From the
Mammalian pyruvate dehydrogenase
(
Pyruvate dehydrogenase (E
We recently coexpressed the
mature forms of human E
To
study dephosphorylation, recombinant E
The
activities of these enzymes were measured using three different assays:
(i) a reconstitution assay, (ii) a DCPIP assay, and (iii) a
The initial rates of phosphorylation
and inactivation of wild-type E
Phosphorylation of key regulatory enzymes is one of the major
mechanisms for regulation of metabolic processes. PDC catalyzes the
decarboxylation of pyruvate and provides acetyl-CoA for the generation
of ATP and also for the synthesis of fatty acids, cholesterol, and some
amino acids. Because of the critical role played by this multienzyme
complex in carbohydrate metabolism, its activity is modulated by the
metabolic state of the cell. One of the major mechanisms of regulating
the activity of PDC is by reversible phosphorylation-dephosphorylation
of the E
Three serine residues
were identified by protease digestion of phosphorylated E
In the present study we took advantage of site-directed mutagenesis
and made E
The rates of
dephosphorylation of the three sites and possible involvement of sites
2 and 3 in dephoshorylation of site 1 have remained
controversial(27, 28, 29) . In previous studies,
the dephosphorylation rates of individual sites were determined by
measuring the radioactivity at each site in tryptic phosphopeptides
generated from the partially and fully phoshorylated E
Availability of the site-specific human E
There was no
indication of interactions among sites 1, 2, and 3 in the E
The phosphorylation of the three serine residues of E
Recent
studies of the phosphorylation site 1 (Ser-293) and site 2 (Ser-303) of
the branched-chain
What could be the
function of three phosphorylation sites when phosphorylation of each of
them is sufficient for complete inactivation of E
A serine to alanine mutation used in the present study does
not represent a significant change in the size of the amino acid side
group, and only a change in the polarity and hydrophobicity of this
residue. Nevertheless such a change at Ser-264 (site 1) decreased the
specific activity of the enzyme to 40-50% of the wild-type enzyme
and caused increases in K
The mechanism of E
We thank Dr. Thomas E. Roche for preparation of highly
purified bovine kidney E
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) (E
) is
regulated by phosphorylation-dephosphorylation, catalyzed by the E
-kinase and the
phospho-E
-phosphatase. Using site-directed
mutagenesis of the three phosphorylation sites (sites 1, 2, and 3) on E
, several human E
mutants were made with single, double, and triple mutations by
changing Ser to Ala. Mutation at site 1 but not at sites 2 and/or 3
decreased E
specific activity and also increased K
values for thiamin pyrophosphate and
pyruvate. Sites 1, 2, and 3 in the E
mutants were
phosphorylated either individually or in the presence of the other
sites by the dihydrolipoamide acetyltransferase-protein
X-E
kinase indicating a site-independent mechanism
of phosphorylation. Phosphorylation of each site resulted in complete
inactivation of the E
. However, the rates of
phosphorylation and inactivation were site-specific. Sites 1, 2, and 3
were dephosphorylated either individually or in the presence of the
other sites by the phospho-E
-phosphatase resulting
in complete reactivation of the E
. The rates of
dephosphorylation and reactivation were similar for sites 1, 2, and 3,
indicating a random dephosphorylation mechanism.
)
(
)is the first component of the pyruvate dehydrogenase
complex (PDC). E
catalyzes the oxidative
decarboxylation of pyruvic acid and reductive acetylation of lipoic
acid residues of dihydrolipoamide acetyltransferase (E
). E
transfers acetyl
moieties to CoA, and lipoic residues are reoxidized by dihydrolipoamide
dehydrogenase (E
) with the formation of NADH.
Mammalian E
is a tetramer
(
) with molecular mass of 154
kDa(1, 2) . It has two active sites(3) , that are
most probably composed of amino acid residues from both E
and E
subunits(4, 5) . Several active site residues (cysteine,
tryptophan, arginine, lysine, and histidine) have been found to take
part in active site formation(6, 7) . Three of these
amino acid residues were identified in bovine E
:
namely, the Cys-62 equivalent of human
-subunit(8) ,
Trp-135 of the human
-subunit,
(
)and Arg-239
of the human
-subunit.
(
)E
is regulated in the PDC by phosphorylation-dephosphorylation
carried out by specific regulatory enzymes: E
-kinase and
phospho-E
-phosphatase,
respectively(9, 10) . Three serine residues were shown
to be phosphorylated and designated site 1 (Ser-264), site 2 (Ser-271),
and site 3 (Ser-203) because of the extent and the rate of
phosphorylation(11) . Phosphorylation of site 1 was correlated
with major inactivation of the enzyme; and half-of-the-site reactivity
was exhibited as phosphorylation of only one serine residue of one of
the two
-subunits was sufficient for complete inactivation of the
enzyme(11) . The roles of sites 2 and 3 remain to be determined.
