From the Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York 14214
Received for publication, August 18, 2000, and in revised form, November 15, 2000
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ABSTRACT |
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Activity of the mammalian pyruvate
dehydrogenase complex (PDC) is regulated by
phosphorylation-dephosphorylation of three serine residues (designated
site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) in the The mammalian pyruvate dehydrogenase complex
(PDC)1 plays an important
role in defining the fate of three-carbon compounds derived largely
from carbohydrates and to some extent from amino acids. PDC comprises
multiple copies of three catalytic enzymes, namely pyruvate
dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and
dihydrolipoamide dehydrogenase (E3); in addition, the complex also
contains a binding protein referred to as E3-binding protein (E3BP),
and two regulatory enzymes, namely pyruvate dehydrogenase kinase (PDK)
and phosphopyruvate dehydrogenase phosphatase. E1 carries out the
decarboxylation of pyruvic acid and the reductive acetylation of lipoyl
moieties covalently linked to E2. E2 then catalyzes the transfer of
acetyl groups to CoA, forming acetyl-CoA and fully reduced lipoyl
moieties. E3 reoxidizes the reduced lipoyl groups of E2 and transfers
the electrons to NAD+, forming NADH. Sixty subunits of
mammalian E2 and 12 subunits of E3BP form the inner core structure,
whereas 20-30 tetrameric E1 ( Regulation of mammalian PDC activity is accomplished in large part by
phosphorylation (resulting in inactivation) of the E1 component by a
family of pyruvate dehydrogenase kinases (PDK 1-4 isozymes) and
dephosphorylation (leading to activation) of phosphorylated E1 by a set
of specific phosphatases (phosphopyruvate dehydrogenase phosphatase
1-2 isozymes) (1, 3-6). The E1 has two active sites formed by contribution of amino acid residues
of both the Materials--
Restriction enzymes were purchased from Promega
and Roche Molecular Biochemicals. Sequenase 2.0 was from
Amersham Pharmacia Biotech. [1-14C]Pyruvate and
[35S]ATP were from PerkinEmler Life Sciences.
Nickel-nitrilotriacetate (Ni2+-NTA)-agarose was obtained
from Qiagen. The protein assay reagent was from Bio-Rad. pPDK1 and
pPDK2 expression vectors and Escherichia coli strain BL21
with pPDHE2/E3BP were generously provided by Dr. Robert A. Harris
(Indiana University School of Medicine).
Site-directed Mutagenesis--
Several mutants of the three
phosphorylation sites localized on the E1
The amplified mutated E1 Protein Expression and Purification--
Recombinant wild-type
and mutant human E1s were overexpressed in E. coli M15 cells
and purified using Ni2+-NTA-agarose chromatography (16).
The purified enzyme preparations had purity of more than 90% as judged
by densitometry of SDS-polyacrylamide gels (results not shown).
Recombinant human E2-E3BP was purified (about 92% purity) from BL21
cells harboring pPDHE2/E3BP as described by Yang et al. (27)
for E2 with some modifications: (i) the resuspension buffer was 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM
EDTA, 1 mM dithiothreitol, and a mixture of protease
inhibitors; (ii) cells were disrupted by a French press; (iii)
polyethylene glycol precipitation was performed dropwise, followed by
measuring activity of bacterial PDC and E2-E3BP in PDC assay (after
E2-E3BP reconstitution with E1 and E3), and precipitated E2-E3BP was
separated from bacterial PDC by centrifugation; and (iv) Sepharose
CL-4B was used for gel-filtration. Recombinant rat PDK1 and PDK2 were purified to about 92-95% purity from BL21 cells transformed with pPDK1 and pPDK2 expression vectors according to Bowker-Kinley et
al. (28) with three modifications: (i) the resuspension buffer was
50 mM potassium phosphate, pH 7.5, 100 mM KCl,
5 mM Determination of E1 Activity and Kinetic Parameters--
E1
activity was assayed by three different assay systems: (i) by the
formation of NADH after reconstitution of E1 with recombinant human
E2-E3BP and recombinant human E3 in the PDC assay (to measure the
overall complex activity) (21); (ii) by the reduction of 2,6-dichlorophenolindophenol (DCPIP), the artificial electron acceptor
in the DCPIP assay (to measure E1-catalyzed reaction only,
decarboxylation of pyruvate, and oxidation of HE=TPP to acetate) (30);
and (iii) by the production of 14CO2 from
[1-14C]pyruvate in the absence of an electron acceptor in
the decarboxylation assay (decarboxylation of pyruvate and release of
acetaldehyde) (31, 32). For the PDC assay, E1 (0.8-1 µg) was
preincubated in the cuvette with a premixed mixture of recombinant
human E2-E3BP and recombinant human E3 in the ratio of 1:3:3 for 1 min
at 37 °C in 50 mM potassium phosphate buffer, pH 7.5, containing 2 mM MgCl2, 2 mM
NAD+, 156 µM CoA, 4 mM cysteine,
0.2 mM TPP. The reaction was then started with the addition
of 2 mM pyruvate, and the formation of NADH was monitored
spectrophotometrically. In one set of experiments (Fig. 2), PDC
activity was measured by the formation of 14CO2
from [1-14C]pyruvate by reconstituted PDC instead of
monitoring the formation of NADH. The DCPIP assay was carried out at
37 °C with E1 (20-50 µg) in potassium phosphate buffer, pH 7.0, containing 5 mM MgCl2, 0.5 mM TPP,
and 100 µM DCPIP; the reaction was started by the addition of 5 mM pyruvate, and the reduction of DCPIP was
monitored at 600 nm. Activity in the decarboxylation assay was measured with E1 reconstituted in PDC and free E1 (1-20 µg) in the absence of
an electron acceptor in 85 mM potassium phosphate buffer,
pH 6.3, containing 3.4 mM MgCl2, 0.34 mM TPP, and 0.82 mM
[1-14C]pyruvate. Apparent Km values
for TPP and pyruvate were determined in the PDC and DCPIP assays by
varying the concentrations of TPP from 0.03 to 100 µM
(PDC assay) or from 0.2 to 200 µM (DCPIP assay) and the
concentrations of pyruvate from 10 to 800 µM (PDC assay)
or from 1 to 1500 µM (DCPIP assay). The linear portions (initial rates) of the time curves were used to calculate the kinetic
parameters from double-reciprocal plots. One unit of enzyme activity is
defined as 1 µmol of product formed/min at 37 °C. Correlation
coefficients in all kinetic experiments were at least 95%. Protein was
measured by the Bradford method using bovine serum albumin as the
standard (33).
