Download PDH02 - OSU Biochemistry and Molecular Biology

The-Keto Acid
Dehydrogenase Complexes
Prepared by
Franklin R. Leach
Department of Biochemistry and Molecular Biology
Oklahoma State University
Stillwater, OK
9/15/02
The-Keto Acid Dehydrogenase Complexes
A Brief Review 2002
Contents
Introduction
Background
Complex Structure
Regulation
Whole Genomes
Components Parts
E1
E2
X
E3
Relation of Lipoic Acid to Oxidative Damage
Relation to Medicine
Therapeutic potential
Ischemic heart disease
Lactic acidosis and related diseases
Maple syrup urine disease and branched chain-related diseases
Leigh's necrotizing encephalomyelopathy
Alzheimer's disease
Primary biliary cirrhosis
Systemic sclerosis
Lipoic Acid Activation and the Lipoamidase Reaction
Recent Results on the Regulatory Enzymes
Web Connections
Literature Cited
Appendix
Symposium Honoring Lester Reed - A Tribute
Recollection - From lipoic acid to multienzyme complexes
Introduction
Remarkable progress in understanding the function and mechanism of action of the keto acid dehydrogenases has been made in the last 50 years. These complexes are the
classical example of a multienzyme complexes. A conference on "-Keto Acid
Dehydrogenase Complexes: Organization, Regulation, and Biomedical Aspects" (1), held
November 16-18, 1988 in Austin, Texas to honor Professor Lester J. Reed, summarized
much of the early progress. This conference celebrated Dr. Reed's 65th birthday. My
interest in this topic is because I did my dissertation research with Lester Reed (1953-57)
and worked on the lipoic acid activating enzyme system. A Tribute to Lester Reed
written by Tom Roche, Head of the Department of Biochemistry at Kansas State
University and a former Reed postdoc, is in the appendix. It shows the extent to which
Reed's laboratory has contributed to our understanding of the -keto acid
dehydrogenases. The complexes have been isolated, their composition and organization
determined, their base sequences are being elucidated, and their amino acid sequences
and crystallographic patterns are being deduced. The mechanisms of regulation of the
activities of these complexes have been established. A FASEB symposium reviews the
topic (2). Several other reviews have appeared (3-6). At the 1994 ASBMB meeting in
Washington, DC, Reed was given the ASBMB-Merck Award and presented a review of
"A Trail of Research: From Lipoic Acid to Multienzyme Complexes". An American
Institute of Nutrition Symposium "-Keto Acid Dehydrogenase Complexes: Nutrient
Control, Gene Regulation, and Genetic Defects" was also held in 1994. A review paper
from that symposium has appeared (7). Reed has recalled "From lipoic acid to multienzyme complexes" for Protein Science (7a). (See appendix). For the Journal of
Biological Chemistry Centennial collection Reed (7b) traced "a trail of research from
lipoic acid to -keto acid dehydrogenase complexes".
The molecular understanding of the -keto acid dehydrogenases began in the 1950s with
the isolation and determination of the structure of lipoic acid (8-10). The next key finding
was the enzymatic mechanism by which lipoic acid was converted to the enzyme-bound
functional form (11, 12). From that point until the current application of molecular
biology techniques the emphasis of study has been on the isolation, characterization,
determination of structure/function relationships, and regulation of the -keto acid
dehydrogenase complexes (13-15). This has matured into recent and current
determinations of the amino acid and base sequences for many components of these
complexes (see refs. 1-7).
The reaction (sum) catalyzed by the -keto acid dehydrogenases is:
TPP, Lipoic acid, FAD
RCOCO2H + NAD+ +CoASH
&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;> RCO-SCoA + CO2 + NADH +H+
The component steps in this overall reaction are:
CH3COCO2- + E1TPP + H+ <==> CO2 + E1 CH3C(OH)=TPP (1)
E1 CH3C(OH)=TPP + E2_LipS2 <==> E1TPP + E2-Lip(SH)-S-COCH3 (2)
E2-Lip(SH)-S-COCH3 + CoASH <===> E2_Lip(SH2) + CH3COSCoA (3)
E2-Lip(SH2) + E3FAD <===> E2-LipS2 + Dihydro-E3FAD (4)
Dihydro-E3FAD + NAD+ <===> E3FAD + NADH + H+ (5)
Lewisite (CHCl=CHAsCl2) is a poison gas that was synthesized too late to be used in
World War I. Rudolph Peters headed the Oxford University laboratory that searched for
antidotes to chemical warfare agents. They developed BAL (British anilewisite, 2,3
dimercaptopropanol, and for security reasons, OX 217) in 1940. The target of the
arsenical was lipoic acid. See (16).
