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BIOCHEMISTRY
AND
MOLECULAR BIOLOGY EDUCATION
Vol. 31, No. 1, pp. 5–15, 2003
Mini-Series: Modern Metabolic Concepts
The Biochemistry of the Pyruvate Dehydrogenase Complex*
Received for publication, September 5, 2002, and in revised form, September 17, 2002
Mulchand S. Patel‡ and Lioubov G. Korotchkina
From the Department of Biochemistry, School of Medicine and Biomedical Sciences, State University
of New York at Buffalo, Buffalo, New York 14214
Keywords: Pyruvate dehydrogenase complex, regulation by phosphorylation, diabetes, genetic defects.
compound to pyruvate (or its equivalent) for the synthesis of
glucose in animals, the flux through PDC is tightly regulated
to meet the specific metabolic and energetic needs of different tissues during the fed and fasting (starvation) states.
This is accomplished by covalent modification of the ratelimiting component of the complex involving sophisticated
interplay among the components of the complex and allosteric modulations by acetyl-CoA and NADH, the products of
the reaction (and also of fatty acid oxidation). It is evident
from these simple considerations that PDC plays a key role
as a gatekeeper of both caloric and glucose homeostasis in
mammals. In this review, we will discuss recent developments concerning the structure-function relationship of this
multienzyme complex from various organisms with emphasis on regulatory aspects of the mammalian complex. Detailed accounts of various aspects of this complex can be
found in several excellent reviews [1–11].
In the Westernized world the daily dietary caloric requirements are roughly provided as follows: 40% from
carbohydrates, 40% from fats, and 20% from proteins. In
some populations in developing countries the daily caloric
contribution from carbohydrate is even higher due to its
readily available sources and relatively low cost. Glucose,
the principal product of carbohydrate digestion, passes
through a series of enzymatic steps first in the non-oxidative glycolytic pathway (from glucose to pyruvate) followed
by efficient oxidative metabolism via the tricarboxylic acid
cycle (from acetyl-CoA to CO2 and H2O) to harness a
portion of its potential energy as ATP. Interestingly, these
two major pathways are directly linked by the pyruvate
dehydrogenase complex (PDC)1 localized in the mitochondrial matrix (Fig. 1). In the fed state acetyl-CoA generated
from pyruvate (derived mostly from glucose and some
dietary amino acids) is also utilized for the biosynthesis of
lipids such as long chain fatty acids and cholesterol by
lipogenic tissues (such as liver and adipose tissues and
under special conditions in mammary glands during lactation and in the brain during the prenatal and early postnatal
development). Additionally, amino acids from excess dietary protein are metabolized by several specialized reactions or pathways generating intermediates that have to be
ultimately converted to pyruvate first and then to acetylCoA via PDC either for complete oxidation to CO2 and H2O
or for lipogenesis. PDC is the only known reaction in most
eukaryotes to generate acetyl-CoA (two-carbon compound)
from pyruvate (three-carbon compound). Since this is a
physiologically irreversible reaction and since there is no
other known reaction or pathway to convert the two-carbon
STRUCTURE AND ORGANIZATION OF PDC
PDC is present in most prokaryotic and eukaryotic organisms. It catalyzes several sequential reactions of oxidative decarboxylation of pyruvic acid by the action of its
three catalytic components: (i) pyruvate dehydrogenase
(E1) catalyzing the decarboxylation of pyruvate followed by
reductive acetylation of lipoyl moieties covalently linked to
the dihydrolipoamide acetyltransferase (E2), the second
catalytic component of the complex; (ii) E2 catalyzing the
formation of acetyl-CoA; and (iii) dihydrolipoamide dehydrogenase (E3) reoxidizing the reduced lipoyl moieties of
E2 with the consequent reduction of NAD⫹ to NADH (Fig.
2) [12]. PDC is a multienzyme complex with a well organized structure that has two different morphologies based
on symmetry of the central E2 core, i.e. octahedral for
Gram-negative bacteria and icosahedral for eukaryotes
(mammals, yeast, plants, and nematodes) and some
Gram-positive bacteria. E2 consists of three well defined
domains connected by flexible linkers: (i) inner domains
interacting noncovalently with each other to form the core
of the complex and each having the catalytic site; (ii)
subunit-binding domain interacting with E1 and E3 (for
PDC from bacteria, plants, and nematodes); and (iii) the
lipoyl domain (containing up to three repeating units, one
for yeast and Gram-positive bacteria, two for mammals,
and three for Gram-negative bacteria; Fig. 3) providing
coupling of the E1, E2, and E3 active sites by transferring
* This work was supported by United States Public Health
Service Grants DK20478 and DK42885.
‡ To whom correspondence should be addressed: Dept. of
Biochemistry, School of Medicine and Biomedical Sciences,
State University of New York at Buffalo, 140 Farber Hall, 3435
Main St., Buffalo, NY 14214. Tel.: 716-829-3074; Fax: 716-8292725; E-mail: [email protected].
1
The abbreviations used are: PDC, pyruvate dehydrogenase
complex; E1, pyruvate dehydrogenase; E2, dihydrolipoamide
acetyltransferase; E3, dihydrolipoamide dehydrogenase; BP, E3binding protein; PDK, pyruvate dehydrogenase kinase; PDP,
phosphopyruvate dehydrogenase phosphatase; TPP, thiamine
pyrophosphate; PPAR␣, peroxisome proliferator-activated receptor ␣; CRE, cAMP-response element; CAT, chloramphenicol
acetyltransferase; MEP, mouse E1␣ promoter site.