Phosphorylation of E1 and Its Mutants--
E1 mutants with site
1, 2, or 3 only (S2A,S3A, S1A,S3A, and S1A,S2A, respectively) were
phosphorylated by PDK1 or PDK2 in the presence of E2-E3BP and 100-500
µM ATP depending upon the site to be phosphorylated.
Phosphorylation was performed at 30 °C in 20 mM
potassium phosphate buffer, pH 7.0, 0.1 mM EDTA, 1 mM MgCl2, 2 mM dithiothreitol. To
measure decarboxylation and DCPIP activity of the phosphorylated mutant
E1s, modified E1s were washed on Millipore Ultrafree (UFV5BQK25)
filters and concentrated. Controls (without phosphorylation) were
treated in the same way. To study inactivation of E1 by phosphorylation
of site 1, 2, or 3 in the presence of TPP, mutants having site 1 only,
site 2 only, or site 3 only were phosphorylated by PDK1 in the presence
of E2-E3BP, and wild-type E1 was phosphorylated mostly at site 1 by
PDK2 in the absence of E2-E3BP-subcomplex. Activity of PDK2 for site 2 is <1% of its activity for site 1 in the absence of E2-E3BP. Site 3 is not modified by PDK2.2 E1s
were incubated in the conditions described above with ATP and different
compounds as specified in the figure legends. Aliquots (0.8-1 µg of
E1) were withdrawn at the indicated times and added to a 1-ml cuvette
containing the components for the PDC assay. PDC was allowed to
reconstitute for 1 min, and the reaction was started by the addition of pyruvate.
The results of all experiments were calculated using SigmaPlot software
(Jandel Scientific, San Rafael, CA).
Activity Changes in Phosphorylation Site Mutant E1s--
Highly
purified recombinant human wild-type and phosphorylation site-specific
mutant E1s were used to determine activities using three different
assays. A comparison of the activities of site 1 E1 mutants (S1A, S1E,
S1Q, and S1D) in the PDC assay, DCPIP assay, and decarboxylation assay
is shown in Fig. 1A. Mutants S1E and S1D with a single negative charge at that site had undetectable activity in the PDC assay (Fig. 1A), which was expected if
these mutations mimicked phosphorylation of site 1 in inhibiting PDC activity (Fig. 1B). The S1Q E1 mutant (without a negative
charge at that site) had only about 3% of the activity in the PDC
assay, whereas S1A mutant E1 had about 58% of the control PDC
activity. These results indicate that the size of the substituted
residue and possibly its orientation and not only the negative charge at site 1 contributes to inactivation of E1. All the site 1 mutants demonstrated considerable levels of activity (46-62%) in the
decarboxylation assay and in the DCPIP assay (63-90% activity). Only
the S1D mutant E1 had a very low activity (about 10%) in the DCPIP
assay, probably due to high inactivation during the catalytic reaction.
The progress curve of the PDC reaction for the S1D mutant E1 was linear
only during the first 30 s, after which the rate of the reaction
decreased significantly (results not shown). We do not have an
explanation for this kinetic behavior, as this effect was not studied
further. Wild-type E1 phosphorylated at site 1 only had ~59%
activity in the decarboxylation assay (Fig. 1B), similar to
that observed for the substitution mutants at site 1 (Fig.
1A). Activity in the DCPIP assay was 45% for the
phosphorylated wild-type E1, and PDC activity was undetectable (Fig.
1B).
As seen from the three different assays, the reduction in the
activities for the site 2 or site 3 E1 mutants was less variable for
each mutant (about 20%) and ranged from 30% to 70% inhibition (Fig.
1C). It should be noted that the substitution of S2 or S3 with glutamate or glutamine inhibited PDC activity by only 50% (Fig.
1C), whereas substitution of serine at S1 with glutamate resulted in complete inactivation (Fig. 1A). In contrast to
the findings with the site 2 or site 3 substitution E1 mutants,
phosphorylation of either site 2 or site 3 alone, drastically reduced
(more than 90%) PDC activity (Fig. 1D), as opposed to only
about 40% reduction in the decarboxylation assay. Substitution of site
2 or 3 with glutamate did not mimic the phosphorylation effect.