Lipoic acid, 1,2-dithiolane-3-pentanoic acid (6,8-dimercapto-octanic acid), functions in
transacylation, redox, and transport reactions. It plays a central role in oxidative
metabolism: the oxidative decarboxylation of pyruvate, branched chain amino acid
metabolism, glycine decarboxylation, and in the citric acid cycle. Lipoic acid is formed
from octanoic acid via an enzymatic S-insertion. Additional details have been learned
about lipoic acid synthesis. The question of why mitochondria synthesize fatty acids has
been answered: the synthesis of lipoic acid. Wada, Shintani, and Ohlrogge (17)
established that pea mitochondria can acyl carrier protein and the enzymes to synthesize
fatty acids. Radioactivity from labeled malonic acid was found in the H protein, a lipoylcontaining enzyme involved in glycine metabolism. In Saccharomyces cerevisiae Brody,
Oh, Hoja, and Schweizer (18) found that absence of the yeast gene ACP1, resulted in a
decreased lipoic acid content. They conclude that the mitochondrial ACP is invovled in
the synthesis of octanoate which is a lipoic acid precursor. Jordan and Cronan (19) found
that the acyl carrier protein of lipid synthesis could donate lipoic acids to the pyruvate
dehydrogenase complex in both E. coli and mitochondria.
Self, Tsai, and Stadtman (20) have prepared the selenotrisulfide derivatives of lipoic acid
and lipoamide. The selenotrisulfide derivative of lipoic acid was an effective substrate for
thioredoxin reductase. The lipoamide derivative was reduced by dihydrolipoamide
dehydrogenase. The selenium analogs of lipoic acid had been used earlier by Reed,
Morris, and Cronan (21) to isolate E. coli mutants. Replacement of either the C-6 or C-8
sulfur atom with Se gave lipoic acid derivatives with unaltered biological properties. The
replacement of both S's with Se producing selenolipoic acid that was a growth inhibitor
of E. coli. When radioactive 75Se was used, the selenolipoic acid was found incorporated
in the -ketoacid dehydrogenases. Resistant mutants were isolated. These mutations were
traced to lipoate-protein ligase and to an unknown function in the synthesis of lipoic acid.
The E. coli LipA is a lipoyl synthase that forms lipoyl groups from octanoyl-ACP (22).
This enzyme as well as biotin synthase contains (2Fe-2S) centers that can combine to
form a (4Fe-4S) center. The iron-sulfur center is involved in the formation of a C-S bond
(23). The enzymology of sulfur activation during thiamine and biotin biosynthesis has
been discussed by Begley, Xi, Kinsland, Taylor, and McLafferty (24).
The reactions for lipoic acid activation and its covalent attachment to the pyruvate
dehydrogenase complex are:
E1 + ATP + Lipoic Acid &emdash;&emdash;&emdash;&emdash;&emdash;> E1lipoyl-AMP + PP
E1-lipoyl-AMP + E2 &emdash;&emdash;&emdash;&emdash;> Lipoyl-E2 + AMP +
E1
Lipoyl-E2 + apo-PDHC
&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;> Lipoyl -PDHC + E2
Where E1 and E2 are the two enzymes of the Streptococcus faecalis lipoic acid-activating
system. The lipoic acid is covalently bound to the -amino group of lysines of E2 of the
pyruvate dehydrogenase complex (PDHC). PDHC consists of three distinct enzymes
designated as E1, E2, and E3 - note that the distinction between the lipoic acid-activating
enzymes and the -keto acid dehydrogenases made by a subscript number for the former
and an on the line number for the latter.
