From the Division of Molecular Biology and
Biochemistry and ¶ Division of Cell Biology and Biophysics,
School of Biological Sciences, University of Missouri,
Kansas City, Missouri 64110-2499
Received for publication, October 12, 2000, and in revised form, February 19, 2001
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ABSTRACT |
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This study was undertaken to examine the
mechanistic significance of two highly conserved residues positioned in
the active site of pyruvate dehydrogenase kinase, Glu-243 and His-239.
We used site-directed mutagenesis to convert Glu-243 to Ala, Asp, or
Gln and His-239 to Ala. The resulting mutant kinases demonstrated a
greatly reduced capacity for phosphorylation of pyruvate dehydrogenase. The Glu-243 to Asp mutant had ~2% residual activity, whereas the Glu-243 to Ala or Gln mutants exhibited less than 0.5 and 0.1% residual activity, respectively. Activity of the His-239 to Ala mutant
was decreased by ~90%. Active-site titration with
[ Mammalian pyruvate dehydrogenase complex
(PDC)1 catalyzes oxidative
decarboxylation of pyruvate with concomitant formation of acetyl-CoA
and NADH. Under physiological conditions this reaction is irreversible
and, therefore, largely defines the metabolic fate of pyruvic acid and
carbohydrate fuels in general (1). In well oxygenated tissues such as
brain, skeletal muscle, heart, and kidney, PDC commits pyruvate to
further oxidation through the Krebs cycle, thus supplying the oxidative
fuel for the generation of ATP. In lipogenic tissues such as liver,
fat, and mammary gland, PDC provides acetyl-CoA primarily for
biosynthesis of fatty acids, allowing the excessive carbohydrates
obtained with diet to be spared. To fulfill these opposing functions,
the activity of mammalian PDC is tightly regulated by reversible
phosphorylation (2). Phosphorylation of the dehydrogenase component of
the complex (E1 component) by dedicated pyruvate dehydrogenase kinase
(PDK) renders the entire complex inactive (3). Phospho-PDC can be re-activated through the action of another dedicated enzyme, pyruvate dehydrogenase phosphatase (4). In mammals, both kinase and phosphatase
components exist in several isozymic forms (four for PDK (5) and two
for pyruvate dehydrogenase phosphatase (6)), which are likely to
contribute to the tissue-specific regulation of PDC. The latter is
evidenced by the tissue-specific distribution of different isozymes (5)
as well as by their different responses to the naturally occurring
inhibitors and activators of the kinase (7) and phosphatase reactions
(6).
Despite the important role that phoshorylation of PDC plays in
regulation of carbohydrate metabolism, little is known about molecular
mechanisms underlying the phosphorylation reaction. Kinase-driven
inactivation of PDC occurs as a result of phosphorylation of three
serine residues usually referred to as phosphorylation sites 1, 2, and
3 (8). This makes PDK a strictly Ser-specific protein kinase.
Surprisingly, PDK (5) along with the homologous mitochondrial protein
kinase that phosphorylates the branched chain On the other hand, the differences between these two groups of enzymes
are also quite apparent. Histidine kinases use ATP to phosphorylate
their own histidine residue positioned within, external to the
nucleotide-binding site histidine-bearing domain (12, 13). It is
generally believed that the phospho-accepting histidine residue
directly attacks the Site-directed Mutagenesis--
Point mutations within the amino
acid sequence of rat PDK2 were introduced using
oligonucleotide-directed mutagenesis (17). The sequences of mutagenic
oligonucleotides were as follows: 5'-CCA CAT GCT CTT TGA
TCT CTT CAA GAA TGC C-3' for Glu-243 to Asp mutant; 5'-CCA
CAT GCT CTT TCA ACT CTT CAA GAA TGC C-3' for Glu-243 to Gln
mutant; 5'-CCA CAT GCT CTT TGC ACT CTT CAA GAA TGC C-3' for Glu-243 to Ala mutant; 5'-CCA CCT CTA CGC CAT GCT CTT TGA
ACT C-3' for His-239 to Ala mutant; and 5'-ATC AAA ATG AGT
GAG CGA GGC GGG GGT-3' for Asp-282 to Glu mutant (the amino
acid residues are numbered according to the sequence of mature rat
PDK2; the altered bases are underlined). Mutagenesis reactions were
carried out on double-stranded DNA of rat PDK2 (5) subcloned into pUC 19 using the ExSiteTM site-directed mutagenesis kit
(Stratagene, La Jolla, CA). The reactions were set up essentially as
recommended by the manufacturer. The mutations as well as the fidelity
of the rest of DNAs were confirmed by direct sequencing (19).