To investigate whether the binding of the mutant E1s in the PDC could
affect its activity, we measured the activities of the site 1 mutant
E1s bound to E2 (in PDC) or in the free state, using the
decarboxylation assay. As expected, wild-type E1-PDC activity as
measured by the 14CO2 formation (25.8 ± 3.4 units/mg of E1; Fig. 2A)
was similar to that determined by NADH formation in the PDC assay
(29.0 ± 0.9 units/mg of E1; Fig. 1A). Site 1 mutants
showed variable inhibitory effects on PDC activity (measured by
14CO2 formation), which were similar to the
ones reported for PDC activity in the PDC assay (NADH formation) in
Fig. 1A. The measurements of E1 activity in the S1
substitution mutants bound to E2 and in the free states showed similar
activities except for the S1D E1 mutant, which had about twice as much
E1 activity when bound compared with the free state (Fig.
2A). Phosphorylation of site 1 in wild-type E1 had similar
levels (about 45%) of E1 activity in the decarboxylation assay when E1
was bound to E2 or was in the free state (Fig. 2B). These
results suggest that the interaction of the site 1 E1 mutants (except
the S1D mutant) with E2 had no effect on their E1 activity in the
decarboxylation assay.
Kinetic Parameters of Phosphorylation Site Mutant E1s--
Kinetic
parameters of the site 1 mutants were determined in the DCPIP assay
(Table I) because three of the four site
1 mutants had no activity or negligible activity in the PDC assay (Fig. 1A). Apparent Km values for pyruvate
increased for E1 mutants with alanine (3.2-fold), glutamate (4.5-fold),
and glutamine (2.6-fold) substitutions at site 1. In contrast, apparent
Km values for TPP remained unaffected for the
glutamate-substituted mutant but only slightly increased for the
glutamine-substituted (1.7-fold), and alanine-substituted (2.7-fold)
site 1 mutant E1s. Kinetic parameters were not determined for the S1D
E1 due to its strong inactivation under the assay conditions.
For the mutants of sites 2 and 3, kinetic parameters were determined
using the PDC assay (Table I). Apparent Km values
for pyruvate did not change for the mutants of sites 2 and 3 (except
about 2.8-fold increase for S3E). In contrast, apparent Km values for TPP increased for the site 2 mutants
(2.4-, 9.8-, and 3.2-fold for S2A, S2E, and S2Q mutant E1s,
respectively) and ~27-fold for S3E (with only about 2% catalytic
efficiency with TPP; Table I). This latter result indicates a possible
effect of glutamate substitution for serine at site 3 on TPP
interaction with this mutant E1.
Earlier mammalian E1 was reported to have a lag-phase in the progress
curve of the PDC reaction (34). Human E1 displayed a lag-phase only in
the presence of low TPP concentrations (below 1 µM); the
lag-phase was suggested to reflect E1 activation during catalysis (34).
Of several mutant E1s investigated, only S1A, S2E, and S3E did not show
the lag-phase in the PDC reaction under the experimental conditions
employed (Table I). The absence of the lag-phase could reflect a change
in the conformation of the E1 active site caused by these mutations.
The information about the presence of the lag-phase presence for S1E,
S1Q, and S1D E1s is not available due to the lack or negligible PDC
activity of these mutant proteins. Human E1 was shown previously to be
subject to substrate-induced inactivation during incubation with TPP
plus pyruvate involving acetylation of the protein groups (35). As shown in Table I, except for the mutant S3E E1 with only about 10%
inhibition, all other mutant E1s tested showed inhibition from 25% to
55%. Mutation of site 3 (S3E) could affect TPP binding, which is
required to achieve the substrate-induced inactivation. It is
noteworthy that inactivation of the site 1 mutant E1s (S1A, S1E, and
S1Q) was only about one-half (~26%) as compared with that observed
for the wild-type E1 (~55%). This may be due to the different
conformation of the active site and/or protection of the protein groups
involved in the substrate-induced inactivation (S1A, S1E, S1Q).
To further investigate the changes in TPP binding, pyrophosphate was
used as a competitive inhibitor of TPP in wild-type and mutant E1s in
the PDC assay (Table I). Glutamate substitution at site 2 or site 3 caused a marked reduction in its affinity for pyrophosphate that
correlated with the increased apparent Km values for
TPP (Table I). The less efficient binding of pyrophosphate may be due
to the presence of the negative charge in the S2E and S3E mutant E1s.
However, the mutant S2Q also showed a 3-fold increase in
Ki. Unfortunately, the Ki value
for the S1E and S1Q mutant E1s could not be determined due to the lack
of activity of this enzyme in the PDC assay.
Substrate Recognition by Phosphorylation Site Mutant E1s--
E1
is able to use Interaction of the Site-specific Mutant E1s with E2--
The
subunit-binding domain in mammalian PDC-E2 binds only E1. The possible
effect of the mutations on the interaction of E1 with E2 was
investigated using competition by mutant E1 with the wild-type E1 for
its interaction with E2 (38, 39). Wild-type and mutant E1s were
preincubated with E2-E3BP in different ratio; aliquots were taken to
measure PDC activity after E1-E2-E3BP reconstitution with E3. This
assay includes interactions of the wild-type and mutant E1s with E2 in
the preincubation mixture and under turnover conditions. As shown in
Fig. 3, the three E1 mutants of site 1 (S1E, S1Q, and S1D), having almost no activity in the PDC assay, competed for the interaction with E2 with less affinity than the wild-type E1 (curves were displaced from the expected line for the
equal binding affinity for E2). Activity was 80% for WT/S1E, 87% for
WT/S1Q, and 83% for WT/S1D at 5/5 ratio of WT/mutant E1 (calculated as
the best fit of the hyperbolic curves by computer) instead of the
theoretical 50%. The same approach was used in the earlier study to
demonstrate the higher affinity of the active E1 for E2 compared with
the phosphorylated E1 (38). We have confirmed this observation with E1
phosphorylated at site 1 (results not shown). E1 mutants of sites 2 and
3 were not investigated using this experimental approach as they retain
nearly 50% of wild-type activity in the PDC assay (Fig. 1).
