(Received for publication, December 5, 1995)
From the
Phosphoribulokinase (PRK) is one of several plant enzymes that
is regulated by thiol-disulfide exchange as mediated by thioredoxin,
which contains spatially vicinal, redox-active cysteinyl residues. In
an earlier study (Brandes, H. K., Larimer, F. W., Geck, M. K.,
Stringer, C. D., Schürmann, P., and Hartman, F.
C.(1993) J. Biol. Chem. 268, 18411-18414), our
laboratory identified Cys-46 of thioredoxin f (Trx), as
opposed to the other candidate Cys-49, as the primary nucleophile that
attacks the disulfide of target proteins. The goal of the present study
was to identify which of the two redox-active cysteinyl residues of PRK
(Cys-16 or Cys-55) is paired with Cys-46 of Trx in the interprotein
disulfide intermediate of the overall oxidation-reduction pathway.
Incubation of a mixture of the C16S mutant of PRK and the C49S mutant
of Trx with Cu results in covalent complex formation
as detected by SDS-polyacrylamide gel electrophoresis. Complexation is
fully reversible by dithiothreitol and is retarded by ligands for PRK.
Under the same conditions, Cu
induces very little
complex formation between the following pairs of mutants: C16S PRK/C46S
Trx, C55S PRK/C49S Trx, and C55S PRK/C46S Trx. When either
5-thio-2-nitrobenzoate-derivatized C16S or C55S PRK, as mimics of the
oxidized (disulfide) form of the enzyme, is mixed with C49S Trx, stable
covalent complex formation occurs only with the C16S PRK. Thus, two
independent approaches identify Cys-55 of PRK in the intermolecular
disulfide pairing with Trx.
Thioredoxins are small (12-14 kDa) ubiquitous proteins
that control the redox state of diverse target proteins by the
mediation of thiol-disulfide exchanges(1, 2) . The
redox-active sulfhydryls of thioredoxin are located in the highly
conserved active-site sequence -Trp-Cys-Gly-Pro-Cys-. The pathway for
the reduction of a protein disulfide by thioredoxin entails
nucleophilic attack by one of the active-site sulfhydryls to form a
protein-protein mixed disulfide followed by intramolecular displacement
of the reduced target protein with concomitant formation of oxidized
thioredoxin (Fig. 1). As the mixed disulfide is disfavored
thermodynamically, its direct characterization has not been possible.
However, the active-site cysteinyl residue of thioredoxin that is
nearest the N terminus (Cys-32 of the Escherichia coli protein) is well established as the primary nucleophile.
Originally, this assignment was deduced from the high chemical
reactivity and the low pK (6.7) of Cys-32 (3, 4, 5, 6) and was subsequently
supported by the three-dimensional structure of E. coli thioredoxin, in which Cys-32 is solvent-accessible and Cys-35 is
inaccessible(7) . More recently, Cys-32 of E. coli thioredoxin, the corresponding residue of human thioredoxin, and
the corresponding residue of the closely related protein glutaredoxin
were shown to engage in mixed disulfide bond formation with low
molecular weight thiols or
peptides(8, 9, 10, 11) . Finally,
the C49S mutant of chloroplastic thioredoxin f (Trx) (
)retains the capacity to activate chloroplastic
fructose-1,6-bisphosphatase, whereas the C46S mutant is totally
inactive in this regard. Thus, Cys-46 of Trx (analogous to Cys-32 of E. coli thioredoxin) is verified as the primary nucleophile in
the reduction of in vivo target proteins, thereby minimizing
the possibility that protein-protein interactions might alter the
relative accessibility and reactivity of the active-site sulfhydryls of
thioredoxin.
Figure 1: Pathway for redox modulation of target proteins (TARG) by thioredoxin (TRX).
In contrast to the rigorous proof that Cys-46 of Trx participates in intermolecular disulfide bond formation, the identity of the pairing residue in any target protein has not been established heretofore. We have addressed this issue with PRK by examining the potential of site-directed mutants, which lack either one of the two redox-active sulfhydryls (Cys-16 or Cys-55), to form stable mixed disulfides with the C49S mutant of Trx. This approach, which should be applicable to other target proteins, clearly identifies Cys-55 as the participant in the intermolecular mixed disulfide.