Expression and Purification of the Mutant Kinases--
PDK2
cDNAs (~1.2 kilobases) carrying the point mutations were cut out
of the pUC 19 DNA with SacI and HindIII
restriction enzymes and re-ligated into the pET-28a expression vector
(Novagen, Madison, WI) between SacI/HindIII sites
of the vector (pPDK2 vector). Plasmids containing the inserts of the
correct size (~1.2 kilobases) were identified by restriction
analysis. Positive plasmids were co-transfected into BL21(DE3) cells
(Novagen) along with pGroESL plasmid carrying the genes coding for
molecular chaperones GroEL and GroES under the control of an
isopropyl-1-thio-
The expression of the mutant kinases was performed essentially as
described previously (14). Their purification was carried out using
TALONTM (CLONTECH Laboratories, Inc.,
Palo Alto, CA) affinity resin as described elsewhere (7). The protein
composition of each preparation was evaluated by SDS/PAGE analysis.
Gels were stained with Coomassie R250. All enzyme preparations used in
the present study were more than 90% pure.
Other Enzyme Preparations--
Human recombinant PDC was
expressed in Escherichia coli as described in Harris
et al. (20). The complex was purified by polyethylene glycol
8000 (Sigma) precipitation, gel filtration on Sepharose 4B (Amersham
Pharmacia Biotech), and high speed centrifugation (20). The E1
component of PDC was expressed and purified as described elsewhere
(21). All enzyme preparations used in this study were more than 90%
pure as judged by Coomassie-stained SDS/PAGE.
Kinase Pull-down Assay--
To construct "bait" vectors for
pull-down experiments, unique NdeI and XhoI
restriction sites flanking the coding region of the kinase cDNA
were introduced by site-directed mutagenesis. Respective cDNAs were
subcloned between NdeI and XhoI sites of pET-28a
vector (Novagen), producing in-frame fusion with the vector sequence
coding for a His6-Tag. "Catch" plasmids were
constructed by subcloning kinase cDNAs flanked by SstI
and XhoI restriction sites into pET-23a (Novagen) cut with
SstI and XhoI, producing in-frame fusion with a
T7-tag sequence coded by the vector. An expression cassette
carrying the T7 promoter, the cDNA of interest, and a
T7 terminator was cut out of catch vectors using
BbsI and DraIII restriction enzymes. After
blunt-ending, it was subcloned into an appropriate bait vector cut with
DraIII, blunt-ended with T4 polymerase, and
dephosphorylated with calf intestinal phosphatase. Thus, the resulting
catch/bait plasmids were carrying two expression cassettes: one
directing synthesis of His6-tagged kinase and the other
directing synthesis of T7-tagged kinase. Expression of the respective proteins was performed in BL21(DE3) cells co-transformed with appropriate catch/bait plasmid and pGroESL vector, essentially as
described above. Respective recombinant kinases were isolated using
metal affinity chromatography on TALONTM resin
(CLONTECH) (7). Isolated proteins were analyzed
using SDS/PAGE and Western blotting with anti-His6-tag
antibodies (CLONTECH) or anti-T7-tag
antibodies (Novagen). Immunoreactive bands were visualized with
125I-protein A (ICN Biomedicals, Inc., Costa Mesa, CA)
followed by autoradiography (22).
Standard PDK Activity Assay--
Kinase activity was determined
by following [32P]phosphate incorporation from
[ ATP Binding Assay--
Nucleotide binding studies were conducted
using a modified vacuum filtration assay developed by Pratt and Roche
(23). Briefly, the recombinant kinases were used in a final
concentration of 0.1 mg/ml in binding buffer (20 mM
Tris-HCl, pH 7.4, 5 mM MgCl2, 50 mM
KCl, 5 mM dithiothreitol). Binding reactions (total volume of 100 µl) were initiated by the addition of
[ ATPase Activity Assay--
ATPase activity was assayed
essentially as described by Singh and Cerione (24). Briefly, reactions
were set up in a final volume of 100 µl containing 50 mM
Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM
dithiothreitol, 5% (v/v) glycerol, and 10 µM
[ PDC Binding Assay--
PDC binding reactions were set up in
100-µl volumes containing 20 mM Tris-HCl, pH 7.8, 5 mM MgCl2, 50 mM KCl, 1 mM dithiothreitol, 1.0 mg/ml recombinant PDC, and various
amounts of PDK2. Kinase was allowed to bind to the complex for 15 min
at room temperature. By the end of the incubation, reactions were
loaded on the columns containing 1 ml of Sephacel S300 (Amersham
Pharmacia Biotech) equilibrated with 20 mM Tris-HCl, pH
7.8, 5 mM MgCl2, 50 mM KCl, 1 mM dithiothreitol, and 0.1% (w/v) Tween 20. Before the
experiment, columns were centrifuged at 2,000 rpm for 1 min in a IEC
Centra CL2 centrifuge (International Equipment Co., Needham Heights, MA) equipped with a bucket rotor. Immediately after loading, columns were centrifuged again at 2,000 rpm for 1 min. The flow-through containing the PDC-kinase complex was collected in Eppendorf tubes. The
resulting preparations of PDC-bound kinase were analyzed further by
Western blotting with anti-PDK2 antibodies essentially as described (22).