This paper is available on line at http://www.bambed.org
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BAMBED, Vol. 31, No. 1, pp. 5–15, 2003
FIG. 1. Regulation of PDC activity
by phosphorylation/dephosphorylation catalyzed by PDKs and PDPs.
Three serine residues of E1 phosphorylation sites are shown as S1,
S2, and S3 in dephosphorylated condition and as P1, P2, and P3 when
phosphorylated.
FIG. 2. Overall PDC reaction. CH3-COH⫽TPP, 2-␣-hydroxyethylidene-TPP; Lip-S2, oxidized lipoyl moiety of E2.
an acetyl group and reducing equivalents. The core of
Gram-negative bacterial PDC is composed of inner domains of 24 subunits of E2 and binds 24 dimers of E1(␣2)
and 12 dimers of E3(␣2) (Table I). PDCs with icosahedral
symmetry have 60-mer E2 cores that bind up to 30 tetramers of E1(␣2␤2) and 12 dimers of E3. Eukaryotic organisms have an additional component in PDC not present in
bacterial complexes, i.e. E3-binding protein (BP) [6, 12].
BP has a domain structure similar to E2 structure except:
(i) only one lipoyl domain is present; (ii) the subunit-binding
domain binds E3 and not E1; and (iii) the inner domain
does not perform a catalytic function (due to the absence
of the critical histidine residue). The PDC core of eukaryotes is composed of the inner domains of E2 and BP
[13]. Ascaris suum PDC has a BP without a lipoyl domain
and only in the anaerobic form of this nematode [14].
Mammalian, plant, and nematode PDCs are regulated by
reversible phosphorylation/dephosphorylation that requires additional regulatory enzymes, pyruvate dehydrogenase kinase (PDK), and phosphopyruvate dehydrogenase phosphatase (PDP) (Table I).
The three-dimensional structures of several components of PDC were determined by x-ray crystallography:
E1 from Escherichia coli [15] and human2; E1␤ subunit
from Pyrobaculum aerophilum [6]; cubic core of E2 (consisting of the inner domains) from Azotobacter vinelandii,
Bacillus stearothermophilus, and Enterococcus faecalis;
subunit-binding domain of E2 from B. stearothermophilus;
E2 lipoyl domains from B. stearothermophilus, E. coli, and
A. vinelandii; and E3 from yeast, A. vinelandii, Pseudomonas putida, and Pseudomonas fluorescens [5, 6, 16]. So far
it is not possible to solve the three-dimensional structure
of the whole PDC because of its huge molecular mass of
8 –10 million Da, hollow structure of its inner core with high
solvent content, and flexibility of E2 domains. However,
the structures of the E2 core and entire PDC from yeast
and mammals were studied by cryoelectron microscopy
(Fig. 4) [17, 18]. Both electron microscopy and crystal
structures of the E2 core revealed the trimer building
blocks in the cubic (8 trimers) or dodecahedral (20 trimers)
structures. The trimers are connected by bridges and form
a structure with large openings and a cavity inside. Substrates of E2 enter the active site from different directions,
i.e. CoA come from inside the core, while lipoyl moieties
come from the outside. Twelve monomers of BP (for eukaryotic PDCs) were suggested to bind within the cavity of
E2 and interact with the E3 dimer inside 12 pentagonal
openings. Yeast E2 core has the size of 250 Å with 40-Å
variations in diameter as found by electron microscopy.
The contraction and expansion of the E2 core was proposed to reflect the conformational mobility of PDC during
2
E. Ciszak, L. G. Korotchkina, P. Dominiak, S. Sidhu, and M. S.
Patel, unpublished observations.
7
FIG. 3. The structural domains of E2 and BP.
TABLE I
Composition of the pyruvate dehydrogenase complex from different species
Oligomeric
structure
Source
Isoenzymes
␣2
␣2
␣2␤2
␣2␤2
␣2␤2
␣2␤2
␣2␤2
E. coli
A. vinelandii
B. stearothermophilus
Mammals
Nematode
Yeast
Plant
␣ somatic/␣ testis
E1-I/E1-II
E2
␣24 (65–66 kDa)
␣60 (49–76 kDa)
␣24
E3 (49–58 kDa)
␣2
BP (45–48 kDa)
␣
PDK (39–48 kDa)
␣2
E. coli
A. vinelandii
B. stearothermophilus
Mammals
Nematode
Yeast
E. coli
A. vinelandii
B. stearothermophilus
Mammals
Nematode
Yeast
Plant
Mammals
Yeast
Nematode
Mammals
Components
E1
␣2 (89–99 kDa)
␣2␤2
␣ (40–43 kDa)
␤ (35–37 kDa)
␣60
␣2
␣2
PDP
Ca (52 kDa)
Rb (96 kDa)
a
b
C, catalytic subunit.
R, regulatory subunit.
C/R
Nematode
Plant (Arabidopsis thaliana)
(maize)
Mammals
Mitochondrial/
plastid
PDK1
PDK2
PDK3
PDK4
PDK
PDK1/PDK2
C: PDP1/PDP2
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BAMBED, Vol. 31, No. 1, pp. 5–15, 2003
FIG. 4. Three-dimensional reconstruction of bovine PDC (A and B) and diagrammatic representation of the structural
domains of E2 subunit (C). Shaded-surface representation of 3-fold axes of symmetry of the three-dimensional reconstruction of the
bovine kidney PDC (A) and with the closest half removed to reveal the linker (blue) that binds E1 (yellow) to the E2 core (green; B). The
inner linker is 50 Å in length. C, the C-terminal self-association domain is responsible for the assembly of the dodecahedral scaffold
to which E1 and BP䡠E3 bind. The N-terminal half of the E2 comprises the L1 and L2 lipoyl domains, the E1-binding domain, and their
associated linkers. The inner linker is revealed in the three-dimensional structure of the PDC. 5-, 3-, and 2-fold axes are indicated.