The reaction involved in lipoic acid removal (lipoamidase reaction) is:
Lipoyl-PDHC + H2O
&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;> apoPDHC + Lipoic acid
Background
The -keto acid dehydrogenases are large enzyme complexes that serve essential roles in
metabolism (25). The pyruvate dehydrogenase (PDHC) provides the link between
glycolysis and the citric acid cycle and produces acetyl-CoA for the citric acid cycle and
acetyl groups for acetylcholine synthesis; in omnivores 50-80% of metabolism goes
through the PDHC (26). The -ketoglutarate dehydrogenase functions in the citric acid
cycle. The branched chain -keto acid dehydrogenase is important in regulation of
nitrogen metabolism (26). These enzyme complexes involve five cofactors: thiamine
pyrophosphate (TPP), lipoic acid (LA) in the form of enzyme-bound lipoamide (the
amide between lipoic acid and the -amino group of lysine), nicotinamide dinucleotide
(NAD+), coenzyme A (CoA), and flavin adenine dinucleotide (FAD) shown in Fig. 1
where E1 is the carboxylase (in the case of pyruvate, pyruvate dehydrogenase, EC #
1.2.4.1), E2 is the transacylase-reductase (in the case of pyruvate, dihydrolipoamide
acetyltransferase, EC # 2.3.1.61), and E3 is dihydrolipoamide dehydrogenase (EC #
1.8.1.4). The activity of the mammalian forms of these enzymes is regulated by inhibition
by products and by a phosphorylation-dephosphorylation cycle (involving insulin among
other factors) (14).
There are a series of 5 reactions indicated by the Arabiac numbers that constitute the
complete reaction sequence. Reaction 1 is the decarboxylation of pyruvate with the
production of CO2 and hydroxyethythiamine pyrophosphate. The hydroxyethylthiamine
pyrophosphate is then oxidzed to acetylthiamine pyrophosphate with the reduction of
lipoic acid. The acetyl group is then transferred to lipoic acid yielding the 8-S-acyl
compound in reaction 2. The third reaction is the transfer of the acyl group to CoA
yielding the acyl CoA derivatives. Lipoic acid is now in the dihydro form and must be
reoxidized. This occurs in reaction 4. The reduced dihydrolipoyl dehydrogeanse is then
oxidzed in reaction 5 producing NADH.
TPP
FAD
Lipoic Acid
NAD+
CoA
The following scheme shows the reaction mechanism in detail.
Complex Structure
There are two polyhedral forms of E2: cubic and dodecahedral (8). The components of
the mammalian complexes and E. coli are summarized in Table 1.
Enzyme
Abbreviation
Mr x 10-6
Subunits
#
#
#
Mr x 10-3
Part A. Mammalian
Bovine heart
Native complex
PDHC
8.5
Pyruvate dehydrogenase
E1
0.154
E1
2
41
E1
2
36
60
60
6
50
2
55
E2
3.1
X
Dihydrolipoyl dehydrogenase
60
E3
0.11
1
12
Kinase
Phosphatase
PDHk
0.1
PDHk
1
48
PDHk
1
48
PDHp
1
97
PDHp
1
50
PDHp
0.15
Part B. Bacteria
E. coli
Native complex
PDHC
4.6
Pyruvate dehydrogenase
E1
0.19
2
99
24
Dihydrolipoyl transacetylase
E2
1.7
24
66
24
Dihydrolipoyl dehydrogeanse
E3
0.112
2
where k is for kinase and p is for phosphatase.
The amino acid sequence of protein X differs from that of E2, but both contain acetylable
lipoamide. Protein X may contribute to assembly of the complex (27). Protein X is also
called E3BP now that its function has been established
There is an unique structural organization of the Saccharomyces cerevisiae pyruvate
dehydrogenase complex (28). The Reed group used truncated E2, BP and various
physical techniques to determine the arrangement. The showed that there were 12 large
openings in the E2 core multimer that permitted entrance of BP into the central cavity.
Various model structures are depicted.
