
glutathione concentrations and on the degree to
which our data on the isolated a domain can be
extrapolated to full-length PDI, these reactions all
appear to be too fast to be discounted.
Four other striking observations can be made from
our analysis. Firstly, the rates of reaction of the yeast
Pdi1p a domain with GSH are more than an order of
magnitude slower than the reactions of the human
PDI a domain. The two enzymes are not equivalent;
hence, extrapolation of experiments on the physio-
logical role of glutathione in native disulfide from
S. cerevisiae (e.g., Frand and Kaiser12 and Cuozzo
and Kaiser 29 ) to mammalian systems must be
performed with care. In addition, a recent publica-
tion on in vivo reduction potential determination30
obtained a value of ca 230 mV for the reduction
potential of the ER of the yeast Pichia pastoris , which
is very different from the value of 180 mV
originally reported for the mammalian ER,26 again
suggesting that care must be taken when extrapo-
lating between species.
Secondly, there is an apparent contradiction in our
results. The rate constant that we obtain for the
246
nucleophilic attack of the C-terminal cysteine on the
mixed disulfide during oxidation is 0.23 s 1, while
the rate constant for nominally the same reaction
during reduction is 430 s 1. We are not able to trap
the mixed disulfide (see Fig. 1d for reasons); hence, it
is possible that one or both of these rate constants
actually represent something else (e.g., an intramo-
lecular conformational exchange). However, the
results are consistent with our hypothesis. In
addition, a closer examination of the reaction
schemes suggests that a large difference in these
nominally identical steps is not only plausible but
also probable. We have previously reported that the
pKa of the C-terminal cysteine of the active site of
PDI is regulated by an intramolecular conforma-
tional change that brings the side chain of R120 into
the active-site locale.22,31 Normally, the pKa of C56 is
high, and it is only due to the infrequent juxtapo-
sition of the positively charged side chain of R120
that the pKa of C56 decreases and a reactive thiolate
is formed. This is the situation that occurs during the
GSSG oxidation of PDI (Fig. 5). In contrast to this,
the species formed during the GSH-mediated
reduction of PDI is the PDIglutathione mixed
disulfide, but it is not the same mixed-disulfide
species. Instead, the leaving group in this reaction is
the C56 thiolate (Fig. 5). This is a highly reactive
species; hence, nucleophilic attack by this group on
the mixed disulfide can proceed rapidly. The pKa of
C56 probably shows great variation during the
catalytic cycle.22 In the extreme case when R120 is
located far from the active-site locale, which is
represented by NMR structure model 1, the calcu-
lated pKa of C56 is 12.8.22 With this pKa, the
proportion of C56 in the active thiolate form at
pH 7.0 would be 0.00016% (i.e., there may be up to a
630,000-fold difference in the proportion of C56
thiolate species between the initially formed mixed-
disulfide state on the oxidation pathway and the
Fig. 5. Reactions schemes for the reduction (upper) and oxidation (lower) of the active site of PDI by glutathione
showing the thiol/thiolate status of C56 after the initial reaction.
Glutathione and PDI
initially formed mixed-disulfide state on the reduc-
tion pathway, although conformational exchange
and proton transfer may rapidly change both states).
This thiol-versus-thiolate effect may be exaggerated
by the position of the sulfur atom of the C56 side
chain, which must, by definition, be juxtaposed to
the mixed disulfide formed during the reduction
reaction but may be more distant or may have a less
optimal geometry to undertake nucleophilic attack
in the mixed disulfide formed during oxidation.
When combined, these could give the 1870-fold
difference in rate constants that we have observed.
Thirdly, to our knowledge, the PDIglutathione
mixed disulfide is a potential species for nucleophil-
ic attack by a peptide cysteine; hence, a three-
covalent-step catalytic cycle, instead of a four-
covalent-step cycle (Fig. 4a), has not previously
been fully considered. Our results show that
mutation of the C-terminal active-site cysteine
results in mutants that have maximally 2.5% of the
activity in the peptide oxidation assay of the wild-
type a domain. While this may be higher than would
be expected by the dogma in the field, it is still
40-fold lower activity than the wild-type enzyme.