Reprinted with permission from Zhou et al. [18]. Copyright 2001 National Academy of Sciences, U. S. A.
catalysis [17]. Recently the structure of the whole PDC
from bovine kidney was constructed based on cryoelectron microscopy (Fig. 4) [18]. Bovine PDC was found to
have 60 monomers of E2, 12 monomers of BP, 22 tetramers of E1, and 6 dimers of E3. The size of the complex in
the presence of E1 molecules is about 500 Å. E1 molecules are organized in trimers bound through the subunitbinding domains of E2 70 Å above the trimers of inner
domains of E2. Lipoyl domains are proposed to rotate
around the subunit-binding domains transferring intermediates between active sites of E1, E2, and E3, and this
movement is provided by the conformational changes of
the E2 core.
E1 of PDC is a dimer of identical subunits in Gramnegative bacteria or a heterotetramer composed of 2 ␣
and 2 ␤ subunits in eukaryotes and Gram-positive bacteria. Both types of E1 have two active sites and require
thiamine pyrophosphate (TPP) and Mg2⫹ as cofactors.
Sequence similarity between the two types of E1 is low;
however, there is a significant similarity in the structure of
the TPP-binding region for several TPP-dependent enzymes, the three-dimensional structures of which are
known (yeast transketolase, Lactobacillus plantarum pyruvate oxidase, yeast, and Zymomonas mobilis pyruvate
decarboxylase, P. putida benzoylformate decarboxylase,
human and P. putida branched-chain ␣-keto acid dehydrogenase, Desulfovibrio africanus pyruvate:ferredoxin oxidoreductase [19, 20], and E. coli PDC-E1 [15]). The common features of the active sites of these proteins are: (i)
active sites are localized on the interface of two subunits;
(ii) two residues of the TPP motif (a sequence of 30 –33
amino acid residues conserved for all known TPP-requiring enzymes) coordinate binding of the pyrophosphate
moiety of TPP through interactions with a divalent cation
(Mg2⫹ or Ca2⫹); (iii) amino acid residues help TPP to maintain the “V” conformation necessary for catalysis (among
them are phenylalanine or tyrosine residues providing a
stacking interaction with the aminopyrimidine ring of TPP
and a hydrophobic residue between two aminopyrimidine
and thiazole rings); and (iv) N1⬘ of the pyrimidine ring binds
to the glutamine residue through a hydrogen bond, which
affects reactivity of the 4⬘-amino group of TPP and activates the C2 hydrogen of the thiazolium ring of TPP. Only
the E. coli PDC-E1 structure has been reported so far. The
human PDC-E1 structure was solved recently.2 The structure-function relationships of E1 from human and E. coli
have been studied intensively using site-directed mutagenesis [21–23]. Mammalian E1 has two isoforms (with
92% homology), a somatic cell form with the gene
(PDHA-1 for human) located on the X chromosome and a
testis-specific form with the gene (PDHA-2 for human) on
chromosome 4 [24]. The second isoform is necessary during spermatogenesis when the X chromosome is inactivated or absent. A. suum has two E1 isoforms, isoform I in
anaerobic adults and isoform II in aerobic larvae [25].
E3 is a homodimer with two active sites localized at the
dimer interface. Each active site contains tightly but noncovalently bound FAD, which participates in the electron
transfer from dihydrolipoamide to NAD⫹ with participation
of an active site disulfide. Each subunit of E3 has four
structural domains: FAD-binding domain, NAD⫹-binding
domain, central domain, and interface domain. Interestingly, E3, the product of the same gene, participates in four
different complexes, i.e. PDC, ␣-ketoglutarate dehydrogenase complex, branched-chain ␣-keto acid dehydrogenase complex, and as H-protein in glycine synthase.
Only in P. putida do three different genes code for three
dihydrolipoamide dehydrogenases. E3 is bound to the
subunit-binding domain of BP in eukaryotic PDC and to
the subunit-binding domain of E2 in other complexes. The
structure of B. stearothermophilus E3 bound to the subunit-binding domain of E2 was solved to 2.6-Å resolution
[16]. The interaction between these proteins was shown to
be electrostatic, i.e. between positively charged residues
of the binding domain and negatively charged residues of
E3. The same subunit-binding domain of E2, however,
9
binds E1 at the different site. While E3 interacts with the
N-terminal part of the subunit-binding domain, E1 binds to
the C-terminal part of the same domain. The binding site
for the A. vinelandii E1 was shown to involve not only the
subunit-binding domain but also the E2 inner domain [9].
PDK is a specific kinase that phosphorylates E1 of PDC
and is present in human, rodent, plant, nematode, and fruit
fly. PDK has several isoforms, four in mammalian PDC
(PDK1, PDK2, PDK3, and PDK4), two in plants, and one in
nematode and fruit fly (Table I) (see Ref. 26). PDK, a
serine-specific kinase, phosphorylates three specific serine residues named site 1, site 2, and site 3 based on the
rate of phosphorylation of mammalian E1 [27], isoform II of
A. suum E1, and fruit fly E1. Only two phosphorylation sites
are present in isoform I of A. suum E1 and in Caenorhabditis elegans, and only one site is in plant E1s. Yeast E1
has phosphorylation site 1 in a sequence that can be
phosphorylated by bovine PDK; however, PDK is not detected in yeast. Mammalian PDKs have low homology with
eukaryotic serine protein kinases and are more related to
bacterial histidine protein kinases [28]. Recently the threedimensional structure of rat PDK2 was solved [29]. The
C-terminal domain of PDK2 appeared to be similar to the
nucleotide-binding domain of bacterial histidine kinases.