Effect of Coenzyme and Substrates on Phosphorylation of Different
Sites in E1--
To probe the possible spatial arrangements of the
three phosphorylation sites in E1 in relation to the active site,
phosphorylation of each individual site was investigated in the
presence of different ligands. Fig. 4
depicts inactivation of E1 harboring mutations at site 1 (E1 S2A,S3A),
site 2 (E1 S1A,S3A), and site 3 (E1 S1A,S2A) by PDK1 in the absence and
presence of varying concentrations of TPP (up to 200 µM).
TPP afforded protection of all three sites, and the level of protection
was in the order site 1 < site 2 < site 3. Site 3 was
protected almost completely by 200 µM TPP. The
inactivation rates (kapp) decreased at 200 µM as follows: ~3.9-fold for site 1, 7.1-fold for site
2, and 41-fold for site 3, suggesting that site 3 is closer to the
TPP-binding site and that phosphorylation of this site may affect the
interaction of E1 with the coenzyme.
Because phosphorylation of pigeon breast muscle E1 was shown to prevent
pyruvate binding (24), it was of interest to investigate if pyruvate
could also protect human E1 from inactivation by phosphorylation. Fig.
5A shows phosphorylation of
site 1 of the wild-type human E1 by PDK2, which phosphorylates only
site 1 in the absence of E2.2 Increasing the concentration
of pyruvate (in the absence of TPP) gradually reduced the rate of
inactivation of E1 by phosphorylation of site 1 (Fig. 5A).
Preparations of E1 contained 4-8% of holoenzyme; however, this low
level of holoenzyme could not be enough to significantly reduce the
amount of pyruvate used in the preincubations (in the absence of E2).
Pyruvate is known to be a PDK inhibitor (40). To avoid the inhibition
of PDK2, the wild-type E1 was preincubated with MgCl2, TPP,
and pyruvate for 10 min prior to initiation of phosphorylation of E1 so
that pyruvate would be used in the nonoxidative catalytic reaction
forming HE=TPP. This treatment, however, did not increase protection
over that seen for TPP alone (compare Although inactivation by phosphorylation of E1 has been known for
more than 30 years, the mechanism by which inactivation is exerted is
not well understood. In the present study, the availability of human E1
with only one functional phosphorylation site as well as each
functional serine replaced by several different amino acid residues has
provided us the unique opportunity to investigate the mechanism of
site-specific inactivation of E1. PDC-E1 catalyzes two successive
steps: (i) the decarboxylation of pyruvate to CO2 to form
the intermediate HE=TPP and (ii) the reductive acetylation of the
lipoyl groups of E2. The latter reaction is the rate-limiting step in
E1 (and hence in PDC) catalysis (42). The affinity for TPP of the
phosphorylated E1 was less than that for the unphosphorylated mammalian
and avian E1s; however, it was not sufficient to account for complete
inhibition of the catalytic function of phosphorylated E1 (13, 21-23).
Phosphorylation of bovine E1 was suggested to induce a conformational
transition displacing a catalytic residue at the active site (13). The
phosphorylated porcine E1 was shown to inhibit the formation of
enzyme-bound 2- In the present study, substitution of serine 264 (site 1) with
glutamate or aspartate in human PDC-E1 resulted in complete inactivation of PDC activity, but only a moderate reduction of E1
activity in both the DCPIP and decarboxylation assays (Fig. 1). In a
similar study, the substitution of serine with glutamate at the
phosphorylation site 1 of the In the earlier studies when E1 was most probably phosphorylated at one
active site due to its half-of-the-site reactivity (7, 20), the
residual activity of E1 in the reactions other than the overall PDC
reaction (22, 23) could arguably be explained by functioning of the
second active site. We excluded this possibility by introducing
glutamate or aspartate at the phosphorylation site 1 of both Phosphorylated pigeon breast muscle E1 was shown to form the charge
transfer complex accompanying TPP binding. In contrast to WT-E1, the
intensity of this band did not change after addition of pyruvate to
phospho-E1, indicating possible interaction between the phosphorylated
serine and the substrate-binding site (23). Besides the disturbance in
the charge transfer complex as a result of pyruvate binding, the other
possible explanation could be the conformational transition of the E1
active site after decarboxylation. Apparent Km
values for pyruvate determined in the DCPIP assay increased not only
for the glutamate- and glutamine-substituted E1 mutants of site 1 but
also for alanine substitution (Table I). Similarly, replacement of
Ser-293 (site 1) with alanine in the BCKDH resulted in an increased
apparent Km for the substrate without affecting the
Vmax (43). The substitution of serine at site 1 or 2 in nematode PDC-E1 with alanine did not change the apparent
Km value for pyruvate but decreased the
Vmax value (44). Probably even if
phosphorylation of site 1 affects the substrate binding (change in
apparent Km values), it would not account for the
complete inactivation. It was suggested that phosphorylation of pigeon
breast muscle E1 may prevent pyruvate binding based on
pyruvate-dependent protection against phosphorylation (24).
In our study, pyruvate decreased inactivation by inhibiting PDK in the
absence of TPP (Fig. 5; see also Ref. 40). A substrate analog,
An interesting finding is that substitution of serine 264 (site 1) with
glutamine forced this mutant E1 to lose its substrate specificity and
use pyruvate and The earlier studies demonstrated a greater affinity of the active E1
form to the E2 sites compared with the phosphorylated form (38, 46).