Analysis of Kinetic and Binding Data--
Raw kinetic and
binding data were analyzed using GraFit version 3 software (Erithacus
Software Ltd, Middlesex, UK). The apparent inhibition constants were
determined by measuring the initial rates of the phosphorylation
reaction at various concentrations of nucleotide substrate and
inhibitor (matrix of 24 different conditions). The resulting matrixes
were analyzed as a set to determine the respective kinetic parameters.
Shown are representative results obtained with one out of three
preparations of each enzyme analyzed for this study.
Other Procedures--
SDS/PAGE was carried out according to
Laemmli (25). Protein concentrations were determined according to Lowry
et al. (26) with bovine serum albumin as standard.
Kinase Activity of Wild-type and Mutant Proteins--
In this
study, we used site-directed mutagenesis to test a hypothesis that the
invariant glutamic acid residue of mammalian PDK (Glu-243 in rat PDK2)
serves as a catalyst in the phospho-transfer reaction. We also analyzed
the functional significance of two additional residues: His-239, which
may be involved in polarization of Glu-243, and Asp-282, which
contributes to the binding of ATP in the active site (14). The latter
residue has been chosen as a control because its substitution produces
a kinase with impaired catalytic ability. However, this effect, as
previously established (14), is achieved by different means, through
the knockout of nucleotide binding. The invariant Glu-243 of rat PDK2
was altered to Ala, Asp, and Gln and His-239 was altered to Ala. The
invariant Asp-282 was changed to Glu. Wild-type PDK2 and respective
mutant kinases were expressed in E. coli cells and purified
to near homogeneity as discribed under "Experimental Procedures."
When the activities of wild-type and mutant enzymes were assayed in the
standard phosphorylation assay (see "Experimental Procedures"), all
mutant kinases showed a markedly decreased ability to phosphorylate PDC
(Fig. 1A). Substitution of
His-239 to Ala resulted in reduced kinase activity (~12% of that of
wild-type enzyme). Kinases with mutations of Glu-243 to Asp, Ala, or
Gln had ~2%, <0.5%, and <0.1% residual activity, respectively.
PDK2 carrying the substitution of Asp-282 to Glu showed less than 0.2%
activity of wild-type enzyme (Fig. 1A). This outcome
strongly suggests that all residues targeted for mutagenesis in this
study are important for kinase function.
Kinetics of Phosphorylation Reaction for Wild-type and Mutant
Kinases--
If Glu-243 and His-239 contribute to the catalysis of
phosphotransfer reaction, substitution of these residues would be
expected to affect the catalytic efficiency and should be manifested as a decrease in the apparent Vmax, with little if
any effect on the apparent Km value for the
nucleotide substrate. To test this hypothesis, we determined the
kinetics of the phosphorylation reaction using kinase mutants showing
substantial residual activity: Glu-243 to Asp and His-239 to Ala.
Determinations were made at a single fixed concentration of PDC of 1.0 mg/ml and several concentrations of ATP varied from 3 to 200 µM (Fig. 1B). As expected, substitution of
either His-239 to Ala or Glu-243 to Asp resulted in a decrease in
apparent Vmax values, 7- and 50-fold,
respectively (Fig. 1B, insert). On the other
hand, the apparent Km value for ATP of the His-239
to Ala mutant was equal to the Km value of the
wild-type enzyme within experimental error (4.4 ± 1.3 versus 6.4 ± 1.2 µM). The Glu-243 to Asp
mutant showed a small increase in the apparent Km
value for ATP (~14.7 ± 2.1 µM).