The mechanism of inactivation is also not well understood. It was found
that phosphorylation of bovine E
inhibited all
partial reactions leading to formation of 2-hydroxyethylidene-thiamin
pyrophosphate (HETPP)(12) . Spectral studies on pigeon breast
muscle E
showed that phosphorylated E
was able to bind thiamin pyrophosphate (TPP)
(with less affinity) and was able to interact with HETPP, but could not
bind the substrate pyruvate(13) .
and E
subunits in Escherichia coli M15
cells, and recombinant human E
was purified using
affinity chromatography (5). This coexpression system has permitted a
study of the regulation of E
by phosphorylation
through the use of site-directed mutagenesis. This paper describes a
study of mutants of the three phosphorylation sites. All three sites
were found to be phosphorylated by the E
-kinase
bound to the E
, independent of the other two sites
with concomitant inactivation of the enzyme and were dephosphorylated
by the phospho-E
-phosphatase with resulting
reactivation of E
. Replacement of serine by
alanine at site 1 resulted in a reduction in specific activity of
native E
.
Materials
Restriction and modification enzymes
were purchased from Boehringer Mannheim. Ni-nitrilotriacetic-agarose
(Ni-NTA-agarose) was obtained from Qiagen. pQE-9 and E. coli M15 cells were kindly provided to us by Dr. Stuart F. J. Le Grice
of Case Western Reserve University, and
pQE-6HE1
, pQE-3-E
,
and
pQE-9-6HE
/E
were recently constructed in this laboratory(5) . Recombinant
human dihydrolipoamide dehydrogenase (E
) was
expressed using a pQE-9-6HE3 expression vector in E. coli M15 and purified in this laboratory.
(
)Bovine kidney E
, the
dihydrolipoamide acetyltransferase-protein X-E
kinase (E
-X-kinase) subcomplex, and the
phospho-E
-phosphatase were a generous gift of Dr.
Thomas E. Roche of Kansas State University. Recombinant E
-kinase was kindly provided by Dr. Robert Harris
of Indiana University. Bovine heart E
-X-kinase was
purified by a modified method (14). [
-
P]ATP
(25-50 Ci/mmol) was purchased from ICN.
Site-directed Mutagenesis
Site-directed
mutagenesis was carried out by polymerase chain reaction (PCR). The
mutations in the E cDNA of the site 1-MS1
(S264A), site 2-MS2 (S271A), or site 3-MS3 (S203A) were made by a
two-stage procedure (15) using
pQE-9-6HE
, single mutagenic primer
(MS1, MS2, or MS3) and two primers for 5` and 3` ends including BamHI sites preceding the first codon and after the stop codon
of E
sequence. The amplified, mutated E
cDNA was digested with BamHI and
ligated to pQE-9 (enabling polyhistidine extension at the NH
terminus of the recombinant protein) yielding
pQE-9-6HE
-MS1 (MS2 or MS3). For
construction of a coexpression vector
pQE-9-6HE
-MS1/E
,
pQE-9-6HE
-MS1 was digested with SalI and PvuI to remove the promoter region and E
cDNA, and ligated with fragment of
pQE-3-E
digested with XhoI and PvuI, having the E
sequence (Fig. 1). In the same way
pQE-9-6HE
-MS2/E1
and
pQE-9-6HE
-MS3/E
were constructed. For construction of
pQE-9-6HE
-MS1,2/E1
(with mutations
at sites 1 and 2), pQE-9-6HE
-MS1 was
used as a template for PCR with a mutagenic primer-MS2, and a
coexpression vector was constructed as described above. For
construction of
pQE-9-6H-E
-MS1,3/E
(with mutations at sites 1 and 3),
pQE-9-6HE
-MS1/E
and pQE-9-6HE1
-MS3/E1
were digested with XhoI
and BglII and fragments carrying the mutations were ligated.
In the same way
pQE-9-6HE
-MS2/E
and pQE-9-6H-E
-MS3/E1
were
combined to obtain
pQE-9-6H-E
-MS2,3/E
(with mutations at sites 2 and 3), and
pQE-9-6H-E
-MS1,2/E
and
pQE-9-6H-E
-MS3/E
were used for construction of
pQE-9-6H-E
-MS1,2,3/E
.
The vectors for expression of the E
mutants were
transformed into E. coli M15 cells containing the pDMI.1
plasmid, producing the lac repressor. The correct orientation and the
sequences of each human E
cDNA in the
constructed vectors were confirmed by restriction enzyme digestion
analyses and sequencing(16) .