Our results are in support of these findings and suggest that amino
acid substituted mutants of site 1 can not easily replace the wild-type
E1 when interacting with E2 (Fig. 3). Inactivation of E1 is not
increased because of its binding onto E2, as E1 in the presence or
absence of E2 showed the same level of reduction in its activity in the
decarboxylation assay (Fig. 2). The recently obtained three-dimensional
structure of the human BCKDH provides evidence for location of the
E2-binding site close to the C termini of Phosphorylation of E1 probably alters its interaction with the lipoyl
domains of E2 more than that with pyruvate. The conformational requirements for the reductive acetylation step could be different from
that of decarboxylation; thus, E1 may undergo a transition before
catalyzing its rate-limiting second step. Berg et al. (48) suggested that the conformational change takes place following pyruvate
decarboxylation that opens the binding cleft on E1 for the lipoyl
domain of Azotobacter vinelandii PDC-E2. An attractive idea
is that phosphorylation may prevent the conformational transition thus
eliminating the reductive acetylation step. We have several indications
for a change in conformational mobility of E1. The differences in
apparent Km values for pyruvate and TPP and the
decrease in the substrate-induced inactivation may reflect a different
conformation of the E1 active site. The absence of the lag-phase for
the alanine substitution of site 1 and glutamate substitution of sites
2 or 3 (Table I) may indicate the inability of the phosphorylated E1 to
undergo the activation during catalysis. Gong et al. (49)
suggested that it is not the decarboxylation but interaction of lipoyl
domain with E1 that facilitates structural changes within E1 and
subsequently the efficient reductive acetylation. The negative charge
and the size of the phosphoryl group located in the substrate channel
could prevent the interaction of E1 with the negatively charged region
(Glu-162, Glu-167) of the lipoyl domain (12).
All mammalian PDCs have three phosphorylation sites and phosphorylation
of each site leads to inactivation of E1. Nematode PDC-E1 has only two
inactivating phosphorylation sites, and their positions in the sequence
are conserved with sites 1 and 2 of mammalian PDC-E1 (44). In contrast,
mammalian BCKDC-E1 has two phosphorylation sites and phosphorylation of
only one of them, Ser-293 (site 1), results in complete inactivation
(and is conserved with site 1 of PDC-E1) (43). Phosphorylation of
Ser-303 (site 2; 3 amino acid residues farther from site 2 of PDC-E1)
does not affect the BCKDC activity. Our study showed that the
substitution of serine at sites 2 or 3 in E1 with glutamate or
glutamine caused only 30-70% reduction of activity in all three
assays (Fig. 1C). However, phosphorylation of site 2 or 3 resulted in drastic reductions (>90%) of PDC activity (Fig.
1D). A possible explanation is the larger size of
PO42 Apparent Km values for TPP determined in the PDC
assay were higher for S2E and S3E mutant E1s compared to that for wild-type E1 (Table I). This correlated well with the results of the
inhibition study with pyrophosphate that demonstrated less affinity for
pyrophosphate of S2E and S3E mutant E1s (Table I). In a separate
experiment, TPP protection from phosphorylation-dependent inactivation
increased from site 1 to site 2 and was maximal for site 3 (Fig. 4). It
is possible that site 2 and especially site 3 are localized in close
vicinity to the TPP-binding site and phosphorylation of site 3 affects
TPP binding.
Concluding Remarks--
Phosphorylation of a serine residue has
been shown to alter the activities of several other enzymes by one of
these mechanisms: (i) a long range conformational change (50), (ii) an
electrostatic repulsion and steric hindrance (51), and (iii) an
impairment of protonation by histidine (52). In the case of mammalian
PDC-E1, site 1 is most likely exposed at the interface of the subunit
of the pyruvate dehydrogenase (E1) component. Substitutions of the
phosphorylation sites were generated by site-directed mutagenesis.
Glutamate (S1E) and aspartate (S1D) substitutions at site 1 resulted in
the complete loss of PDC activity; however, these mutants were variably
active in the decarboxylation and 2,6-dichlorophenolindophenol assays.
S1Q had only 3% of wild-type PDC activity. The apparent
Km values for pyruvate increased for the mutants of
site 1 when determined in the 2,6-dichlorophenolindophenol assay. The
substitutions at sites 2 and 3 caused only moderate reductions in
activity in the three assays. S3E had a 27-fold increase in the
apparent Km for thiamine pyrophosphate and 8-fold
increase in the Ki for pyrophosphate. Site 3 was
almost completely protected from phosphorylation by thiamine
pyrophosphate. The results show that the size rather than negative
charge of the substituted amino acid residue affects the active site of
E1 and that modification of each of the three serine residues affect
the active site in a site-specific manner for its ability to bind the
cofactor and substrates.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
2)
components bind this core and 6 E3 homodimers bind to the E3BP subunits
(1, 2).
subunit of the E1 component has three
phosphorylation sites, named site 1 (Ser-264), site 2 (Ser-271), and
site 3 (Ser-203), and phosphorylation of any one of these three sites
results in inactivation (7-9). In vivo inactivation of PDC
correlates mostly with phosphorylation of site 1 (10). Phosphorylation
of site 1 can occur in E1 alone, whereas further phosphorylation at
sites 2 and 3 in E1 requires the presence of E2 component (7, 11). E2
activates PDKs by increasing their catalytic efficiency and mediating
colocalization of E1 and PDK (12). The rates of phosphorylation (and
hence inactivation) of the three sites are site-specific (9). However, the rates of dephosphorylation are similar for the three sites (9).