Active-site Titration with [
In contrast, the substitution of Asp-282 severely affected
nucleotide binding. The Asp-282 to Glu mutant did not exhibit any binding of [ PDC Binding--
It is generally believed that the kinase
component is an integral part of PDC, and every complex contains 2-3
tightly bound molecules of kinase (27). Association with the complex
alone accounts for more than a 10-fold increase in the kinase activity (28). Therefore, an essential decrease in activity would be expected if
the mutations affected the interaction between kinase and PDC directly
or indirectly. To explore this possibility, we measured the PDC-kinase
interaction using gel filtration through Sephacel S-300 columns (see
"Experimental Procedures"). This procedure allows for fast
separation of free and complex-bound kinase, thus decreasing the
possibility of kinase dissociation during the procedure. As shown in
Fig. 3A and B,
under these conditions binding of wild-type PDK2 to the complex was
readily detectable. To evaluate the ability of PDK2 active-site mutants
to interact with the protein substrate, we conducted a series of
experiments to determine their relative affinities for PDC (Fig.
3C). It appeared that all mutant kinases tested bind PDC at
levels similar to the wild-type kinase. This suggests that the ability
to catalyze the phosphotransfer reaction or the ability to bind the
nucleotide substrate has no profound effect on the protein-protein
interactions involved in kinase binding. On the other hand, we have
noticed that all mutant proteins had a somewhat higher affinity for the
protein substrate. The latter was especially apparent for the Asp-282
to Glu mutant, which consistently showed 2-3-fold higher affinity for
PDC. The rationale for this phenomenon is currently unknown.
Inter-subunit Interaction--
In solution, PDK exists as a dimer
(3). Dimerization was suggested to be very important for kinase
function, allowing the kinase to move around PDC, phosphorylating
multiple copies of the E1 component without dissociating from the
complex (30). Thus, if the mutations somehow compromise the
inter-subunit interactions within the dimer, this might have a
deleterious effect on kinase activity. To explore this possibility, we
employed a genetic approach, co-expressing His6-tagged and
T7-tagged kinase molecules in E. coli. This
allowed the use of the His6-tagged kinase as a bait to pull
down T7-tagged kinase on an affinity resin. The resulting preparations can be characterized for the presence of
T7-tagged species by Western blot analysis with
anti-T7-tag antibodies. This way, it is possible to
establish the formation of mixed species and draw conclusions about the
strength of inter-subunit interaction for different mutant kinases.
When the respective constructs were expressed in E. coli,
His6-tagged species were purified on TALONTM
resin and probed with anti-T7-tag antibodies using Western
blot analysis; it was found that all preparations tested contained T7-tagged species, which cannot directly bind
TALONTM resin (Fig. 4,
panels A and B). The latter strongly suggests that the wild-type PDK2 as well as kinases carrying amino acid substitutions within the kinase domain exist as dimers. Furthermore, the ratios between T7-tagged and His6-tagged
species in each preparation were very similar, indicating that the
ability to catalyze phosphotransfer reaction or the ability to bind
adenyl nucleotides is not required for dimer formation.
ATPase Activity in Preparations of PDK--
Many phosphokinases
(31), including some protein kinases (32, 33), have been reported to
possess an intrinsic hydrolytic activity toward ATP. If PDK uses
general base catalysis to activate the hydroxyl group of the serine
residue for the direct attack on the ATP
In several studies, the specificity of ATP hydrolytic
reaction was demonstrated using compounds acting as potent inhibitors of ATP binding (34, 35). Unfortunately, to date, there are no PDK
inhibitors available that are specific for the nucleotide binding
domain. However, we reasoned that some of ATP-binding site inhibitors
designed for enzymes with nucleotide binding domains arranged similarly
to the nucleotide binding domain of histidine kinases and, therefore,
to the nucleotide binding domain of PDK might inhibit PDK activity (18,
36). Indeed, one of these compounds, radicicol (monorden) (36),
potently inhibited phosphorylation of PDC when tested in standard
kinase assay (Fig. 5, panel
A). Inhibition was competitive with respect to ATP, with the
apparent Ki value of 23.3 ± 1.8 µM, making radicicol an invaluable tool as an inhibitor
of the nucleotide binding domain of PDK.