This reduction in catalytic activity comes from at
least two separate effects. (i) The reactivity of the
N-terminal cysteine towards GSSG is reduced by
mutating the C-terminal cysteine. This effect is
greater for the C56S mutants that show around a
10-fold decrease in the rate of reaction of C53
towards GSSG, while the C56A mutant shows less
than a 2-fold effect. This reduction in the rate of
reaction for the C56S mutant may arise from local
conformational changes arising from serine acting
as a strong breaker of regular secondary structure
and/or from hydrogen bonding by the serine (e.g.,
with C53). (ii) Since both C56S and C56A mutants
show a greater effect on their activity in peptide
oxidation than on their rates of reaction with GSSG,
Glutathione and PDI
there must also be a difference between the rate of
reaction of a peptide cysteine with oxidized PDI
and the rate of reaction of a peptide cysteine with
the PDIglutathione mixed disulfide, with the rates
for reaction with the mixed disulfide being signif-
icantly lower. This effect is also visible in the rates of
reaction of GSH with oxidized PDI and with the
mixed disulfide reported here. For the human PDI a
domain, these rates are around 2.5-fold different
(11,200 M 1 s 1 versus 4600 M 1 s 1), while for the All constructs include an N-terminal hexa-histidine tag with the
yeast Pdi1p a domain, they are around 4-fold
different (720 M 1 s 1 versus 170 M 1 s 1). This
effect could arise due either to the difference in the
leaving group and/or to the fact that glutathione
has a net negative charge, and thus a thiol
approaching the PDIglutathione mixed disulfide
would be more likely to see an increase in its pKa
due to electrostatic effects and hence a reduction in
its reactivity.
Fourthly, several studies have used C-terminal
active-site PDI family member mutants to examine
their substrate specificity in vivo (for examples, see
Kadokura et al.32 and Jessop et al.33). While this
methodology only traps a subset of potential
substrates,34 it is a powerful and useful technique
for screening. However, our results indicate that
such studies need to be examined with care, since
mutating the C-terminal active-site cysteine changes
the reactivity of the N-terminal active-site cysteine
and, in other superfamily members, mutation of a
C-terminal active-site cysteine has been shown to
alter the substrate specificity of the enzyme as well.35
Overall, the data generated in this study on the
reactivity of PDI towards glutathione require us to
reevaluate several different aspects of the field of
catalyzed disulfide bond formation, including the
potential physiological role of GSSG in the ER.
Materials and Methods
Generation of expression vectors
Expression vectors for the a domain of human PDI
(D18-A136) with an N-terminal His-tag in frame with the
cloned gene were generated previously.36 The resulting
gene products included the sequence MHHHHHHM
prior to the first amino acid of the domain sequence. Site-
directed mutagenesis was performed as recommended by
the manufacturer using the QuickChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA, USA). All
plasmids generated were sequenced to ensure that there
were no errors in the cloned genes (see Table 2 for
plasmids used in this study).
Protein expression and purification
Protein production was carried out in Escherichia coli
strain BL21(DE3) pLysS. Strains were grown in LB media
247
Table 2. Plasmids used in this study
Name Construct
pLWRP69 Human PDI a domain (D18-A137)
pAKL2 Human PDI a W128F
pOLR144 Human PDI a C53M
pMJP70 Human PDI a C56S
pAKL118 Human PDI a C56S/W128F
pAKL126 Human PDI a C56A/W128F
pKEHS226 Yeast Pdi1p a domain (Q23-P141)
sequence MHHHHHHM.
at 37 C and 200 rpm and induced at an OD600 of
0.3 for 3 h with 1 mM isopropyl -D-thiogalactoside. Cells
were pelleted by centrifugation (6500 rpm for 10 min).
The pellet was resuspended in 1/10 vol of buffer A
(20 mM sodium phosphate, pH 7.3), and 1/1000 vol
of 10 mg ml 1 DNase (Roche Diagnostics GmbH,
Mannheim, Germany) was added. The cells were lysed by
freeze-thawing twice, and cell debris was removed by
centrifugation (12,000g for 20 min). The supernatant had
been filtered through a 0.45-m filter before it was
applied to an immobilized metal-affinity chromatography
column (HiTrap Chelating Sepharose Fast Flow; Amer-
sham Biosciences, Uppsala, Sweden), precharged with
Ni2+, and equilibrated in buffer A. After loading, the
column was washed in 20 mM sodium phosphate, 50 mM
imidazole, and 0.5 M NaCl (pH 7.3), and then in buffer A,
before the His-tagged proteins were eluted using 20 mM
sodium phosphate and 50 mM ethylenediaminetetraacetic
acid (EDTA) (pH 7.3). The eluant was diluted five times
into buffer A and then applied to a 6-ml Resource Q anion
exchanger (Amersham Biosciences), from which the
proteins were eluted with a linear gradient from buffer
A to buffer A plus 0.5 M NaCl over 9 column volumes.
Eluted fractions were checked for purity by SDS-PAGE,
and fractions containing pure protein were pooled and
buffer exchanged into buffer A using an Ultrafree
Centrifugal Biomax 5000 nominal-molecular-weight
membrane filter (Millipore, Bedford, MA, USA). All of
the proteins used in this study gave a single band on
Coomasie-stained SDS-PAGE. The concentration of each
protein was determined spectrophotometrically using a
calculated absorption coefficient: PDI a domain
(19,720 M 1 cm 1; Mr = 14,342), PDI a domain W128F
(14,120 M 1 cm 1; Mr = 14,303), PDI a domain C56S
(19,720 M 1 cm 1; Mr = 14,326), PDI a domain C56S/
W128F (14,120 M 1 cm 1; Mr = 14,287), PDI a domain
C56A/W128F (14,120 M 1 cm 1; Mr = 14,271), and Pdi1p
a domain (9860 M 1 cm 1; Mr = 14,275) at 280 nm. All
purified proteins were analyzed for authenticity by
matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry, and all experimentally determined
masses were the same as the expected mass (within the
mass accuracy limit of the spectrometer).