However, PDK catalyzes the phosphate transfer directly to
the serine residue of the substrate and not through a
histidine residue of the kinase itself as bacterial histidine
kinases do, and hence PDK is related to the eukaryotic
serine kinases and ATPases by catalytic mechanism. Each
PDK isoenzyme is a dimer composed of identical subunits.
PDKs were shown to bind the lipoyl domains of E2 and BP
[6]. In mammalian PDC there are two lipoyl domains in E2
(L1 and L2) and one in BP (L3) (Fig. 3). Different isoenzymes of PDK have preference to the lipoyl domains:
PDK1 binds to L1 and L2; PDK2 and PDK3 prefer L2 but
can also bind L1 less efficiently; and PDK4 binds to L3 and
less to L1 [6]. The number of PDK molecules in the PDC is
as low as one to three molecules per complex. Transfer of
PDK from one lipoyl domain to another is necessary to
achieve phosphorylation of multiple E1 molecules. Moreover, PDK is activated when bound to the lipoyl domain of
E2 (the degree of activation is different for different isoenzymes of PDK). The activation may be achieved by the
conformational changes of PDK when bound to the lipoyl
domain and co-localization of PDK and its substrate E1 on
E2 because activation by a free lipoyl domain is less compared with E2. E2 does not only bind and transfer PDK in
PDC but also regulates the activity of PDK through the
state of the lipoyl groups. PDK activity is stimulated with
the reduction and reduction/acetylation of the lipoyl
groups of E2 occurring during PDC reaction [6]. The stimulation is explained by an allosteric effect of a reduced or
acetylated form of lipoate of the lipoyl domain involved in
the PDK binding. The extent of stimulation depends on the
PDK isoenzyme with the maximum stimulation found for
PDK2.
PDP is a heterodimer composed of two nonidentical
subunits, the catalytic subunit belonging to the phosphatase 2C class of phosphatases and the regulatory subunit,
which is a flavoprotein (Table I). Two isoenzymes of the
catalytic subunit of PDP are identified in mammalian tis-
TABLE II
Tissue distribution of mRNAs of mammalian PDK isoenzymes
Tissue distributions are based on citations in Ref. 7.
Tissue
PDK1
Heart
Skeletal muscle
Pancreas
Liver
Kidney
Testis
Brain
Spleen
Lung
⫹⫹⫹⫹⫹
⫹⫹⫹
⫹
⫹⫹
PDK2
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹
PDK3
PDK4
⫹
⫹⫹⫹⫹
⫹⫹⫹⫹⫹
⫹⫹
⫹⫹⫹⫹
⫹⫹
⫹
⫹⫹⫹
⫹⫹
⫹
⫹⫹⫹
sues (PDP1 and PDP2) with different tissue distribution,
activities, and regulation [7]. The regulatory subunit of
PDP1 affects the sensitivity of the catalytic subunit to
Mg2⫹. Mammalian PDP1 binds to the L2 of E2 with Ca2⫹
playing a bridging role [6].
REGULATION OF PDC
Short-term Regulation of PDC Activity—The central role
of PDC in glucose homeostasis necessitates the high level
of regulation of its activity in mammalian tissues. Regulation of PDC activity within minutes and hours will be referred to as a short-term regulation, while regulation exerted at a transcriptional level within days to weeks is
considered as a long-term regulation. The short-term regulation of PDC by metabolite effectors and hormones is
achieved through phosphorylation of E1 by PDK, leading
to inactivation of E1 (and hence PDC), and dephosphorylation and reactivation is accomplished by PDP (Fig. 1). As
discussed above four isoenzymes of PDK and two isoenzymes of the catalytic subunit of PDP are present in mammalian tissues. Isoenzymes of PDK and PDP have different
specific activities, sensitivity to different effectors, and tissue-specific distribution (Table II) [6, 7]. The highest
amount of PDK1 mRNA is present in heart, and decreasing
levels are present in skeletal muscle, liver, and pancreas.
PDK2 is expressed in many tissues with low amounts in
spleen and lung. PDK3 mRNA is maximal in testis and less
in lung, brain, and kidney. PDK4 mRNA was detected in
skeletal muscle and heart with low levels in lung, liver, and
kidney. A high level of PDP1 is present in skeletal muscle,
while levels of PDP2 are higher in liver and adipose tissue
[7].
The presence of several phosphorylation sites of E1
adds to the complexity of the mechanism of regulation of
PDC activity. In mammalian PDC three phosphorylation
sites are phosphorylated differently by different isoenzymes of PDK [26]. PDK1 phosphorylates all three sites,
whereas PDK2, PDK3, and PDK4 can modify only sites 1
and 2 when PDKs are bound to E1 in PDC. The activities of
PDKs toward site 1 are: PDK2 ⬎ PDK4 ⬎ PDK1 ⬎ PDK3;
the activities for site 2 are: PDK3 ⬎ PDK4 ⬎ PDK2 ⬎
PDK1. In the absence of E2 PDKs are also active. The
maximum activity in the absence of E2 was demonstrated
toward site 1; however, site 2 could be phosphorylated by
PDK4, and site 3 could be phosphorylated by PDK1 in the
absence of E2. Phosphorylation of each site results in E1
inactivation. The mechanism of inactivation of E1 by phosphorylation of three sites is site-specific [30]. Modification
10
of site 1 prevents interaction of the E1 active site with the
substrates of the reaction, pyruvate and more with the
lipoyl domain of E2, whereas phosphorylation of site 3
affects E1 interaction with TPP.