Figure 1:
Construction of a
pQE-9-6HE1-MS1/E1
expression
vector.
Overexpression and Purification of Recombinant Wild-type
and Mutant E
Overexpression and
purification of recombinant wild-type and mutant Es
s using affinity chromatography on
Ni-nitrilotriacetic-agarose (Ni-NTA-agarose) column were performed as
described recently(5) . Ten mg of recombinant E
s were obtained from 4 liters of bacterial
culture.
Measurements of Activity and Kinetic
Parameters
Protein concentration was determined by the method of
Bradford using bovine serum albumin as a standard(17) . Activity
of E was determined in three assay systems: (i) by
reduction of NAD
after reconstitution with purified
bovine E
-X-kinase subcomplex and human recombinant E
according to the method of Roche and Reed (18);
(ii) by the rate of reduction of the artificial electron acceptor
2,6-dichlorophenolindophenol (DCPIP) at 600 nm(19) ; and (iii)
by the production of
CO
from
[1-
C]pyruvate(20) . The last assay was
performed in the presence and absence of K
Fe(CN)
to study oxidative and nonoxidative decarboxylation of pyruvate,
respectively. One milliunit of enzyme activity is defined as 1 nmol of
substrate used or product formed per min at 37 °C. K
values for TPP and pyruvic acid were
determined by the reconstitution assay and assay with DCPIP by varying
the concentration of TPP from 0.05 to 200 µM and the
pyruvic acid concentration from 1 to 300 µM. Linear parts
of the time curves were used for determination of kinetic parameters
using double recipropcal plots.
Phosphorylation and Dephosphorylation of Recombinant
Proteins
Phosphorylation of wild-type and mutant Es was performed as described
previously(21) . To measure activity changes, nonradioactive ATP
was used during incubation and aliquots were withdrawn at specified
times from the reaction mixture to a 1-ml cuvette to measure activity
using the reconstitution assay. To determine incorporation of
P from [
-
P]ATP into protein, E
-X-kinase subcomplex was first incubated with
nonradioactive ATP (5-150 µM) for 5 min (to exclude
phosphorylation of a trace amount of E
bound to E
), followed by the addition of
[
-
P]ATP (5-150 µM,
1.25-13.0 µCi) and recombinant E
or E
mutants. In the experiments with purified
recombinant E
-kinase, preincubation was excluded
and reaction was started by the addition of
[
-
P]ATP (20-200 µM)
(12.5 µCi). The aliquots taken at different time intervals were
applied to dry paper discs presoaked in trichloroacetic acid and the
samples were processed as described previously(21) .
P incorporation was determined by scintillation counting (21) and expressed as mole of
P incorporated per
mole of tetrameric E
. The rates of incorporation
(mol
P/mol E
min) were
determined from the initial slopes of the time courses of
P incorporation. k
of inactivation
(min
) were calculated from the semilog plots of
inactivation during phosphorylation. To demonstrate the incorporation
of radioactivity in the
subunit, E
proteins
were separated by SDS-PAGE and dry gels were autoradiographed.
proteins
were phosphorylated by the E
-X-kinase from bovine
kidney in the presence of 50-200 µM [
-
P]ATP for 45-100 min, and the
phosphorylation was stopped by the addition of 8 mM glucose
and 0.5 units of hexokinase to scavenge ATP. To study
P
release E
s were
extensively washed on Microcon filters to remove labeled glucose
6-phosphate. This step was excluded during reactivation experiments as
it gave the same results. Dephosphorylation was performed in the
presence of 10 mM MgCl
, 100 µM CaCl
, and 1 µg of
phospho-E
-phosphatase per 10 µg of
phosphorylated E
at 30 °C for up to 60 min.
Aliquots taken at different time intervals were analyzed for both
activity and
P release in soluble fraction after protein
precipitation with trichloroacetic acid. k
of
dephosphorylation and reactivation were calculated from semilog plots
of the time courses of
P release and increase of the
specific activity.
Activity Determination of E
To examine the role of the three phosphorylation
sites, seven different mutants were made by site-directed mutagenesis.
Ser-264, Ser-271, Ser-203, corresponding to phosphorylation sites 1, 2,
and 3 in the E Mutants
sequence, were changed to
alanine, either individually or in combination as: MS1 (E
with mutated site 1), MS2, MS3, MS2,3, MS1,3,
MS1,2, and MS1,2,3. Wild-type E
and E
mutants were individually overexpressed in E. coli and purified using Ni-nitrilotriacetic-agarose
chromatography as described previously(5) . Approximately 10 mg
of each enzyme were obtained from 4 liters of bacterial culture with a
purity of about 94% based on densitometry of the SDS-PAGE gel.