and the
subunits (13-19). E1 exhibits half-of-the-site reactivity, as phosphorylation of only one serine residue of one of the two
subunits is sufficient for inactivation of the enzyme (20). Phosphorylation of E1 causes a reduction in its
affinity for thiamine pyrophosphate (TPP) as well as substrate binding,
and impedes the formation of the 2-
-hydroxyethylidene-TPP (HE=TPP)
intermediate of the E1-catalyzed reaction (13, 21-24). The mechanism
of inactivation by phosphorylation of E1 is not understood. In an
earlier study, we investigated several human E1 mutants with single,
double, and triple mutations by replacing serine with alanine and
characterized their site-specific rates of phosphorylation and a random
mechanism of dephosphorylation (9). To probe the mechanism of
inactivation of human E1 by phosphorylation, we have generated a family
of mutant human E1s in which one of the three serine residues is
replaced individually by amino acid residues of different size and
charge. The results of this study indicate that phosphorylation of site
1 leads to the steric interference in catalysis and phosphorylation of
site 3 may affect coenzyme binding.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit were made: serine
264 (site 1) was replaced with alanine (S1A), glutamate (S1E),
glutamine (S1Q), and aspartate (S1D); serine 271 (site 2) was replaced
with alanine (S2A), glutamate (S2E), and glutamine (S2Q); and serine
203 (site 3) was replaced with alanine (S3A) and glutamate (S3E).
Site-directed mutagenesis was performed by a two-stage polymerase chain
reaction (25) using pQE-9-6HE1
for substitution of Ser-264 (site
1), Ser-271 (site 2), and Ser-203 (site 3). pQE-9-6HE1
was
constructed previously in this laboratory (16). A single mutagenic
primer and two primers of 5' and 3' ends including BamHI
sites preceding the first codon and after the stop codon of the E1
sequence were used to amplify E1
cDNA. The following mutagenic
primers (antisense) were used (mutated bases are shown in bold):
5'-GGGTCACTCATAGCGTGTCCGTGG-3' for S1A,
5'-GGGTCACTCATCTCGTGTCCGTGGTAA-3' for S1E,
5'-GGGTCACTCATCTGGTGTCCGTGGTAA-3' for S1Q,
5'-GGGTCACTCATGTCGTGTCCGTGGTAA-3' for S1D,
5'-CGTGTACGGTAAGCGACTCCAGGG-3' for S2A,
5'-CGTGTACGGTACTCGACTCCAGGG-3' for S2E,
5'-CGTGTACGGTACTGGACTCCAGGG-3' for S2Q,
5'-CTCTCAACAGCCGTTCCCATTCC-3' for S3A, and
5'-CTCTCAACCTCCGTTCCCATTCC-3' for S3E.
cDNAs were digested with
BamHI and ligated to PQE-9 (containing a polyhistidine
extension at the amino terminus). The complete coding sequence of each
mutant E1 cDNA was verified by DNA sequencing (26). To overexpress
the mutant E1s, coexpression vectors carrying both E1
and E1
were constructed and transformed into E. coli M15 cells
containing pDMI.1 plasmid, which encoded the lac repressor
as described previously (16). Some E1 mutants (S2A,S3A, S1A,S3A,
and S1A,S2A E1) used in this study were constructed previously (9).
-mercaptoethanol, and a mixture of protease
inhibitors; (ii) a French press was used to disrupt the cells instead
of sonication; and (iii) Ni2+-NTA-agarose was used instead
of Talon resin. Recombinant human E3 was overexpressed and purified to
about 96% purity as described by Liu et al. (29).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (43K):
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Fig. 1.
Activities of the wild-type and
phosphorylation site mutant E1s in three different assays.
A, activities of E1s were measured in the PDC assay by
formation of NADH (black bars), decarboxylation
(DECARB) assay by formation of 14CO2
from [1-14C]pyruvate in the absence of electron acceptor
(hatched bars), and DCPIP assay by reduction of
DCPIP (open bars) as described under
"Experimental Procedures." *, PDC activity was undetectable. One
hundred percent of activity corresponds to 29.0 ± 0.9 units/mg of
E1 in the PDC assay, 122 ± 11 milliunits/mg in the
decarboxylation assay, and 242 ± 10 milliunits/mg in the DCPIP
assay. B, the S2A,S3A E1 was phosphorylated on site 1 only
by PDK2 in the presence of E2-E3BP and 0.1 mM ATP for 30 min and its activity was measured in the PDC (black
bars), decarboxylation (hatched bars),
and DCPIP (open bars) assays. *, PDC activity was
undetectable. One hundred percent of activity corresponds to 19.3 ± 1.8 units/mg of S2A,S3A in the PDC assay, 80 ± 2 milliunits/mg
in the decarboxylation assay, and 157 ± 4 milliunits/mg in the
DCPIP assay. C, activities of E1s were measured in the PDC
assay (black bars), decarboxylation assay
(hatched bars), and DCPIP assay (open
bars). One hundred percent of activity corresponds to
28.2 ± 1.2 units/mg of E1 in the PDC assay, 135 ± 5 milliunits/mg in the decarboxylation assay, and 248 ± 30 milliunits/mg in the DCPIP assay. D, E1 with site 2 only
(S1A,S3A) and E1 with site 3 only (S1A,S2A) were phosphorylated by PDK2
and by PDK1, respectively, in the presence of E2-E3BP and 0.5 mM ATP for 60 min and activity was measured in the PDC
(black bars) and decarboxylation (hatched
bars) assays. One hundred percent of activity corresponds to
20.0 ± 0.7 units/mg of S1A,S3A in the PDC assay and 63 ± 3 millinits/mg in the decarboxylation assay; and 100% of activity
corresponds to 14.1 ± 0.5 units/mg of S1A,S2A in the PDC assay
and 37 ± 8 milliunits/mg in the decarboxylation assay. The
results are means ± S.D. of three to five independent
determinations.