The products of the PDC reaction (and more importantly
the products of fatty acid and ketone body oxidation),
NADH and acetyl-CoA, increase activity of PDK through
reduction/acetylation of lipoyl moieties of the lipoyl domains of E2 to which PDKs are bound. Hence the NADH/
NAD⫹ and acetyl-CoA/CoA ratios determine the portion of
PDC present in active (dephosphorylated) form. The highest stimulation is achieved for PDK2. The least sensitive
isoenzyme is PDK3 [6, 26]. Activities of PDKs also depend
on the ATP/ADP ratio as ADP and phosphate anion are
inhibitors of PDK activity. PDK2 and to a lesser extent
other PDK isoenzymes are inhibited by pyruvate, the substrate of the PDC reaction [6]. Inhibition by pyruvate is
synergistic with ADP inhibition. The mechanism of inhibition was suggested to be formation of the dead end complex PDK-ADP-pyruvate that prevents ADP dissociation
and hence PDK activity [6].
Activities of PDP isoenzymes are regulated by the concentrations of divalent metal ions Mg2⫹ and Ca2⫹ [6]. PDP
is a Mg2⫹-dependent enzyme, and hence changes in Mg2⫹
concentration along with the ATP concentration in the cell
affect PDP activity. PDP2 is less sensitive to Mg2⫹ concentration than is PDP1. Sensitivity of PDP1 to Mg2⫹ concentration changes when it is bound to the regulatory
subunit of PDP. Polyamines (spermine) increase the activities of both PDP2 present alone and PDP1 associated
with a regulatory subunit. Ca2⫹ ions (increased in conditions of energy deficit) elevate activity of PDP1 by facilitating its binding to E2. The short-term hormonal regulation of PDC is achieved by insulin, which increases activity
of PDP resulting in dephosphorylation and activation of
PDC. The signal from insulin is transferred by protein kinase C␦ that translocates to mitochondria where it phosphorylates and activates PDP [31].
Plants have both mitochondrial and plastid PDC, and
only mitochondrial PDC is regulated by phosphorylation
[32]. Interestingly, activity of PDC in plants depends on
light. Photosynthesis and photorespiration cause a decrease in mitochondrial PDC activity due to phosphorylation by the elevated amount of ATP. In contrast, the increase of pH and Mg2⫹ concentration in plastid in light
during photosynthesis enhances plastid PDC activity. Developmental conditions of nematodes are reflected in the
regulation of PDC. Nematodes have two isoforms of E1,
E1-I present in anaerobic adult muscle and E1-II in aerobic
larvae [25]. The two E1 isoforms have different stoichiometry of phosphorylation and inactivation. Three phosphorylation sites in E1-II isoform are modified similarly to
mammalian E1. Anaerobic E1-I isoform has only two phosphorylation sites that are more resistant to inactivation.
This fact together with the reduced sensitivity of nematode
PDK to the elevated NADH/NAD⫹ and acetyl-CoA/CoA
assist in maintaining the PDC activity at a high level in
anaerobic conditions when in the absence of the tricarboxylic acid cycle the products of the PDC reaction are
used for fatty acid biosynthesis.
One wonders why it is critical to have such a tight and
BAMBED, Vol. 31, No. 1, pp. 5–15, 2003
extensive regulation of PDC function with so many phosphorylation sites as well as multiple isoforms of kinases
and phosphatases that are regulated by such a variety of
control factors. This may at first seem like overkill in regulation of PDC! Based on the available information, the
following explanations are considered for such an elaborate regulation of PDC. First, during the transition from the
fed to fasting state, the organism needs to conserve the
three-carbon compounds (such as pyruvate, lactate, and
alanine) from further degradation so that they can be used
for gluconeogenesis (Fig. 5). The phosphorylation mechanism renders the complex completely inactive by phosphorylating even only one of the three phosphorylation
sites present in the ␣ subunit of E1 (two ␣ subunits present
in E1 provide six potential sites for phosphorylation, and
phosphorylation of any one site is sufficient to inactive E1
and hence PDC). There are two possible reasons for the
multiple sites of phosphorylation: (i) the gradual reactivation by dephosphorylation of multisite phosphorylated E1
by the phosphatases during the conditions such as the
transition from the fasted to fed state and (ii) providing a
fall back control to continue to maintain this crucial regulation if one phosphorylation site is modified by genetic
mutation.
Second, the importance of the presence of multiple
forms of specific kinases with differences in their regulation is linked to their tissue-specific distribution. Since all
tissues are not created equal in their roles in maintaining
glucose homeostasis, the distribution of kinases in different tissues (such as increases in PDK2 and PDK4 activities
in liver during fasting and in diabetes) plays critical roles in
regulating the carbon flux through PDC. Additionally, the
sensitivity of PDKs to the changes in the NADH/NAD⫹,
acetyl-CoA/CoA, and ATP/ADP ratios due to increases in
the oxidation of fatty acids (and ketone bodies in extrahepatic tissues after an extended fasting period) not only
speeds up the action of specific kinases but also maintains
control over an extended period.