Regulation
The -keto acid dehydrogenase complexes are regulated by end-product inhibition by
NADH and the appropriate acyl CoA. In addition there is regulation by phosphorylationdephosphorylation (14,15). This covalent modification cycle is in turn regulated by many
components, as is shown in Fig. 2; this occurs for the pyruvate and branched chain
ketoacid complexes. The pyruvate dehydrogeanse complex of yeast is regulation by
phosphorylation (29). Olson and his group at UT San Antonio have reviewed the
regulation of pyruvate dehydrogenase multienzyme complex in the Annual Review of
Nutrition (30).
51
24
Figure 2. Regulation of the mammalian and yeast pyruvate dehydrogenase complexes by
phosphorylation/dephosphorylation.
Whole Genomes
A cluster of genes that encode the branched-chain -keto acid dehydrogenase from
Streptomyces avermitilis has been cloned and sequenced (31). ORF1 has E1 with 1,146
nucleotides encoding a 381 amino acid protein of MW 40,969 Da. ORF2 (E1) 1,005
nucleotides would code for a 334 amino acid protein of MW 35,577. The inner genic
distance is 73 nucleotides. The ATG start codon of ORF3 overlaps the stop codon of
ORF2; ORF3 has part of an E2-like sequence. The sequence and organization of the
genes encoding enzymes involved in pyruvate metabolism in Mycoplasma capricolum
has been analyzed (32). Three operons were found: 1) naox encoding a NADH-oxidase
and lplA coding for lipoyl protein ligase, 2) odpA for E1 and odpB for E1, and 3)
odp2 encodes E2 with a single lipoyl domain and dldH a modified E3 that contains a
lipoyl domain.
The cloning, structure, chromosomal localization, and promoter of human 2-oxoglutarate
dehydrogenase gene has been reviewed by Koike (33). The cDNA contains a 3006-bp
open reading frame encoding a 40-amino acid leader peptide and a 962-amino acid
mature protein with Mr of 108,878. There are 22 exons spanning 85 kb. The gene is
located on chromosome 7 at p13-p14. There are two 10-bp cis-acting elements and two
trans-acting elements with a nuclear factor binding to region -63 to -24 that includes the
two cis-acting elements involved in the control of synthesis.
The gene and subunit unit organization of the bacterial pyruvate dehydrogenase
complexes has been reviewed by Neveling, Bringer-Meyer, and Sahm (34).
Componet Parts
E1. The E1 component contains TPP and catalyzes the decarboxylation of the -keto
acid with the generation of reduced and acylated E2. A tightly bound enzyme
intermediate in the process is 2-(1-hydroxyethylidene)-thiamine pyrophosphate when the
substrate is pyruvate (35). The nucleotide sequence for the ace E gene of Escherichia coli
has been determined (36). The ace E structural gene contains 2,655 base pairs coding for
885 amino acids excluding the initiator. The relative molecular mass of 99,474, the
amino-terminal residue, and carboxyl-terminal sequence predicted from the nucleotide
sequence are in excellent agreement with published information on E1. The upstream
gene A produces protein A of Mr 27,049 which has the helix-turn-helix structure
characteristic of a positive regulator (37).
There is little similarity between the sequences of the E1 enzymes of E. coli and bovine
heart. The amino acid sequences of the E1b subunit [the  subunit of E1 for the branch
chain complex] of rat liver (30), the E1b subunit of human liver (39), the E1p subunit
of human liver (40-42), the E1b subunit of the bovine liver (43), the E1b and E1b of
Pseudomonas putida (44), and the E1p subunit of human liver (42) have all been
determined from cDNAs. All three reported human E1 cDNA sequences have
significant differences still to be be resolved (4). The ace E gene encoding the E1 for
pyruvate (36) and the suc A gene (45) encoding the 2-oxoglutarate dehydrogenase have
been sequenced for E. coli. The yeast E1p has 333 amino acids and 36,486 kD (46). A
cDNA has been cloned and its amino acid sequence deduced for the E1p from
Arabidopsis thaliana (47). It has about 50 % sequence identity and the phosphorylation
site and active site cysteines are conserved.
Tripatara, Korotchkina, and Patel (48) analyzed human point mutations in E1 and found
R349 is critical for activity, M181 is involved in TPP binding, and P188 is necessary for
structural integrity of E1.