CO
assay in the presence and absence of
K
FeCN
as an electron acceptor. The results of
the reconstitution assays are shown in Fig. 2. Similar results
were observed using the other two assays (results not shown). Each
mutant exhibited activity in all three assays. There was little
decrease in the specific activity of the E
phosphorylation sites 2 and/or 3 mutants, but the E
specific activity was reduced by 50-70%
(relative to wild-type E
) for all mutants having
an alteration at site 1.
Figure 2:
Activity of the E mutants determined by the reconstitution assay. Values are the
means ± S.E. of two to five measurements for two preparations of
each enzyme. 100% corresponds to 14.9
units/mg.
shows the K values for TPP and pyruvic acid measured separately by the
reconstitution assay and DCPIP assay. In each site 1 mutant (MS1,
MS1,3, MS1,2, and MS1,2,3) a substantial increase in K
values for both pyruvate and TPP was
observed which is not seen in site 2 or 3 mutants (E
, MS2, MS3, and MS2,3).
Phosphorylation of E
Fig. 3shows the time dependent inactivation of the
wild-type and mutant E Mutants by the E
Kinase in the Presence of E
s during incubation with ATP
and the E
-X-kinase subcomplex (see
``Experimental Procedures''). Based on the results, the E
s can be divided into 4 groups: Group 1, having
site 1 intact (wild-type E
, MS2, MS3, and MS2, 3);
Group 2, with mutation of site 1, but intact site 2 (MS1 and MS1, 3);
Group 3, with mutation of sites 1 and 2, but site 3 intact (MS1, 2);
Group 4, with mutations of all three phosphorylation sites (MS1, 2, 3).
Native E
, MS2, MS3, and MS2,3 (Group 1) behaved
similarly to one another during inactivation at ATP concentrations of
1-10 µM (Fig. 3A). Complete
inactivation was achieved after 10 min with 10 µM ATP.
However, the inactivation of the MS1 and MS1,3 (Group 2) was complete
only after 30 min incubation with 40 µM ATP (Fig. 3B), and the MS1,2 (Group 3) needed higher ATP
concentrations, up to 400 µM, and more than 60 min for
complete inactivation (Fig. 3C). Thus, all E
s were inactivated by E
-X-kinase-dependent phosphorylation. However, the
range of ATP concentration necessary for complete inactivation depended
upon the mutant used, reflecting site-specificity. Mutants of Groups 2
and 3 achieved inactivation by phosphorylation at higher ATP
concentration than mutants of Group 1.
Figure 3:
Time course of E inactivation during phosphorylation by the E
-X-kinase subcomplex. A, inactivation of
Group 1 enzymes; B, inactivation of Group 2 enzymes; and C, inactivation of Group 3 enzyme. Ten µg of each E
were incubated at 30 °C with 8 µg of the E
-X-kinase and various ATP concentrations: 0
µM (*); 1.0 µM (
); 1.6 µM (
); 2.5 µM (
); 4.0 µM ( ); 10
µM (
); 25 µM (
); 40 µM (
); 100 µM (┌); and 400 µM (
). Aliquots (1 µg of E
) were
taken to measure activity by the reconstitution
assay.
To compare the regulatory
behavior of different mutants the inactivation and the corresponding
incorporation of P were determined at a fixed ATP
concentration (40 µM) (Fig. 4). The level of
incorporation (Fig. 4B) depended upon the number of
sites available and duration of incubation. E
with
all the three potential phosphorylation sites available showed the
maximum level of incorporation (2.7 mol of
P/mol of E
); MS3, MS2, and MS1 with only 2 sites available
showed less phosphorylation (1.1-1.7 mol of
P/mol of E
) and minimum incorporation (0.4-0.6 mol of
P/mol of E
) corresponded to
phosphorylation of individual sites 1 (MS2,3), 2 (MS1,3), and 3
(MS1,2). The maximal level of incorporation achieved at 270 µM ATP was: 3.0 for E
; 1.5-1.8 for mutants
with two sites; and 0.5-1.0 for mutants with only one
phosphorylation site available (results not shown). There was no
incorporation of
P into the MS1,2,3 mutant and as expected
there was no inactivation of this enzyme (Fig. 4). For this
reason MS1,2,3 was used as a control in all the experiments concerning
phosphorylation. Data presented in Fig. 4(B and C) showed that all three phosphorylation sites can incorporate
P during incubation with the E
-X-kinase subcomplex and
[
-
P]ATP.