Third, the changes in metabolites such as NADH, acetylCoA, and ATP in the mitochondria due to increased oxidation of fatty acids, an alternate fuel source for acetylCoA, result in drastic changes in the ratios of NADH/
NAD⫹, acetyl-CoA/CoA, and ATP/ADP in the mitochondria
that allosterically stimulate the activities of most PDKs.
Although the same metabolite changes also regulate the
activity of PDC at one of its component enzymes, the
degree of inhibition exerted is generally not sufficient to
shut off the complex; whereas the very same changes in
the metabolite levels through the allosteric activation of
PDKs amplify the process of inactivation! This type of
amplification control is linked to the availability of other
oxidizable fuels in the mitochondria of different tissues. For
example, the levels of the active state of PDC in the liver
and skeletal muscles are rapidly decreased during the fed
to fasting transition, hence enabling these tissues to oxidize fatty acids and sparing complete oxidation of glucose-derived three-carbon metabolites (Fig. 5). In contrast, the levels of the active state of PDC in the brain is
maintained fairly constant during the fed-fasting-refeeding
transitions under normal dietary conditions (meaning the
capacity of the brain to oxidize glucose to CO2 and water
11
FIG. 5. Changes in plasma hormonal levels, intramitochondrial metabolites, and glucose metabolism
in mammalian liver, skeletal muscle, and brain during the fed and
fasted states. The central role of PDC
in glucose disposal during the fed
state and in glucose sparing during
the fasted state is highlighted. The relative flux of glucose, fatty acids (FAs),
and ketone bodies in the liver, skeletal
muscle, and brain are shown by heavy
and thin arrows. TCA, tricarboxylic
acid cycle; VLDL, very low density
lipoproteins; OAA, oxaloacetate; PEP,
phosphoenolpyruvate; AAs, gluconeogenic amino acids; I, insulin; G,
glucagon.
is not affected during an overnight fast or short fasts of
1–2-day duration) (Fig. 5). This allows the brain to continue
uninterrupted oxidation of glucose for energy production.
However, when the plasma concentrations of ketone bodies are sufficiently increased over an extended period of
fasting (early starvation period) or in uncontrolled diabetes,
the brain is gradually able to oxidize increasing amounts of
ketone bodies. That generates the metabolites (such as
acetyl-CoA and NADH) that reduce PDC activity via their
actions on PDKs and hence spare glucose oxidation while
maintaining energy homeostasis in the brain.
Fourth, the general structural and catalytic similarities of
PDC with the other two related complexes in the mitochondria such as the ␣-ketoglutarate dehydrogenase
complex and the branched-chain ␣-keto acid dehydrogenase complex shed light on the need for regulation or an
absence of regulation of these complexes based on their
importance in the oxidative metabolism. For example,
there is a complete lack of regulation by phosphorylation/
dephosphorylation of the ␣-ketoglutarate dehydrogenase
complex because of its central location in functioning of
the tricarboxylic acid cycle. Since the tricarboxylic acid
cycle is involved in the oxidation of all metabolites gener-
ated from the initial metabolism of carbohydrates, fats,
and amino acids, which are capable of feeding into the
cycle for further oxidation, the elaborate regulation of this
complex would not provide any advantage for fuel-specific
switching in the tissue. In contrast, the regulation of the
branched-chain ␣-keto acid dehydrogenase complex by a
phosphorylation/dephosphorylation mechanism does provide a control for oxidation of the three essential amino
acids (leucine, isoleucine, and valine), which play a pivotal
role in nitrogen metabolism in the body. Thus, it is no
surprise that this complex is regulated by phosphorylation
of one functional site only on its E1 component by a
specific kinase and a specific phosphatase with only a
limited effect, if any, of metabolite-mediated control of this
process. Hence, compared with the elaborate control
scheme of regulation of PDC, the branched-chain ␣-keto
acid dehydrogenase complex is controlled by a less elaborate system for its regulation and may reflect a limited,
specific role of this complex in the scheme of overall
metabolism for energy homeostasis.
Long-term Regulation of PDC Activity—Although the
short-term regulation of PDC by covalent modification is
the key to the maintenance of glucose homeostasis during
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BAMBED, Vol. 31, No. 1, pp. 5–15, 2003
FIG. 6. Schematic presentation of
specific cis-acting elements and
nuclear protein-binding domains of
the proximal promoter regions of
the human PDHA-1 and PDHA-2
genes. ChoRE, carbohydrate-response element.
the transition between the fed and fasted states, alterations in total PDC activity (and hence PDC content) in
response to dietary and hormonal changes over longer
periods (long-term regulation) are also observed. For instance, total PDC activity in liver and adipose tissue increased by ⬃1.5- and 3.5-fold, respectively, in rats fed a
high sucrose diet for 1–2 weeks compared with that of
chow-fed controls [33]. The levels of the component proteins of PDC measured by immunological analysis in livers
from high sucrose-fed animals closely correlated with
changes in total PDC activity [34]. In contrast, total PDC
activity was significantly decreased in livers from high
fat-fed rats. Interestingly, total and active PDC levels were
not affected in heart and skeletal muscle of rats fed the
high sucrose or high fat diets. Hypothyroidism in the rat
caused about one-third reduction in total PDC activity in
the liver, and this change was correlated with a similar
reduction in amounts of E1 proteins (the other components
of PDC were not measured) [33]. These stable changes in
PDC activity are explained by long-term regulation occurring at the transcriptional level. Several genes encoding for
PDC components were shown to be regulated through
different cis-acting elements in their promoter regions [4].