View larger version (28K):
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Fig. 2.
Comparison of E1 activities of wild-type and
site 1 mutants in the free state and in reconstituted PDC.
A, after the wild-type and site 1 mutant E1s were
reconstituted with E2-E3BP and E3, PDC activity was measured by
14CO2 production (black
bars). Decarboxylation activity (in the absence of added
electron acceptor) was determined for E1s, which were reconstituted to
PDC (hatched bars). Decarboxylation activity was
measured for free E1 (open bars). PDC was
reconstituted in 50 mM potassium phosphate buffer, pH 7.5, with 2 mM dithiothreitol. One µg of E1 was used to assay
PDC activity. Decarboxylation activity of E1 in reconstituted PDC or in
the free form was measured with 4 and 10 µg of E1, respectively. One
hundred percent of activity corresponds to 22.6 ± 4.2 units/mg of
E1 in PDC reaction, 210 ± 8 milliunits/mg in E1 reaction in
reconstituted PDC, and 150 ± 5 milliunits/mg in E1 reaction in
the free state. *, PDC activity was 0.3-0.5%. B, the
wild-type E1 was phosphorylated on site 1 only by PDK2 (in the absence
of E2-E3BP) in the presence of 0.25 mM ATP for 30 min and
its activity was measured as in A. The results are
means ± S.D. of three to four independent determinations.
Catalytic parameters of wild-type and site-specific mutant E1s
determined in PDC and DCPIP assays
-ketobutyrate and
-ketoisovalerate as substrates
in the DCPIP assay (36) and only
-ketobutyrate in the PDC assay (due
to specificity of the E2 component) (37). We therefore investigated the
possible changes in substrate recognition by the E1 mutants (Table
II). The mutants of site 1 (except S1D) had higher activity with
-ketobutyrate than wild-type E1. The most
striking observation was a 5-fold higher activity of the S1Q mutant E1
compared with wild-type E1. The DCPIP activity of S1Q with
-ketobutyrate as a substrate (169 milliunits/mg) was as high as the
activity with pyruvate as a substrate (153 milliunits/mg), whereas the
activity of the wild-type E1 with
-ketobutyrate as a substrate (34 milliunits/mg) was much less than with pyruvate (242 milliunits/mg)
(Table II). The E1 mutants of sites 2 and 3 displayed activity nearly
similar to the wild-type E1 with
-ketobutyrate as a substrate,
whereas their activity with pyruvate was less than that observed for
the wild-type E1. When
-ketoisovalerate, the
-keto acid of
isoleucine, was used as a substrate, all of the mutants displayed
significantly decreased levels of activity; the greatest decrease
occurred with the substitution generating a negative charge (residual
activity: 7.7% for S1E, 9% for S1D, 33.6% for S2E, and 20.9% for
S3E) (Table II). The general direction of the reduction in activities
of the mutants of sites 2 and 3 with
-ketoisocaproate was similar to
that observed for these mutant E1s with pyruvate as a substrate (Table
II).
Activities of wild-type and mutant E1s in the DCPIP assay with
different substrates
-ketobutyrate, and 83 milliunits/mg with
-ketoisovalerate as substrates. The results are means ± S.D.
of four to five independent determinations.
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Fig. 3.
Substitution of the site 1 mutant E1s with
wild-type E1 in binding to E2-E3BP. Ten µg of E1s were
preincubated with 1 µg of E2-E3BP and 3 µg of E3 (at a 330:1:150
molar ratio calculated for E1 as a tetramer, E2 as a 60-mer, and E3 as
a dimer) at room temperature for 5 min in 50 mM potassium
phosphate buffer, pH 7.5. Aliquots (0.8 µg of E1) were withdrawn to
measure PDC activity. The ratio of wild-type/mutant E1 was gradually
increased keeping the total amount of E1 constant (except for a
negative control with different concentrations of the wild-type E1 in
the absence of the mutant E1, in A). The dashed
line represents the theoretical activity assuming equal
binding affinities of the wild-type and mutant E1. The results are
means ± S.D. of three to four independent determinations.
View larger version (14K):
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Fig. 4.
Protection of sites 1, 2 and 3 from
phosphorylation by TPP. E1s with site 1 only (S2A,S3A), site 2 only (S1A,S3A), or site 3 only (S1A,S2A) were phosphorylated by PDK1 in
the presence of E2-E3BP and 200 µM ATP in the absence
( ) or presence of TPP at concentrations of 5 µM (
),
20 µM (
) and 200 µM (
). Aliquots were
taken to measure PDC activity. PDK1/E1 ratio was 1/200 (w/w) for site 1 and 1/25 for sites 2 and 3.
and
in Fig.
5B). HE=TPP is known to bind E1 very tightly (41); however,
we could not exclude that some amount of HE=TPP formed was further
converted to TPP and acetaldehyde or acetoin. To eliminate inhibitory
effect of pyruvate on PDK2 and conversion of HE=TPP,
-ketoisovalerate was used as a substrate, which showed no inhibitory effect on PDK activity under the conditions used here (compare
and
in Fig. 5B).