Figure 4:
A,
inactivation of Es during phosphorylation by the E
-X-kinase subcomplex in the presence of 40
µM ATP. Activity was measured in the reconstitution assay.
100% activity of each curve corresponded to the activity of each E
at zero time. B,
P
incorporation from [
-
P]ATP (40
µM) into E
s by the E
-X-kinase subcomplex. Ten µg of the E
and 8 µg of the E
-X-kinase were used in each reaction.
Incorporation was measured by counting paper discs in a scintillation
counter. Phosphorylation in control tubes containing only the E
-X-kinase (without E
) was
subtracted. C, autoradiography of the E
after incorporation of
-
P followed by
separation of proteins in SDS-PAGE.
The difference in the rate of
inactivation of E during incubation at a fixed ATP
concentration (40 µM) is depicted in Fig. 4A by comparing inactivation of the enzymes in Groups 1, 2, and 3.
Group 1 (wild-type E
, MS2, MS3, and MS2,3)
represents phosphorylation mostly of site 1 in the presence or absence
of sites 2 and 3; Group 2 (MS1 and MS1,3) represents phosphorylation of
site 2 in the absence or presence of site 3, and Group 3 (MS1,2) shows
phosphorylation of site 3 only. The inactivation of the enzymes of
Group 1 was complete after 10 min incubation, Group 2 was inactivated
in 30 min, while Group 3 having only site 3 required more than 100 min
for complete inactivation. Results presented in Fig. 4showed
that phosphorylation with concomitant inactivation of the enzymes was
found for each of the three sites, but the rates of these processes
were different for each site.
and the mutant E
s are compared in . The rates were
similar for all species having site 1 (Group 1). Group 2 (without site
1 but with site 2 available) showed slower rates of phosphorylation and
inactivation than Group 1. The lowest rates of phosphorylation and
inactivation were found for Group 3 with only site 3 available.
Phosphorylation of E
Fig. 5shows
the incorporation of Mutants by the E
Kinase in the Absence of E
P and inactivation of wild-type E
and E
mutants during
incubation with E
-kinase and ATP. To achieve
phosphorylation of E
by the E
-kinase in the absence of E
it was necessary to increase the concentrations of both the E
-kinase and ATP. Complete inactivation of
wild-type E
occurred after 15 min incubation in
the presence of 200 µM ATP. Mutants MS3, MS2, MS2,3 were
phosphorylated with concomitant inactivation similar to wild-type E
indicating that site 1 can be phosphorylated in
the absence of E
. However, mutants having only
site 2 (MS1,3), site 3 (MS1,2), or sites 2 and 3 (MS1) exhibited low
levels of phosphorylation (10-15% of that for wild-type E
and mutants with site 1), and low levels of
inactivation (approximately 10% of that for wild-type E
and mutants with site 1). Autoradiography (Fig. 5C) showed a low level of phosphate incorporation
into sites 2 and 3, when only the E
-kinase was
present, and this was much less than phosphorylation of the E
s by the E
-X-kinase
subcomplex (Fig. 4C).
Figure 5:
A, inactivation of Es
by phosphorylation by the E
-kinase in the presence
of 200 µM ATP. Activity was measured in the reconstitution
assay. 100% activity for each curve corresponded to the activity of
each E
at zero time. B,
P
incorporation from [
-
P]ATP (200
µM) into E
s by the E
-kinase. Seven µg of the E
and 2 µg of the E
-kinase were used in
each reaction. Incorporation of
P was measured by
scintillation counting of paper discs. Phosphorylation in control tubes
containing only the E
-kinase (without E
) was subtracted. C, autoradiography of E
after incorporation of
-
P
followed by separation of proteins in SDS-PAGE. Upper band represents autophosphorylation of the E
-kinase, and the lower band represents
P incorporation into E
subunit.
shows the initial
rates of incorporation of P and inactivation during
phosphorylation of E
s by the E
-kinase. The difference between the rates of
inactivation and phosphorylation for sites 2 and 3 compared to that of
site 1 was much larger during phosphorylation by the E
-kinase alone than during phosphorylation by the E
-kinase in the presence of E
. The corresponding k
of
inactivation (and rates of phosphorylation) were: in the absence of E
for site 1, 0.347 min
(0.125
mol of
P/mol of E
); for site 2, 0.002
min
(0.005 mol of
P/mol of E
); for site 3, 0.003 min
(0.007 mol of
P/mol of E
); and
in the presence of E
for site 1, 0.732
min
(0.241 mol of
P/mol of E
); for site 2, 0.096 min
(0.053 mol of
P/mol of E
); and
for site 3, 0.022 min
(0.015 mol of
P/mol of E
).