PRK, a homodimer with a subunit molecular weight of 39,232(16, 17) , is representative of plant enzymes that are regulated during light-dark transitions(18) . Photon flux drives the reduction of thioredoxin (as mediated by ferredoxin-thioredoxin reductase), which in turn reduces the regulatory disulfide of target proteins(18, 19, 20) . Having recently identified Cys-46 as the primary nucleophile of Trx (13) , we wished to complete the description of the molecular pathway for activation (reduction) of PRK by ascertaining which of its two regulatory cysteinyl residues (Cys-16 or Cys-55) is involved in the protein-protein mixed disulfide intermediate. As the instability of the putative intermediate precludes its direct characterization, we reasoned that a stable complex should result by combining mutants of Trx and PRK, each retaining the cysteinyl residue necessary for intermolecular disulfide bond formation but lacking the spatially proximal cysteinyl residue responsible for intramolecular disulfide bond formation.
Reaction mixtures were prepared with all four
possible combinations of the relevant mutants of PRK and Trx: C16S
PRK/C49S Trx, C16S PRK/C46S Trx, C55S PRK/C49S Trx, and C55S PRK/C46S
Trx. Cupric sulfate was added to the reaction mixtures to catalyze
disulfide bond formation between those pairs of sulfhydryls that become
juxtaposed within the protein-protein complexes. SDS-polyacrylamide gel
electrophoresis reveals a prominent 54-kDa species, consistent with
covalent coupling between PRK (40-kDa subunit) and Trx (13.5-kDa),
derived from the reaction mixture of C16S PRK/C49S Trx (Fig. 2A). The 54-kDa species is lacking in the
reaction mixtures of C55S PRK paired with either mutant of Trx and is
barely discernible in the reaction mixture of C16S PRK/C46S Trx. Thus,
the observed covalent coupling involves Cys-55 of PRK and Cys-46 of
Trx. When the C16S PRK/C49S Trx reaction mixture is incubated with DTT
prior to electrophoresis, the complex is not present, verifying that
stable complexation is via a disulfide linkage. Covalent complexation
does not occur merely in the presence of oxygen (all buffers are
air-saturated), so either a catalyst (e.g. Cu) or stronger oxidant would appear to be
required. Interestingly, Cu
-o-phenanthroline
(generally considered a more efficient catalyst than Cu
alone for oxidation of sulfhydryls)(21) ,
dehydroascorbate, and oxidized glutathione are totally ineffective in
promoting formation of the 54-kDa species. These negative observations
provide indirect evidence that the oxidation catalyzed by
Cu
occurs within the preformed noncovalent C16S
PRK
C49S Trx complex in which solvent accessibility to the site of
oxidation is limited.
Figure 2:
Polyacrylamide gel electrophoresis of
PRK/Trx reaction mixtures. Denaturing (+ SDS), silver-stained gels
are shown in A, B, and C, and a
nondenaturing, Coomassie Blue-stained gel is shown in D. In
all cases, Trx was used at a 2-fold molar excess relative to the
concentration of PRK subunits. A, incubations of mixtures of
PRK mutants and Trx mutants in the presence of Cu at
time zero (lane 1) and after 24 h (lanes 2-6).
Samples applied (equivalent to 50 ng of PRK) are as follows: lane
1, C16S PRK/C49S Trx (control); lane 2, C16S PRK/C49S
Trx; lane 3, C16S PRK/C46S Trx; lane 4, C55S PRK/C49S
Trx; lane 5, C55S PRK/C46S Trx; lane 6, C16S PRK/C49S
Trx + 0.1 M DTT. B, time course for covalent
complex formation between C16S PRK and C49S Trx in the presence of
Cu
with or without an added ligand for PRK. Samples
applied (equivalent to 40 ng of PRK) are as follows: lane 1,
time zero; lane 2, 3 h; lane 3, 10 h; lane
4, 24 h; lane 5, 24 h in the presence of 1.5 mM MgATP; lane 6, 24 h in the presence of 2.6 mMD-ribulose 5-phosphate (each ligand at 20
K
). C, covalent complex derived
from C49S Trx plus TNB-derivatized C16S PRK. Samples applied
(equivalent to 25 ng of PRK in lanes 1-6 and 25 ng of
Trx in lane 2) are as follows: lane 1, C16S PRK; lane 2, C49S Trx; lane 3, TNB-derivatized C16S PRK; lane 4, C49S Trx + TNB-derivatized C16S PRK; lane
5, sample shown in lane 4 after incubation with 10 mM DTT; lane 6, C49S Trx + TNB-derivatized C55S PRK. D, covalent complex derived from C49S Trx plus TNB-derivatized
C16S PRK. Samples applied (equivalent to 350 µg of PRK) are as
follows: lane 1, TNB-derivatized C16S PRK; lane 2,
C49S Trx + TNB-derivatized C16S PRK; lane 3, C16S PRK; lane 4, TNB-derivatized C16S PRK after treatment with 10
mM DTT; lane 5, C49S Trx
C16S PRK complex after
treatment with 10 mM DTT.