The addition of radicicol to the standard ATPase assay inhibited
~25-30% of ATP hydrolytic activity in preparations of wild-type PDK2. The specific activity of radicicol-sensitive ATPase was ~1.0
nmol of Pi released/min/mg (Fig. 5, panel B),
suggesting that PDK2 possesses intrinsic ATPase activity that comprises
~2.5% of kinase activity. Having analyzed the ATPase activity in
preparations of wild-type PDK2, experiments were performed to establish
whether the mutations within the active site of kinase affect the ATP hydrolytic activity. When kinases carrying the substitutions of Glu-243
and His-239 were assayed similarly to the wild-type PDK2, there was
little if any radicicol-sensitive ATP hydrolysis that could be assigned
to PDK2, indicating that these residues are essential for ATPase
activity. To further explore the possible mechanistic significance of
Glu-243 and His-239 in an ATP hydrolytic reaction, we prepared several
highly purified preparations of PDK2 carrying substitutions of Glu-243
to Gln and His-239 to Ala. These preparations had a drastically reduced
radicicol-insensitive ATPase activity. Subsequent analysis of these
preparations revealed that His-239 to Ala enzyme had ~7% of the
activity of the wild-type PDK2, whereas the activity of Glu-243 to Gln
enzyme was very close to the background values, less than 0.5% of the
wild type (Fig. 5, panel B). These results strongly suggest
that the same residues of PDK2 are involved in catalysis of both
hydrolytic and phosphotransferase reactions. To further analyze the
relationship between kinase and ATPase activities, we characterized the
effect of the substrate of kinase reaction (E1 component of PDC) on the
rate of ATPase reaction. As shown in Fig. 5, panel C,
inhibition of the total ATPase activity caused by the addition of E1
component was comparable with that caused by the addition of radicicol.
This further confirms that PDK2 possesses an intrinsic ATPase activity
and also shows that the kinase preferentially catalyzes the
phosphorylation reaction under conditions when the native
phosphoacceptor is provided.
Thus, it appears that both Glu-243 and His-239 are crucial for kinase
activity. In conjunction with the results showing that Glu-243 and
His-239 do not contribute to ATP binding, inter-subunit interaction,
and binding to PDC, these data strongly suggest that Glu-243 and
His-239 are required for catalysis of the phosphotransfer reaction. In
accord with this idea is the observation that PDK possesses a weak ATP
hydrolytic activity, which can be detected only in the absence of
physiological substrate, the E1 component of PDC. Furthermore,
the ATPase activity appears to depend on the presence of both Glu-243
and His-239. Substitution of either Glu-243 or His-239 resulted in a
decrease in ATPase activity that closely corresponded to the decrease
in kinase activity.
In conclusion, it is interesting to note that bacteria contain another
protein of the same lineage, the so-called anti-sigma factor SpoIIAB
(29). Its sequence is very similar to the bacterial histidine
kinases but, like PDK, contains a properly spaced glutamic acid in the
N box. Accordingly, SpoIIAB phosphorylates its substrate SpoIIAA on
Ser-58 (29). This suggests that the ability of this type of
catalytic domain to phosphorylate exogenous protein substrates on
serine residues might be largely defined by the presence of glutamic
acid in the N box.
-32P]ATP revealed that neither Glu-243 nor
His-239 mutations affected nucleotide binding. All mutant kinases
showed similar or even somewhat greater affinity than the wild-type
kinase toward the protein substrate, pyruvate dehydrogenase complex.
Furthermore, neither of the mutations affected the inter-subunit
interactions. Finally, pyruvate dehydrogenase kinase was found to
possess a weak ATP hydrolytic activity, which required Glu-243 and
His-239 similar to the kinase activity. Based on these observations, we propose a mechanism according to which the invariant glutamate residue
(Glu-243) acts as a general base catalyst, which activates the hydroxyl
group on a serine residue of the protein substrate for direct attack on
the
phosphate. The glutamate residue in turn might be further
polarized through interaction with the neighboring histidine residue
(His-239).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-ketoacid
dehydrogenase complex (BCKDC) and BCKDC kinase, respectively) (9), does
not show any sequence similarity to the other Ser/Thr-specific protein
kinases. Instead, it resembles histidine kinases (10), a diverse group
of enzymes involved in regulation of various signal transduction
pathways in bacteria. All structural elements characteristic of
histidine kinases (so-called boxes H, N, G1, G2, and G3) can be readily
identified in the amino acid sequence of PDK (11). Recently, the
three-dimensional structures of two histidine kinases involved in the
regulation of chemotaxis and osmolarity (CheA (12) and EnvZ (13),
respectively) have been determined by x-ray crystallography and NMR.