Dephosphorylation of E
Fig. 6shows the
reactivation of the phosphorylated wild-type and mutant E Mutants by the
Phospho-E
-phosphatase
s during dephosphorylation by the
phospho-E
-phosphatase. All E
s
with sites 1, 2, and 3 present either individually or in combination
with the other sites were dephosphorylated by the
phospho-E
-phosphatase (Fig. 6B)
with similar rates of P
release as reflected in . The calculated rates of dephosphorylation for sites 1,
2, and 3 were: 0.111 min
, 0.104
min
, and 0.102 min
, respectively.
Dephosphorylation resulted in complete reactivation of all the mutant
enzymes (Fig. 6A). The rates of reactivation were
similar for the E
proteins with only site 1
(MS2,3), site 2 (MS1,3), or site 3 (MS1,2) (Fig. 6A, inset; also see ). The reactivation was slower
for the mutants with two phosphorylation sites (MS1, MS2, MS3) than the
mutants with only one site (MS2,3, MS1,3, MS1,2), and the reactivation
was slowest for E
with three potential
phosphorylation sites than for the mutants with two sites (). The reactivation curves for the mutants with two sites
and especially for wild-type E
were sigmoidal, and
longer incubation times were necessary to achieve complete
reactivation. The different behavior of the mutants with different
number of phosphorylation sites can be explained by the fact that
activity depends upon dephosphorylation of all of the available sites.
Figure 6:
A,
reactivation of Es during dephosphorylation by the
phospho-E
-phosphatase. Activity was measured in
the reconstitution assay. Inset shows the semilog plots of
reactivation of MS2,3, MS1,3, and MS1,2 by the
phospho-E
-phosphatase. Ao, inactivation
(%) at zero time; At, inactivation (%) at time intervals
indicated. This is a representative experiment of the results presented
as the means ± S.E. (n = 4) shown in Table II. B,
P release from phosphorylated E
s by the phospho-E
-
phosphatase. E
proteins were phosphorylated by the E
-X-kinase in the presence of 50 µM [
-
P]ATP for 45 min. Ten µg of the
phosho-E
and 1 µg of the
phospho-E
-phosphatase were used in each
dephosphorylation reaction.
P release was determined by
taking aliquots at different time intervals, protein precipitation with
trichloroacetic acid, and measuring the radioactivity by counting the
soluble fraction in a scintillation counter. The level of radioactivity
released with acid treatment at zero time (before the
phospho-E
-phosphatase addition) was subtracted
from each point. C, the semilog plots of
P
release from phosphorylated MS2,3, MS1,3, and MS1,2 by the
phospho-E
-phosphatase. Bo,
P
bound with the protein (nmol/mg E
) at zero time; Bt,
P bound with the protein (nmol/mg E
) at time intervals indicated. This is a
representative experiment of the results presented as the means
± S.E. (n = 5) shown in Table
II.
subunit catalyzed by the E
-kinase and the
phospho-E
-phosphatase, respectively. The
activities of these two enzymes are regulated by the concentration
ratios of ATP/ADP, NAD
/NADH, acetyl-CoA/CoA, and by
pyruvate, TPP, Mg
, and
Ca
(2, 22) .
from bovine and porcine PDC (11, 23). These
three serine residues are referred to as site 1, site 2, and site 3
that correspond to Ser-264, Ser-271, and Ser-203 in the human E
sequence, respectively(24) . It was
shown that phosphorylation of site 1 caused the major inactivation of
the enzyme(11, 25) . However, the role of sites 2 and 3
in enzyme inactivation is not well
understood(26, 27, 28) . Also, it is not clear
whether sites 2 and 3 can undergo phosphorylation in the absence of
site 1. It was suggested that sites 2 and 3 are important for
dephosphorylation and reactivation of E
by the
phospho-E
-phosphatase(28, 29) . As
all three sites were shown to undergo phosphorylation at different
rates, it was difficult to determine the possible role of sites 2 and 3
in the presence of the highly reactive site 1(23, 26) .
mutants having only site 1 (MS2,3),
site 2 (MS1,3), site 3 (MS1,2) or all possible combinations of two
sites. Using these mutant enzymes it was possible to study
phosphorylation of each site separately or in combination with the
other sites and determine the extent of inactivation. All the mutants
having one or two phosphorylation sites were phosphorylated by the E
-kinase bound with E
. This
shows a random rather than sequential mechanism of phosphorylation.