The crystal structure of 2-oxoisovalerate heterotetrameric (22) E1 has been solved (49)
for the enzyme from Pseudomonas putida. This is available as PDB file 1QS0. The TPP
cofactor is bound at the phosphate end by the -subunit and the aminopyrimidine end by
the -subunit. The amino acid around the binding site include Y133, R134, G182,
L184, D213, A215, N242, W244, I246 H312, I60', Y88', and H131'. The
lipoyl moiety of the E2 visit either H312 or H131', residues that are probably involved
in the catalytic mechanism.
The determination of the E1 structure allows for the first time the construction of a model
of the 2-oxo acid dehydrogenase multienzyme complexes. The core in P. putida
branched-chain dehydrogenase is a cubic arrangement of 24 E2 subunits. During a
functional cycle, the lipoyl domain swings between the three enzyme components
communicating among the three active sites. At the beginning of the cycle, the disulfide
at the tip of the lipoyl domain is in the S-S or oxidized form. The 2-oxo acid is
decarboxylated by E1 using the TPP cofactor. The substrate is oxidized to an acyl group
and the lipoyl is reduced and then acylated. The CoA enters the active site of E2 from
inside the complex and then accepts the acyl group. The lipoyl moiety is fully reduced. It
is in turn oxidized by E3. See
http://www.bmsc.washington.edu/people/hol/WimFigs5.html for the illustrations.
The crystal structure of the human branched-chain -ketoacid dehydrogenase has also
been solved at a resolution of 2.7 Å (50). This E1b is a 170 kDa 22 heterotetramer.
The  subunit is a 45.5 kDa protein containing 400 amino acid residues and the  subunit
is a 37.8 kDa protein. This structure differs from the P. putida one by having a 30-resiude
N-terminal tails that intertwin in a firm handshake whereas the P. putida structure extents
to opposite sides on the tetramer far from crossing paths.
There are two K+ binding sites involving L164, T165, Q167, S161, and S162 for
the first and G128', N183, L130, C178, T131, and D181.
The small C-terminal domain of the human  subunit is 16 residues lower than the
counterpart from P. putida this gives a longer last helix and an irregular tail. There is an
important mutation in this region Y393N- causes one form of MSUD. The TPP binding
site involved E76', S162, Y102', A195, G194, L74', Y113, R114, R220,
H291, and I226

E2. The E2 component is the central part of the complex and both E1 and E3 bind to it.
E2 contains covalently bound lipoyl moieties and participates in the generation of the
acyl groups and their subsequent transfer to CoA. Three domains are defined in E2 as
seen in Fig. 3 below There are one, two, or three lipoyl domains each consisting of about
100 amino acid residues at the N-terminus depending on the E2 (51). The domains are
joined by flexible regions rich in Ala and Pro. The NMR spectrum of a 32-residue
synthetic peptide corresponding to the flexible region is similar to those of the intact
complex (52). Thus, the three flexible regions are exposed to solvent and enjoy
considerable conformational flexibility. There are separate domains on E2 for the binding
E1 and E3 (53). The acyl transferase activity is located toward the C-terminus. The lipoyl
moieties are bound through the -amino group of lysine and there is considerable
sequence conservation around the actual lipoic binding sites among the various E2s
consensus sequence for 12 different lipoyl domains from E. coli (54), Azotobacter
vinelandii (55), human and bovine branched-chain dehydrogenase (56), bovine kidney
(57), bovine heart (58), and chicken liver H protein (59) is:
L4E9S5D9 K12A10S8M6 E7V6P8
The specific peptide sequences on E1 that are phosphorylated are shown below (14).
Tyr-His-Gly-His-Ser-Met-Ser(P)-Asn-Pro-Gly-Val-Ser(P)-Tyr-Arg
Tyr-Gly-Met-Gly-Thr-Ser(P)-Val-Glu-Arg
.
Figure 3. The domains of the E2 enzymes.
The actual sequences for several enzymes is shown in Table 2 below.
Table 2. Amino Acid Sequences Among the Lipoyl Moities.