Interestingly, culturing of hepatoma HepG2 and insulinoma MIN6 cells in medium containing high glucose levels
resulted in increased total PDC activity, and this increase
was correlated with the levels of E1␣ and E1␤ mRNA levels
[35]. The elevated glucose presumably increases transcription of E1␣ and E1␤ in situ due to the presence of
carbohydrate-response elements in the promoters of the
E1␣ and E1␤ genes [35].
The major determinant of the PDC activity under different physiological conditions is the amount of PDK. An
increase in the expression of PDK isoforms in different
pathophysiological conditions is well documented and intensively studied [7]. Of the four isoenzymes of PDK, the
highest degree of regulation is observed for PDK4 and to
a lesser extent for PDK2. However, the abundance of
PDK2 in all tissues makes its regulation very important.
Starvation increases the amounts of PDK2 and PDK4 in
liver, kidney, lactating mammary gland, and fast-twitch
muscle and PDK4 levels in heart and slow-twitch muscle
[7]. During starvation, mRNA, but not the protein of PDK4,
is higher in white adipose tissue and brain. Brain is the only
tissue in which PDC activity does not change during fasting because brain depends on glucose as a major source
of energy. Diabetes increases the expression of PDK4 in
heart and skeletal muscle and slightly increases the expression of PDK2 in skeletal muscle [7]. Insulin treatment
of diabetic animals rapidly reverses the changes in PDK4
activity [7]. In adipose tissue PDC activity is highly regulated by insulin, which activates PDP after refeeding of
starved animals or by administration of insulin to diabetic
animals.
Hyperthyroidism results in increased amounts of PDK4
protein in heart and muscle and of PDK2 protein in fasttwitch muscle [36]. Starvation, diabetes, and a high fat diet
are conditions leading to higher levels of free fatty acids.
Fatty acids are known to activate peroxisome proliferatoractivated receptor ␣ (PPAR␣). An agonist of PPAR␣ (WY14,643) is found to up-regulate expression of PDK4 in rat
skeletal muscle and liver and in cultured hepatoma cells
[7, 11]. Glucocorticoid (dexamethasone) causes the same
response in hepatoma cells. Both compounds increased
PDK4 mRNA without altering its stability. Insulin, on the
other hand, diminishes the levels of PDK4 and PDK2
mRNAs. It is proposed that elevated levels of fatty acids in
certain physiological conditions increase expression of
PDK4 and PDK2 through activation of PPAR␣. The expression of PDK4 and PDK2 is suggested to be regulated to
some extent by the drop in insulin [37]. Recently expression of PDP2 was reported to decrease in diabetes and
starvation, resulting in lower rates of dephosphorylation
[38]. Up-regulation of PDK expression and down-regulation of PDP expression leads to hyperphosphorylation
(phosphorylation on multiple sites) of E1 and inactivation of
13
PDC. Phosphorylation of any one of the E1 phosphorylation sites (site 1 is modified first in the cell) is sufficient for
inactivation; however, hyperphosphorylation results in a
longer duration of inactivation. Complete dephosphorylation of more than one site requires a longer period for
reactivation after changing physiological conditions.
REGULATION OF PDC GENES
Gene organization and functional analysis of the promoter-regulatory regions of the three human PDC genes
(E1␣, E1␤, and E3) and the mouse Pdha-2 gene have been
reported [33]. Two genes have been found to encode the ␣
subunit of the E1 component of PDC in most mammals.
Human PDHA-1 and rodent Pdha-1, both localized on
chromosome X, are expressed in somatic cells, whereas
human PDHA-2 (on chromosome 4) and rodent Pdha-2 (on
chromosome 19, band B in the mouse) are expressed in
testis only [24]. Of interest are the promoters of the
PDHA-1 and PDHA-2 (and Pdha-2 of mouse) genes (Fig.
6). The presence of several cis-acting elements as well as
seven distinct nuclear protein-binding domains in the
⫺763/⫹33 promoter region of the human PDHA-1 gene
have been identified by sequence analysis and DNase
footprinting analysis, respectively [39]. A detailed mutational analysis of the ⫺100 bp promoter region of the
human PDHA-1 gene indicated that the overlapping CRE/
TATA-like sequence (⫺24 to ⫺34 bp) functioned as the
CRE-like sequence and had a minimal affinity for the
TATA-binding protein, indicating this promoter functions
as a TATA-less promoter. Furthermore, it was observed
that the CRE site is functionally important, and the GCGG
sequence at the ⫺52 to ⫺49 bp region was indispensable
for the minimal promoter activity.3 A transgene containing
⫺763/⫹33 bp of the human PDHA-1 promoter/structural
chloramphenicol acetyltransferase (CAT) gene was responsive to diet-induced expression of CAT activity in
kidney and adipose tissue but not in the brain of transgenic
mice [40]. However, this transgene construct did not contain sufficient sequence information to direct regulated
CAT expression in several tissues. Subsequently it was
shown that the ⫺1.7/⫺2.2 and ⫺5.2/⫺5.9 kb regions of
the PDHA-1 promoter may possess negative regulatory
elements that are likely to function in a tissue-specific
manner. In another study, the ⫺100 bp promoter region of
the human PDHA-1 gene was shown to be responsive to
glucose in transiently transfected HepG2 cells. Mutational
analysis of the human PDHA-1 gene promoter region uncovered the presence of two sequences from ⫺78 to ⫺73
bp (CCCCTG) and from ⫺8 to ⫺3 bp (GCGGTG) that are
responsible for the glucose-induced increases in promoter
activity [35]. Of these two sequences representing new
variations of the carbohydrate-response element identified
in other glucose-regulated genes, the former sequence
(CCCCTG) exhibited a large effect on promoter activity. It
should be noted that glucose-mediated stimulation of human PDHA-1 promoter was relatively low (3–5-fold only)
compared with that of the L-pyruvate kinase gene and the
S14 gene. This could be due to the lack of two copies of
3
J. Tan, R. Dey, H.-S. Yang, and M. S. Patel, unpublished
observations.
the perfect CACGTG sequence separated by an ideal fivenucleotide spacer in the human PDHA-1 promoter [35].