These structures reveal a very characteristic nucleotide binding domain
that, in contrast to the catalytic domains of Ser/Thr-specific protein kinases, folds as an
/
sandwich consisting of five strands and three helices with unique left-handed connectivity. Furthermore, certain amino acids of boxes N, G1, G2, and G3 are intimately involved
in the anchoring of the nucleotide substrate in the active site of the
kinase molecule (13). When we probed the corresponding residues of PDK2
by site-directed mutagenesis, the resulting mutant kinases were
catalytically defective due to the impaired ability to bind the
nucleotide substrate, strongly suggesting that the nucleotide binding
domain of PDK is folded similarly to the nucleotide binding domain of
histidine kinases (14).
phosphate of ATP (12). PDK, in contrast,
catalyzes the transfer of
phosphate to the side chain of the serine
residue of the exogenous substrate, E1. To date, there is no evidence
for existence of a phospho-enzyme intermediate formed during catalysis
by either PDK (15) or a related branched chain
-ketoacid
dehydrogenase complex kinase (16). This prompted us to suggest that
PDK, in contrast to histidine kinases, uses a general base catalysis to
promote a direct nucleophilic attack on the
phosphate by the
hydroxyl group of a serine residue of the protein substrate (14). Here
we report the first evidence that the invariant glutamic acid (Glu-243
in PDK2) serves as a general base catalyst of phosphorylation reaction.
The results reported here are also consistent with the idea that, at
least in mammalian kinases, the nucleophilicity of Glu-243 might be further increased through the interaction with the neighboring histidine residue (His-239 of PDK2).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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REFERENCES
-D-galactopyranoside-inducible promoter
(the respective plasmid was obtained as a generous gift from Dr.
Anthony Gatenby at DuPont Central Research and Development, Wilmington,
DE). Double-transformants were selected on yeast-tryptone agar
containing kanamycin and chloramphenicol (50 µg/ml each) (7). Several
individual colonies from every transformation were tested for their
ability to produce significant amounts of soluble, recombinant kinase.
Clones expressing the greatest amount of the soluble kinase were used
for further analysis.
-32P]ATP into the E1
subunit of PDC, essentially
as described previously (14). Phosphorylation reactions were incubated
at 37 °C in a final volume of 100 µl containing 20 mM
Tris-HCl, pH 7.8, 5 mM MgCl2, 50 mM
KCl, 5 mM dithiothreitol, 1.0 mg/ml PDC, and nucleotide substrate (the specific activity of [
-32P]ATP was
~200-500 cpm/pmol). For assaying recombinant kinases, the respective
proteins were reconstituted with recombinant human PDC before the
assay. Reconstituted preparations were kept on ice for 30 min. The
final protein concentrations of the recombinant proteins in the assay
mixture were as follows: the E1-E2·E3BP subcomplex at 1.0 mg/ml and
the corresponding wild-type kinase at 5 µg/ml. When we tried to apply
the above assay for the analysis of kinase mutants with minute
activity, it was difficult to obtain reliable estimates even when
reactions were conducted for 30 min. To improve the sensitivity of the
assay, the amount of kinase protein added to the reaction was increased
(see legend to Fig. 1). Under these conditions, there was a linear
incorporation of [32P]phosphate into PDC with time for at
least 30 min of the reaction for all mutants tested. The rates of
phosphorylation reactions were proportional to the amount of added
kinase, indicating that the protein substrate is not limiting under the
conditions used. Protein-bound radioactivity was determined as
described previously (14). The activity of wild-type kinase was
determined in the standard assay and was calculated based on
incorporation of [32P]phosphate during the first 30 s of the reaction. The activities of mutant kinases were calculated
based on incorporation of [32P]phosphate during 30 min of
the reaction. The concentrations of nucleotide substrate (ATP) and the
concentrations of inhibitors used in particular experiments are given
in the legends to figures. Radicicol was added from a solution made
with dimethyl sulfoxide. The final concentration of dimethyl sulfoxide
in the reaction mixture was 1% (v/v) at all concentrations of
inhibitor tested. Under these conditions, dimethyl sulfoxide had no
effect on the kinase activity as established in preliminary
experiments. Phosphorylation reactions in this case were initiated by
the addition of wild-type PDC or reconstituted PDC kinase after
equilibration at 37 °C for 30 s. The volume of PDC added was
-32P]ATP (with a specific radioactivity of 200-500
cpm/pmol). The final concentration of ATP in the binding buffer was
varied from 2.5 to 50 µM. The reactions were incubated at
room temperature for 2 min. Protein-bound radioactivity was determined
essentially as described previously (14). All binding experiments were
conducted in triplicate.