Escherichia coli (40)
E2pL 1 E Q S L I T V E G D K* A S M E V P S P Q A
E2pL 2 E Q S L I T V E G D K* A S M E V P A P F A
E2pL 3 E Q S L I T V E G D K* A S M E V P A P F A
E2oL 1 D E V L V E I E T D K* V V L E V P A S A D
Azotobacter vinelandii (41)
E2p---L 1 Q G L V V L E S A K* A S M E V P S P K A
E2pL 2 Q S L I V L E S D K* A S M E I P S P A
E2p------L 3 Q S L I V L E S D K* A S M E I P S P A A G
Human and bovine (42)
E2bL 1 F D S I C E V Q S D K* A S V T I T S R Y D
Bovine kidney (43)
E2oL 1 I E T D K* T S V Q V P S P A N G
Bovine heart (44)
E2pL 1 V Q T D K* A T V G F
E2p-----------L 2 K* A T I G F
Chicken liver H protein 45)
D D E F G A L E S V K* A A S E L Y S P L T
NMR analysis has been applied to the lipoyl domains (60 and 61). There are two
antiparallel -sheets of four strands each. The lipoyl-lysine residue is found in a type-I
turn connecting two -strands. There is a high structural similarity of the lipoyl domains
in spite of only 25 % sequence identity.
The inner lipoyl domain of E2 is invovled in the interaction of pyruvate kinase with the
complex (62). The lipoyl-lysine residues have been postulated to swing between E1 and
E3 subunits while accomplishing their functions. X-ray crystallographic evidence from
an examination of the Bacillus stearothermophilus complex structure has supplied
additional information for a model of Perham and Hol and their colleagues (63) that
shows how the lipoyl group can visit the active sites of the E2 and E3 during catalysis.
The lipoyl domain bearing subunit, E2, serves as the central protein in forming the
multienzyme complex, communicates between subunits, is involved in active-site
coupling, conformational mobility, substrate specificity, and metabolic regulation (64).
Jones, Stott, Reche, and Perham (65) found that the lipoyl domains of the E2 subunit
undergo conformational changes when interacting with their homologous E1 but not
heterologous E1s. It is evident that recognition of the protein domain is the ultimate
determinant of whether reductive acylation of the lipoyl group occurs, and this is ensured
by a mosaic of interaction with the E1.
Roche's group (66) evaluated the contribution of particular amino acids of the L2 lipoyl
domain of human PDHC. The specificity loop contains L140, S141, and T143 whose
mutagenesis influencing enzyme activity. Other residues that markedly reduce activity
are E162, D172, A174, and E179. The lipoylated K is K173. The influential residues are
spread over >24Å of one side of the L2 domain and this side would support extensive
contacts between the E1 and L2 domain. Thus surface residues contribute to the unique
surface patterns that enable recognition.
The structure of the lipoyl domains has been determined by multidimensional NMR.
Perham and his group (67) have concluded that there is a greater restriction in the motion
of the lipoyl-lysine swinging arm of the E. coli pyruvate dehydrogenase complex than
previously thought. Reductive acetylation of the lipoyl moiety gave larger chemical shifts
than expected and multiple resonant forms. These observation imply a change in
conformation upon acetylation and multiple conformations which may stabilized this
catalytic intermediate.
The E2 of maize pyruvate dehydrogenase complex contains a single lipoyl domain (68).
There are two distinct E2s 50-54 kDa and 76 kDa. Arabidopsis thaliana aslo contains a
single lipoyl domain E2 suggesting that all plant mitochondrial PDHs contain an E2 with
a single lipoyl domain.
The nucleotide sequences have been determined for aceF encoding the E2p of E. coli
K12 (69), the sucB encoding the E2o of E. coli K12 (70), the E2b of human and bovine
(56), the E2b from placenta (71), the E2p of human (72), the E2b of P. putida (73), and
the E2p of A. vinelandii (55). The amino acid sequence of the lipoyl domain has been
determined for Bacillus stearothermophilus (74). The E2 inner core domain of E2s
(bovine E2b, human E2p, E. coli E2p, and E. coli E2o) is conserved (75). The crystal
structure of the catalytic domain of A. vinelandii E2p has been determined (76). His
B610, Ser C558, Tyr B608, Ile C571, Phe C568, and Leu C580 are closer to the CoA
binding site. Further details have been published (77). CoA and lipoate are found in
extended conformation at the two opposite entrances of the 30 Å long channel which runs
at the interface between two 3-fold-related subunits and forms the catalytic center. The
reactive groups of both (-SHs) are within hydrogen bond distance of the side chain of His
610. There is suggestion of a direct hydrogen bond between Ser 558 to one of the two
peptide bonds in CoA. Site-directed mutagenesis studies (78) have revealed that in
bovine E2b His 391 and Ser 338 chagnes modified catalytic activity. Ala 348 presumably
contacts CoA and plays a key role in the substrate preference.