A time course of
Cu-catalyzed oxidation of C16S PRK/C49S Trx and the
inhibitory effects of ATP and ribulose-5-phosphate are shown in Fig. 2B. As both regulatory cysteinyl residues of PRK
are located at the active site, the inhibition by MgATP and ribulose
5-phosphate of intermolecular disulfide bond formation between C16S PRK
and C49S Trx is readily explained. The inability to drive the
complexation to completion even with a 5-fold molar excess of Trx (data
not shown) probably reflects undefined Cu
-catalyzed
oxidations of PRK distinct from protein-protein disulfide bond
formation. For example, we note inactivation of the PRK control
(including Cu
but excluding Trx) at about the same
rate as that of the C16S PRK/C49S Trx reaction mixture (data not
shown). Furthermore, in both cases, these inactivations are fully
reversible with DTT so that kinase activity cannot be used to monitor
the extent of complex formation.
Even though the results of the
Cu-catalyzed oxidations clearly invoke Cys-55 of PRK
as participatory in the normal mixed disulfide intermediate and confirm
our earlier identification of Cys-46 of Trx as the bridging partner, a
caveat arises. Is the noncovalent complex generated from mimics of the
reduced forms of both Trx (C49S) and PRK (C16S) equivalent to the
normal complex of the thiol-disulfide exchange pathway, in which one of
the two proteins would be oxidized and the other reduced? To circumvent
this concern, we prepared mixed disulfides of the C16S and C55S mutants
of PRK by treatment with DTNB; the derivatized mutants can be viewed as
mimics of the oxidized (disulfide) form of PRK. With each mutant, only
1 molar eq of TNB was released concomitant with complete loss of kinase
activity. Original levels of activity (specific activities of C16S and
C55S are 95 and 20% of the wild-type enzyme, respectively) were
restored by incubating the derivatized proteins with 10 mM DTT. These data (not shown) verify the formation of the desired
protein-TNB mixed disulfide. Upon combining TNB-derivatized C16S PRK
with a 2-fold molar excess of C49S Trx, the former is totally consumed
and replaced by a 54-kDa species, which undergoes dissociation in the
presence of DTT (Fig. 2C). The absence of a band
coinciding with free PRK subunit demonstrates that both binding sites
for Trx of dimeric PRK are occupied in the covalent complex prior to
its dissociation by SDS. In stark contrast, TNB-derivatized C55S PRK
does not give rise to detectable levels of the covalent complex. Thus,
an approach totally independent of Cu
-catalyzed
oxidation invokes the same residue (Cys-55) of PRK in formation of the
mixed disulfide with Trx.
The C16S PRKC49S Trx complex is also
well resolved from C16S PRK by polyacrylamide gel electrophoresis under
nondenaturing conditions (Fig. 2D). Its slower
mobility, relative to PRK, shows that dissociation of PRK subunits does
not occur under the conditions used to prepare the covalent complex.
Based on analogies with thioredoxin, we had presupposed that Cys-16
of PRK, rather than Cys-55, engages in mixed disulfide bond formation.
Cys-16 (like Cys-32 of E. coli thioredoxin) displays an
abnormally low pK(22) and hyperreactivity
toward numerous sulfhydryl
reagents(23, 24, 25) , whereas Cys-55 (like
Cys-35 of E. coli thioredoxin) is far less accessible for
chemical modification(26, 27, 28) .
Apparently, the sulfur atom of Cys-55 is either more exposed in the
disulfide form of PRK than in the free sulfhydryl form or the
interaction of Trx with PRK induces a conformational change that
renders this sulfur susceptible to attack by Cys-46 of Trx.
In
summary, this study marks the first example of a complete molecular
description of the thiol-disulfide exchange pathway between any
thioredoxin and a target enzyme, provides a facile avenue for large
scale preparation of a PRKTrx covalent complex for future
biochemical and structural characterization, and validates an approach
that could be of general utility in the characterization of other
target protein-thioredoxin complexes.