-Ketoisovalerate did not have any
additional protection over that afforded by TPP alone (compare
and
in Fig. 5B).
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Fig. 5.
Effect of different compounds on
phosphorylation of site 1. The wild-type E1 was phosphorylated at
site 1 only by PDK2 in the absence of E2-E3BP and in the presence of
different compounds. Aliquots were taken to measure PDC activity.
A, , no addition;
, 52 µM ATP;
, 52 µM ATP and 50 µM pyruvate;
, 52 µM ATP and 100 µM pyruvate;
, 52 µM ATP and 200 µM pyruvate. B,
, no addition;
, 52 µM ATP;
, 52 µM ATP, 1 mM MgCl2 and 0.2 mM TPP;
, 52 µM ATP, 1 mM
MgCl2, 0.2 mM TPP, and 0.05 mM
pyruvate;
, 52 µM ATP and 0.2 mM
-ketoisovalerate;
, 52 µM ATP, 1 mM
MgCl2, 0.2 mM TPP, and 0.2 mM
-ketoisovalerate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxyethyl-TPP carbanion from E1-TPP in the
presence of either pyruvate (forward reaction) or acetylhydrolipoate
(reverse reaction) (22).
subunit of the branched-chain
-keto acid dehydrogenase component (BCKDH) of the BCKDH complex (BCKDC) resulted in abolition of its activity (43). These authors concluded that the negative charge of the phosphoryl group accounted for the complete inhibition. However, our results with PDC-E1 show that
inactivation was not caused only by the introduction of the negative
charge but also by an increase in the size of the amino acid at
position 264; as substitution of serine with glutamine reduced PDC
activity to about 3% of control level.
subunits by site-directed mutagenesis, i.e. modifying both
active sites. The moderate reduction of activity in the individual E1-catalyzed reactions may indicate the effect of phosphorylation on
the reductive acetylation step. However, we cannot exclude the
possibility that the decrease in activity levels could be explained by
the differences in the catalytic rates of different reactions: 29 units/mg in the PDC assay, 242 milliunits/mg in the DCPIP assay, and
122 milliunits/mg in the decarboxylation assay.
-ketoisovalerate (not an inhibitor of PDK activity under our
experimental conditions), did not afford protection from
phosphorylation (Fig. 5). The recent crystal structure of
Pseudomonas putida BCKDH suggested that a serine may be
involved in the substrate binding through a water molecule (45). It is
possible that Ser-264 (site 1) of human PDC-E1 is involved in
stabilization of the substrate binding and any substitution, even with
alanine, would affect the substrate binding; however, it would not
eliminate E1 activity (Fig. 1A).
-ketobutyrate with nearly the same rates in the
DCPIP assay. An increase in the size of the amino acid at position 264 increased E1 activity with
-ketobutyrate (Table II). Substitution of
site 1 with glutamine may result in a conformational change of the
substrate channel, allowing it to better accommodate a larger
substrate,
-ketobutyrate. The branched-chain keto acid,
-ketoisovalerate, seems to be a poor substrate for S1E and S1Q due
to the interference of the bulky, substituted substrate.
subunits, which is far
from the active sites and possibly from the phosphorylation site 1 (47).
group or compensation in the
protein structure occurring during protein folding of the substitution
mutant E1s. This situation is different for site 1 phosphorylation,
which correlates with serine replacement with glutamate and aspartate
(Fig. 1A). This observation lends support to the importance
of site 1 and its major contribution to the inactivation during phosphorylation.
and
the
subunits and may be at the substrate channel directing and
positioning the lipoyl moiety in the E1 active site. Hence, the
phosphorylation of this site might interfere with the second partial
reaction catalyzed by E1, using E2 as a substrate. Serine at site 1 by its juxtaposition in the substrate channel eliminates PDC activity by
its replacement with a large group with or without a negative charge.
Sites 2 and 3 are exposed only after binding of E1 onto E2, and hence
are most likely localized differently and may have different mechanism
of inhibition. Site 3 seems to be close to the TPP pyrophosphate
moiety-binding site, and its phosphorylation may affect TPP binding.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Murray Ettinger (Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, NY), Dr. Thomas E. Roche (Kansas State University, Manhattan, KS), and Dr. Natalia S. Nemeria (Rutgers, State University of New Jersey, Rutgers, NJ) for helpful discussions and critical reading of a draft manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by U. S. Public Health Service Grant DK20478.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.
To whom correspondence should be addressed: Dept. of Biochemistry,
School of Medicine and Biomedical Sciences, State University of New
York, 140 Farber Hall, 3435 Main St., Buffalo, NY 14214. E-mail:
mspatel@buffalo.edu.
Published, JBC Papers in Press, November 22, 2000, DOI 10.1074/jbc.M007558200
2 L. G. Korotchkina and M. S. Patel, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
PDC, pyruvate
dehydrogenase complex;
E1, pyruvate dehydrogenase;
E2, dihydrolipoamide
acetyltransferase;
E3, dihydrolipoamide dehydrogenase;
E3BP, E3-binding
protein;
PDK, pyruvate dehydrogenase kinase;
TPP, thiamine
pyrophosphate;
HE=TPP, 2--hydroxyethylidene-TPP;
Ni2+-NTA-agarose, Ni2+-nitrilotriacetate-agarose;
DCPIP, 2,6-dichlorophenolindophenol;
BCKDH, branched-chain
-keto acid
dehydrogenase;
BCKDC, branched-chain
-keto acid dehydrogenase
complex;
WT, wild-type.
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