In contrast to regulation of expression of the PDHA-1
gene promoter, expression of Pdha-2 in mice is tightly
regulated so that its transcription occurs only during specific stages of spermatogenesis [24, 41]. Extensive analysis of a core mouse Pdha-2 promoter region has revealed
four regions of protection. Two of these regions contain
potential motifs for Sp1 and activating transcription factor/
cAMP-response element-binding protein (ATF/CREB) and
the remaining two binding sites harbor novel cis-motifs
designated MEP-2 (mouse E1␣ promoter site 2) and
MEP-3. MEP-2 forms a complex with a putative testisspecific binding factor (␶-MEP-2BF) [41]. Analysis of the
proximal promoter of human PDHA-2 gene showed the
presence of four testicular nuclear protein-binding domains within the ⫺106/⫹9 region and three cis-acting
motifs, namely MEP-2, Sp1, and Ets (Fig. 6). Functional
studies also identified the presence of both enhancer and
repressor elements (Ets) in the PDHA-2 promoter that are
only expressed in mature sperm [42].
MOLECULAR DEFECTS IN PDC COMPONENTS
PDC deficiency is one of the major genetic disorders
resulting in congenital lactic acidosis [43]. More than 150
cases of PDC deficiency have been reported with heterogeneous clinical manifestations that are limited largely to
the central nervous system due to the brain’s dependence
primarily on glucose oxidation for energy production [4, 9,
43, 44]. Given that the human PDC is composed of several
different gene products, a genetic defect involving any one
of them (except PDKs) could lead to the impairment in its
function. Approximately 80% of all reported cases of PDC
deficiency involve defects of the E1 component, all of
which are found in the ␣ subunit, and the remainder are
distributed among the other components except PDKs. A
PDK deficiency would not cause lactic acidosis, and the
presence of four isoenzymes with overlapping tissue distribution may compensate for deficiency of one isoform.
Perhaps only patients with partial defects in which there is
some residual PDC activity can survive. This point is well
illustrated by null mutation of the Pdha-1 gene in mice
using homologous recombination techniques resulting in
embryonic lethality [45]. Since the human PDHA-1 gene is
localized on chromosome X, the pattern of inactivation of
chromosome X in different cells and tissues in affected
female patients contributes to the observed variability in
residual PDC activity with associated clinical manifestations. In summary, PDC deficiencies further illustrate the
importance of PDC in glucose metabolism in the developing mammalian brain.
A large number of specific mutations associated with the
PDC components have been identified, almost all of which
are within the E1␣ coding region [4, 9, 43, 44]. Interestingly, about one-half of E1␣ mutations result from missense codon changes preserving the primary structure
and some residual catalytic function of the protein. Several
of these missense mutations (at Met-181, Pro-188, Arg349, His-15, and Arg-234, based on numbering of the
residues in mature ␣ subunit protein) were recreated by
overexpressing human mutant E1 in E. coli to characterize
14
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the biochemical basis for altered function [21, 22]. Interestingly, many of these mutations alter the binding of TPP
in the E1 active site. To minimize recurrence of lactic
acidosis, rational treatments have included the use of ketogenic diets (high in fat content and extremely low or even
absent in carbohydrates in a diet), daily administration of
large amounts of thiamine (to increase intramitochondrial
TPP levels for some mutant proteins), and administration
of dichloroacetate (to inhibit PDK and possibly increase
the residual PDC in a more active state) [43]. Unfortunately,
all of these interventions have met with variable and very
limited successful clinical outcomes.
CONCLUSION
The central role of PDC in glucose homeostasis has
been recognized for several decades and has been emphasized in textbooks of biochemistry as a pacesetter for
glucose metabolism during fed/fasting/refeeding transitions normally occurring during the 24-h period. The carbon flux through PDC is meticulously controlled by an
elaborate mechanism via phosphorylation/dephosphorylation that is not only extremely sensitive to intramitochondrial metabolic parameters such as the ratios of NADH/
NAD⫹, acetyl-CoA/CoA, and ATP/ADP but also to the
elaborate interactions among the component proteins of
PDC such as E2, PDKs, PDPs, and E1. We now know that
protein-protein interactions in PDC contribute significantly
to the elaborate regulation. Moreover the structural and
functional diversity of the regulatory components, PDKs
and PDPs, as reviewed in this article, illustrate further the
complexity necessary for tissue-specific regulation of PDC
and its importance in the maintenance of glucose homeostasis in mammals. Finally, genetic defects of PDC, although rare, further illustrate the importance of PDC in
both glucose and energy homeostasis not only in adult life
but also from early embryonic development.
Mammalian life does not exist in the complete absence
of PDC!
Acknowledgments—We are grateful to Dr. Richard Hanson of
the Department of Biochemistry, Case Western Reserve University School of Medicine and Dr. Murray Ettinger of this department for critical reading of the manuscript and helpful
discussions.
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