The solution structure of the lipoyl domain of the 2-oxoglutarate dehydrogenase complex
from Azotobacter vinelandii has been determined (79). The lipoyl domains are solvent
exposed. A recognition of the lipoyl domain-containing surface loop underlies the
substrate channelling in the PDHC (80). Trp-22 plays a central role in anchoring two
four-stranded  sheets which positions the lipoyl attached to lys-43 at the tip of an
exposed loop (81).
The flexibility of the linker segment of E2 is thought to be a detriment toward
crystallization. Smaller parts of E2 such as 24-mer and 60-mer have been analyzed using
NMR and x-ray crystallography. The E2o of E. coli occurs as trimer when expressed with
a C-terminal [His6] tag (82). Using molecular replacement the structure has been solved
to 3.0 Å resolution. The conserved (5 sequences) amino acids in the E2o lipoyl domain
are P-X3-ES-X13-G-X5-E-X4-IETDK-X3-V-X5-G and for the catalytic domain part 1 are
M-X-R-X-R-X3-A-X-RL-X-E and part 2 D-X3-AV-X4-GLV-X-PV-X-R.
X, aka E3BP. Associated with the E2 core is another lipoyl-containing component,
Protein X, that undergoes reduction and acetylation (83). The region of protein X that
contains the lipoyl moiety is structurally and antigenically related to the lipoyl-bearing
portions of E2. However, the bulk of protein X is distinct in sequence from the structure
of E2 (84, 85). Protein X is found in liver, kidney, heart, adipose tissue, spleen, skeletal
muscle, testes, uterus, red blood cells, and brain of the rat. Thus, it is not just a tissuespecific isozyme of E2 (86). The outer domain of X binds and facilitates regulation of the
catalytic subunits of the kinase (87). It is believed that E3 associates with X. The
conditions for reconstitution of mammalian pyruvate and 2-oxoglutarate dehyrogenase
complexes have been established (88). Protein X is selectively cleaved using arg C and
reconstitution activity is decreased. Adding a large excess of E3 gives a more effective
reconstitution. Reed and colleagues (89) find that E3BP anchors E3 homodimers inside
each of the 12 pentagonal faces of the 60-mer E2. Steric hinderance by the lipoyl and E3
binding domains limits the binding of E2. A novel dihydrolipoyl dehydrogenase binding
protein that lacks the amino terminal lipoyl domain has been found in Ascaris suum (90).
The stoichiometry of E3BP interaction is 12 mol per mol of PDHC and 60 mol of E2 per
mol of PDHC (91). The lipoyl domains of E3BP can substitute for the lipoyl domains of
E2 in overall complex catalytic activity. It may have an unique catalytic function.
E3. One dihydrolipoyl dehydrogenase gene, lpd, codes for the E3 that serves each of the
-keto acid dehydrogenases. The gene is comprised of 1,419 base pairs (473 codons
excluding the initiating AUG). The composition, Mr (50,554 or 51,274 if the FAD
cofactor is included), the amino-terminal sequence, and the carboxyl-terminal sequence
predicted from the nucleotide sequence are in excellent agreement with previous
biochemical studies on the enzyme (92). E3 is similar in structure to glutathione
reductase (EC # 1.6.4.2), a related disulfide oxido-reductase (93). The crystal structure of
glutathione reductase has been determined (94). The sequence of cDNAs encoding E3
has been determined for porcine and human (95) and yeast (96, 97) genes. The lipoamide
dehydrogenase gene of A. vinelandii has been cloned in E. coli (98). This protein has
40% conservation of amino acid residues when compared with the E. coli enzyme. When
compared with the three-dimensional structure of glutathione reductase, all of the
essential residues in four domains are conserved. E3 may have other functions such as in
sugar transport in E. coli (99-101). E3 has been reviewed by Patel and his colleagues
(102). A lipoamide dehydrogenase that contains a lipoyl domain has been found in
Neisseria menginitidis (103´). This protein is membrane-associated and may be invovled
in transport processes.