Phosphorylation of each site alone resulted in enzyme inactivation. In
an earlier study only 0.7-6.4% of inactivation was suggested for
site 2 phosphorylation, and phosphorylation at site 3 was considered to
be non-inactivating(26) . Although the phosphorylation of a
single site resulted in complete inactivation, the rates of
phosphorylation and inactivation were site-specific. High rates were
observed for the E
mutants having site 1 alone or
in combination with site 2 or 3 (Group 1: MS2,3, MS2, MS3); low rates
were observed for the E
mutants having site 2
alone or with site 3 (Group 2: MS1, MS1,3), and very low rates were
detected for the mutant with site 3 alone (Group 3: MS1,2) (). Different rates of phosphorylation were proposed
earlier based on determining site occupancy by tryptic digestion and
high-voltage paper electrophoresis(30) .
proteins(27, 28) . Using this approach the
relative rate of dephoshorylation was found to be higher for site 2
than site 3, and also higher for site 3 than site
1(27, 28) . Also, the rate of dephosphorylation of the
fully phosphorylated E
(three site occupancy) was
higher than that of a partially phosphorylated
enzyme(27, 28, 29) . Using selective
thiophosphorylation of sites 2 and 3 (which remained resistant to
hydrolysis by the phospho-E
-phosphatase), it was
shown that the modification of sites 2 and 3 did not affect the rate of
dephosphorylation of site 1 of bovine E
,
suggesting a random dephosphorylation of the three sites(27) .
In contrast, reactivation of phosphorylated porcine E
was found to be faster for the partially phosphorylated enzyme
than for the fully phosphorylated E
(28, 29) . Based on these findings
it was proposed that the mechanism of dephoshorylation is not purely
random and that dephosphorylation of site 1 in fully phosphorylated E
is not independent of that of site
2(28) .
mutants has allowed us to examine possible
interactions among the three sites in dephoshorylation. We observed
that the mutant phospho-E
s having only one site
available for phoshorylation (e.g. sites 1, 2, or 3) displayed
similar rates of dephosphorylation as well as reactivation (Fig. 6, ). Furthermore, the rates of
dephosphorylation of the E
mutant proteins with
only two sites available for phosphorylation were similar to that of E
proteins with either any one site or all three
sites available for phosphorylation (Fig. 6, ),
supporting the earlier finding of a random mechanism for
dephosphorylation of the three sites. Although we did not measure the
rate of dephosphorylation of each site in the mutant E
s (MS1, MS2, and MS3) having any two sites
present, the observed similarity in the dephosphorylation rates of the
three E
mutants with any two phosphorylation sites
intact (MS1, MS2, MS3) indicates that the presence of any one site did
not influence dephosphorylation of the other site present (Fig. 6). The reactivation of the different mutant E
s during dephosphorylation was different,
however. The reactivation of the E
mutants with
only one site phosphorylated was faster than that of the E
mutants with any two sites which in turn was
faster than that for the E
with three sites. These
differences in the reactivation rates can be explained by the presence
of two instead of one site (or three instead of two sites) which are
required to be dephosphorylated to activate the enzyme. As shown above,
phoshorylation of any one site results in inactivation of the enzyme (Fig. 4). Our findings clearly indicate that sites 1, 2, and 3
are dephosphorylated independently of each other.
subunit. Individual sites seemed to behave
the same either in the presence or absence of other sites. However,
phosphorylation of one Ser residue (Ser-264, Ser-271, or Ser-203) per E
molecule was enough for complete inactivation of
the enzyme. We found that the half-of-the-site reactivity is true not
only during phosphorylation of site 1 as was shown
earlier(11, 25) , but is also the case during
phosphorylation of sites 2 or 3. Maximal level of incorporation in
different experiments with two preparations of the enzymes was not more
than 1 mol of
P/mol of E
for mutants
with one site available, not more than two for mutants with 2 sites
present while maximum number of phosphoryl groups incorporated in E
was not more than three which corresponds to the
previously reported data on highly purified porcine PDC(25) .
This was suggested previously that three phosphoryl groups can be
incorporated in one
-subunit(25) . It is possible if one
takes into consideration that tetrameric E
is a
highly cooperative enzyme, showing interaction between its two active
sites during its interaction with the substrate and cofactor, and also
during catalysis(31) . Two active sites of this enzyme were
proposed to display an alternating site catalytic mechanism where an
event in one active site depends upon the reaction catalyzed in the
other active site(31) . It has half-the-site reactivity when
interacting with HETPP (32). The intersubunit interactions are revealed
also in the different reactivities of essential amino acid residues,
belonging to the neighboring active site(31) . The kinetic
model, including a conformational transition from one active site after
its modification to the other has been proposed by Khailova et
al.(31) . The existence of an interaction between the two
active sites can explain the observed regulation by phosphorylation of
one of the two
-subunits by the E
-kinase.
subunit was found to be different when
catalyzed by the E
-kinase not bound to E
(11, 30) . We confirmed this
observation by using the phosphorylation site E
mutants. We observed that site 1 undergoes phosphorylation by the E
-kinase only leading to its inactivation, but
this requires higher ATP and kinase concentrations compared to
phosphorylation of the site 1 in E
by the E
-kinase in the presence of the E
. For sites 2 and 3 phosphorylation and
inactivation were much lower ( Fig. 6and ). This
phenomenon has been attributed to a conformational change induced in E
or E
-kinase after
association with the E
(30) .