-32P]ATP (specific radioactivity of 200-400
cpm/pmol). Reactions were initiated by adding the respective
preparation of recombinant kinase to a final protein concentration of
0.1 mg/ml. Assays were conducted at room temperature. At the indicated
times, reactions were terminated by the addition of 1 ml of ice-chilled
5% (w/v) Norit A (Sigma) in 50 mM
NaH2PO4. The mixture was centrifuged, and 100 µl of supernatant was mixed with 5 ml of scintillation fluid.
The release of [32P]phosphate as the outcome of ATP
hydrolysis was measured in a scintillation counter. Radicicol (final
concentration 200 µM) was added from the solution made
with dimethyl sulfoxide. The final concentration of dimethyl sulfoxide
in the reaction mixture was less than 1% (v/v). Reactions made without
radicicol received an appropriate amount of dimethyl sulfoxide for
control. When E1 component was used as phosphate acceptor, its final
concentration was 1.0 mg/ml.
RESULTS AND DISCUSSION
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ABSTRACT
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RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Kinetics of PDC phosphorylation by wild-type
and mutant PDKs. Panel A, average activities of various
PDK preparations analyzed in this study. Experiments were conducted
with 200 µM ATP and 1.0 mg/ml recombinant PDC as
described under "Experimental Procedures" section. Respective
enzymes, except for the wild-type PDK2, were used at final
concentrations of 20 µg/ml. The activity of wild-type kinase was
calculated based on incorporation of [32P]phosphate
during the first 30 s of the reaction. Activities of mutant
kinases were calculated based on the incorporation of
[32P]phosphate during 30 min of the reaction. Panel
B, representative results showing ATP dependence of PDC
phosphorylation by wild-type PDK2 ( ) and His-239
Ala (
) and
Glu-243
Asp (
) mutants. The insert shows respective
curves for His-239
Ala (
) and Glu-243
Asp (
) mutants. ATP
concentration was varied from 3 to 200 µM.
-32P]ATP--
If
His-239 and Glu-243 contribute to the catalysis of phosphotransfer
reaction, it would be expected that the respective mutant proteins
would still be capable of binding ATP at levels similar to the
wild-type enzyme. The results of kinetic experiments described above
are consistent with this hypothesis. However, due to the limits of
detection of the phosphorylation assay, we could not apply this
approach to the mutant proteins having very low kinase activity.
Furthermore, a drastic reduction in the activity of the mutant proteins
could be attributed to the fact that only a small portion of the
protein in each preparation binds ATP and phosphorylates PDC, whereas
the majority cannot bind the nucleotide substrate. Thus, to determine
whether all kinase molecules were capable of binding ATP, we performed
an active-site titration with [
-32P]ATP as a ligand.
The binding experiments showed that both Glu-243 and His-239 mutants
bind [
-32P]ATP at levels comparable with the wild-type
enzyme with the Kd value of ~4 µM
(Fig. 2). The stoichiometry of binding for the wild-type enzyme and for the His-239 to Ala mutant was close to
the unity. For kinases carrying the substitutions at Glu-243, the
stoichiometry of nucleotide binding was ~0.8-0.9 mol of
[
-32P]ATP per mol of kinase, suggesting that neither
Glu-243 nor His-239 contribute to the binding of nucleotide substrate
to a great extent.
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Fig. 2.
[ -32P]ATP binding by the
wild-type PDK2 and mutant enzymes. Recombinant PDK2, wild-type
(
), His-239
Ala (
) Glu-243
Ala (
), Glu-243
Gln
(
), or Asp-282
Glu (
) mutants were incubated with the
indicated concentrations of [
-32P]ATP (specific
radioactivity ~200 cpm/pmol) for 2 min. Free and protein-bound
nucleotides were separated using a vacuum-filtration assay as described
under "Experimental Procedures." The specific binding was
determined after subtraction of nonspecific binding from the total
binding. The nonspecific binding was determined in the presence of
1,000-fold excess of cold ATP.
-32P]ATP in excess of the background
values, in agreement with its role in providing the major specific
interaction between the adenine base and the protein (14).
Interestingly, Asp-282 to Glu is a conserved substitution that does not
change the charge of the side chain. Nevertheless, this mutation has a
great impact on the nucleotide binding. The latter is entirely
consistent with the available structural information (12, 13). The
nucleotide-binding sites of histidine kinases appear to be very tightly
packed and the additional methylene group of glutamic acid should put
severe conformational constraints on the positioning of ATP within the active site.