Kinetics of oxidation and reduction
The rates of oxidation and reduction of the active site of
the a domains of human PDI or yeast Pdi1p were
determined by the change in fluorescence of the
248
tryptophan adjacent to the active site using a KinTek
SF-2004 stopped-flow fluorometer, an excitation of
280 nm, a bandpass emission of N 320 nm at 25 C,
and 10 M PDI a domain W128F mutant or Pdi1p a
domain with 120 mM GSSG or 550 mM GSH. To ensure
complete oxidation or reduction of the active site prior
to the kinetic studies, we incubated purified protein
(up to 76 M) for 30 min in a 5-fold molar excess of DTT
or GSSG (both from Sigma, St. Louis, MO, USA) at room
temperature, and then the excess of DTT or GSSG was
removed and the enzymes were buffer exchanged into a
reaction buffer [0.1 M disodium phosphate, 0.1 M citric
acid, 0.1 M boric acid, and 1 mM EDTA (pH 7)] with
Biomax Ultrafree 0.5 centrifugal filter concentrators
(Millipore). Each data point was collected using two to
four kinetic traces from each diluted sample and with
multiple diluted samples. Hence, the standard deviation
represents both errors in measuring rate constants from
a single loading of a diluted sample and errors in
dilution to prepare multiple samples. The data are not
all collected on the same day, but there is no evidence of
day-to-day variations in the data, with the exception that
that absolute magnitude of the fluorescence goes down
slightly as the bulb ages. This does not affect the
kinetics. The kinetic traces were analyzed with IGOR
(Wavemetrics, Portland, OR, USA) using preset single-
exponential or double-exponential functions or user-
defined functions such as sequential reactions. The
equations for a sequential reaction (A to B to C) are as
follows:
AA0 exp k1 t
BA0 k1 expk1 t expk2 t = k2 k1
CA0 k2 1 expk1 t k1 1 expk2 t = k2 k1 by grants from the Academy of Finland (to L.W.R.),
where k1 and k2 are the first-order or pseudo-first-
order rate constants, and t is time. Upon analysis of
fluorescence, the fluorescence of the sample at time
t (Ft) is:
Ft AFA BFB CFC
where FA, FB, and FC are the fluorescence intensities of
species A, B, and C, respectively.
Analysis of the concentration dependence of the
reduction of the active site by GSH was performed with
IGOR (Wavemetrics) using preset or user-defined func-
tions. The simplest kinetic scheme that gave apparently
random residuals by eye was used for all subsequent
analyses.
Control experiments indicated that the effects of
photobleaching on the determined kinetics for reactions
up to 5 s in length are negligible.
Parallel experiments were also performed by quenched-
flow measurement using a KinTek RQF3 quenched-flow
apparatus. Protein (40 M) was allowed to react with
GSSG or GSH for a variable amount of time before being
quenched with 200 mM N-ethyl maleimide. The quenched
protein sample was then purified using a PepClean C-18
spin column (Pierce, Rockford, IL, USA) before electro-
spray ionizationmass spectrometry analysis (Micromass
Glutathione and PDI
LCT, Manchester, UK). For a time point longer than 10 s,
manual mixing of the reagents was performed. When we
were unable to observe trapping of a mixed disulfide
using this methodology, other trapping methods were
used prior to mass spectrometric analysis. These included
varying the quencher concentration, using other chemical
quenchers such as iodoacetamide, and combining
quenchers with chemical denaturants such as urea or
guanidinium hydrochloride, acids such as trifluoroacetic
acid or hydrochloric acid, and acids in combination with
acetonitrile. While the mixed-disulfide state was observed,
none of these conditions gave reproducible trapping of the
mixed-disulfide state.
The quenched-flow kinetic traces were analyzed using
IGOR (Wavemetrics) using preset single-exponential
functions.
Oxidase assay
The method of Ruddock et al. using a fluorescent
decapeptide was used to determine the oxidase activity of
each of the purified human PDI family members. Substrate
peptide (3.2 M) and reduced enzyme (0.22 M) were
incubated in a reaction buffer [0.1 M disodium phosphate,
0.1 M citric acid, 0.1 M boric acid, and 1 mM EDTA (pH 7)]
at 25 C for 2 min before GSSG was added to the reaction
mixture and the measurement was started.23
Acknowledgements
We thank Robert Freedman for valuable com-
ments on this manuscript. This work was supported
Biocenter Oulu (to L.W.R.), University of Oulu
(to L.W.R. and A.-K.L.), Finnish Concordia Fund
(to A.-K.L.), Gustaf Komppa Foundation (to A.-K.L.),
and Orion-Farmos Research Foundation (to A.-K.L.).
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