-ketoacid dehydrogenase complex (33) by
site-directed mutagenesis showed that phosphorylaton of only site 1 or
mutation of serine 293 to glutamate caused enzyme inactivation.
However, phosphorylation of site 2 or replacement of serine 303 to
glutamate did not effect enzyme activity, indicating non-involvement of
the site 2 with respect to regulation of the activity by
phosphorylation. This contrasts with our findings on PDC E
, as phosphorylation of sites 2 and 3 caused
complete inactivation of E
. This specificity may
reflect the differences between the amino acid sequences surrounding
sites 1 and 2 of the two multienzyme systems.
(and hence the whole multienzyme complex)? It is plausible that
since the metabolism of pyruvate via the PDC is a critical step in
maintenance of glucose homeostasis that the presence of multiple sites (e.g. sites 2 and 3) is developed as a safety mechanism for
regulation of the complex activity if an unexpected mutation occurs at
site 1.
values for TPP
and pyruvate (). This suggests that either site 1 is closer
to the active site than the other sites; or that it lies on the pathway
of the main catalytic conformational change. The activities of E
measured by the DCPIP and
CO
assays were also decreased for E
with
mutation at site 1 to the same extent as compared to activity
measurement after reconstitution of E
with the E
-X and E
. This finding
excludes the possibility of the S264A mutation affecting the
interaction of E
with E
.
inactivation by
phosphorylation is not well understood. It was found that
phosphorylation of pig heart E
inhibited all the
partial reactions (forward and backward) leading to the formation of
HETPP(12) . Butler et al.(34) showed that
phosphorylation affected TPP binding to E
, but
this effect was not significant since the transition state analog of
TPP, thiamine thiazolone pyrophosphate, could interact with
phosphorylated E
. It was suggested that
phosphorylation produced a conformational change in E
that displaced a catalytic group (or groups) at the active
site(34) . Spectral studies of pigeon breast muscle
phospho-E
showed that it bound TPP in the active
conformation with the formation of a charge transfer complex, to
interact with HETPP by the alternating site mechanism, but could not
bind the substrate pyruvate(13) . It was suggested that a
negative phosphoryl residue may compete for the active site arginine
and thereby block the substrate binding. Phosphorylation of a serine
residue has been shown to regulate the activity of other enzymes in
different ways: (i) enhancing the activity of glycogen phosphorylase by
means of a long-range conformational change upon
phosphorylation(35) ; (ii) abolishing isocitrate dehydrogenase
activity as a result of electrostatic repulsion and steric hindrance
between the phosphoryl moiety and the carboxylic group of the
substrate(36) ; and (iii) attenuating the activity of Syrian
hamster 3-hydroxy-3-methylglutaryl-CoA reductase by impairing the
ability of the catalytic histidine to protonate CoA(37) . Which
one of these possibilities is attributed to phosphorylation and hence
inactivation of E
remains to be determined.
Table: K values for pyruvate and
TPP for E
and phosphorylation site mutants
Table: Rates of phosphorylation and inactivation of
E and its mutants by the
E
-X-kinase subcomplex or by the
E
-kinase alone and of dephosphorylation and
reactivation by the phospho-E
-phosphatase
, pyruvate dehydrogenase; E
,
dihydrolipoamide acetyltransferase; E
,
dihydrolipoamide dehydrogenase; PDC, pyruvate dehydrogenase complex; E
-X-kinase, dihydrolipoamide
acetyltransferase-protein X-E
kinase; TPP, thiamin
pyrophosphate; HETPP, 2-hydroxyethylidene-thiamin pyrophosphate;
Ni-NTA-agarose, Ni-nitrilotriacetic-agarose; DCPIP,
2,6-dichlorophenolindophenol; PAGE, polyacrylamide gel electrophoresis.
, E
-protein X-kinase subcomplex, and
phospho-E
-phosphatase. We are indebted to Dr.
Robert A. Harris for a preparation of recombinant E
-kinase. We are grateful to Drs. Edward Niles,
Murray Ettinger, and Cecile Pickart of this department for critical
reading of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.