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Fig. 3.
Binding of wild-type PDK2 and respective
mutant kinases to recombinant PDC. Panel A, SDS/PAGE
analysis of wild-type PDK2, recombinant PDC, and PDC-kinase complex
centrifuged through Sephacel S300 columns. Panel B, Western
blot analysis of corresponding preparations with monoclonal antibodies
against His6-tag. Immunoreactive bands were visualized by
[125I]protein A staining followed by autoradiography.
Panel C, concentration-dependence curves for binding of
wild-type PDK2 ( ), His-239
Ala (
), Glu- 239
Asp (
),
Glu-243
Gln (
), and Asp-282
Glu (
) mutants to recombinant
PDC. Binding curves were constructed based on the results of scanning
densitometry of the respective Western blots stained with
125I-protein A. Data was analyzed using UN-SCAN-IT
gelTM software (Silk Scientific, Inc., Orem, UT). Results
are expressed as percent of wild-type PDK2 bound to PDC.
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Fig. 4.
Western blot analysis of differentially
tagged preparations of PDK purified by metal affinity
chromatography. Panel A, affinity-purified kinase
preparations probed with antibodies raised against T7-tag.
Panel B, corresponding preparations probed with
anti-H6-tag antibodies. Immunoreactive bands were
visualized by 125I-protein A staining followed by
autoradiography.
phosphate, it also must be
able to utilize the hydroxyl of water as a phosphoryl acceptor instead
of an amino acid hydroxyl group, although at a slower rate. To
investigate this possibility, we determined ATPase activity in various
preparations of PDK. When wild-type enzyme was incubated in a reaction
mixture containing the standard components of the ATPase assay, and
aliquots were removed and assayed for the release of
[32P]Pi, there was a linear formation of
Pi with time. Furthermore, when Mg2+ was
omitted from the standard ATPase assay, no activity was observed, suggesting that, indeed, preparations of PDK contain some ATP hydrolytic activity (data not shown). However, these results do not
exclude the possibility that the source of the observed ATPase activity
is a contaminating ATP hydrolase.
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Fig. 5.
ATP hydrolytic activity in preparations of
PDK2. Panel A, inhibition of pyruvate dehydrogenase
kinase activity by radicicol. Kinase activity was determined without
radicicol ( ) or in the presence of radicicol at concentrations of 25 µM (
), 50 µM (
), or 100 µM (
). Panel B, radicicol-sensitive ATP
hydrolytic activity in preparations of wild-type, Glu-243
Gln,
and His-239
Ala PDK2. Total ATP hydrolysis was measured in the
absence of radicicol with 10 µM [
-32P]ATP as a substrate. Nonspecific hydrolysis
was determined in the presence of 200 µM radicicol. Shown
is radicicol-sensitive ATPase activity (difference between total and
nonspecific activity) intrinsic to PDK2. Panel C,
radicicol-sensitive (
) versus E1-sensitive (
) ATPase
activity in preparations of wild-type PDK2. Radicicol-sensitive ATPase
activity was determined essentially as described in the legend to
panel B. E1-sensitive ATPase activity was determined as a
difference between total ATPase activity and ATPase activity measured
in the presence of E1 component (final concentration 1.0 mg/ml).
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FOOTNOTES |
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* This work was supported by United States Public Health Services Grants GM 51262 and DK 56898.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.
§ Current Address: A. V. Palladin's Institute of Biochemistry, National Academy of Sciences of Ukraine, 9 Leontovich St., Kiev, P. O. Box 252030, Ukraine.
To whom correspondence should be addressed: Div. of Molecular
Biology and Biochemistry, School of Biological Sciences, University of
Missouri, 5110 Rockhill Rd., Kansas City, MO 64110-2499. Tel.: 816-235-2595; Fax: 816-235-5595; E-mail: popovk@umkc.edu.
Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M009327200
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ABBREVIATIONS |
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The abbreviations used are: PDC, pyruvate dehydrogenase complex; PDK, pyruvate dehydrogenase kinase; PDK1, PDK2, PDK3, and PDK4, isozymes 1, 2, 3, and 4 of pyruvate dehydrogenase kinase; E1, pyruvate dehydrogenase component of PDC; E2, dihydrolipoyl acetyltransferase component of PDC; E3, dihydrolipoamide dehydrogenase component of PDC; E3BP, E3-binding protein component of PDC; PAGE, polyacrylamide gel electrophoresis.
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