Relation of Lipoic Acid to Oxidative Damage
Dihydroipoic acid reduces neuronal injury after cerebral ischemic (104). Expression of
fos and jun genes were delayed in the presense of dihydrolipoic acid but were accelerated
in presence of lipoic acid (105) suggestion a role of redox. Dihydrolipoic acid prevents
hypoxic/reoxygenation and peroxidative damage in rat heart mitochondria (106). Both
lipoate and dihydrolipoate prevented singlet oxygen-induced damage of DNA (107).
Lipoic acid can prevent symptoms of vitamin E deficeincy (108) suggesting that many of
the reductive (antioxidative) elements are in communication. Lipoate prevents serum
albumin glycation (109). It improves memory in aged mice (110). The naturally
occurring optical isomer of lipoic acid is more active in improbing blood during
reoxygenation (111). Lipoic acid protects against cerebral ischemia-reperfusion (112).
Ischemic-reperfusion injury in humans occurs in conditions such as strokes, cardiac
arrest, subarachnoid hemorrhage or head trauma. Oxidative injury due to oxygen free
radicals occurs. Lipoic acid protects rats against reperfusion injury following cerebral
ischemia (113). Lipoic acid is also neuroprotective in focal cerebral ischemia in rodents
(114). In insulin-resistant rat skeletal muscle addition of lipoic acid enchaned insulinstimulated glucose metabolism (115).
Ziegler and colleagues (116) evaluated the efficacy and safety of oral treatment with the
antioxidant lipoic acid in non insulin dependent diabetes mellitus patients with autonomic
neuropathy. Treatment with lipoic acid was well-tolerated and may slightly improved the
patients with cardiovascular autonomic neuropathy symptoms.
Packer and Cardenas (117) edited a monograph that considered Biothios in Health and
Disease. This is an excellent summary of the status in 1992 (published in 1995).
Relation to Medicine
Structural basis in humans. Malfunctioning of any of the three -keto acid
dehydrogenases leads to clincal manifestations. Deficiency of the pyruvate
dehydrogenase complex predominantly leads to lactic acidosis, impaired neurological
function, and delayed growth and development. Deficiency in the branched chain
enzymes leads to maple urine disease. There is an association between both Alzheimer
disease and Parkinson disease and the-ketoglutarate dehydrogenase genes. The
structural basis of these effects have been reviewed by Hengeveld and de Kok (117a).
Therapeutic potential. The role of lipoic acid in liver metabolism and disease has been
reviewed (118). It serves as an antioxidant at 600 mg doses. It has been used to treat
alcohol-induced damage to the liver, mushroom poisoning, metal intoxication, and CCl4
poisoning. Lipoic acid has a well-defined molecular role in biochemistry and a still
diffusely defined role in pharmacology.
Ischemic Heart Disease. Clofibrate, an antilipidemic agent, is useful in primary
prevention of ischemic heart disease. The branched-chain ketoacid dehydrogenase
complex, BCKADHC, is displaced from serum albumin by clofirate and enzyme activity
in the heart is increased activity by clofibrate treatment (119, 120). Feeding clofibrate to
rats increases the activity of BCKADHC 3-fold (121). Administration of clofibrate to rats
both activates and induces the BCKADHC. The increased synthesis would require
additional lipoic acid and possibly the activating enzymes. Induction has been
demonstrated for carboxylase (E1) and dihydrolipoamide transacetylase (E2), but not
dihydrolipoamide dehydrogenase (E3) (122). The BCKADHC increased by clofibrate is
mitochondrial and not peroxisomal. Clofibrate causes peroxisomal proliferation (123).
The activation is due to kinase inhibition (124).
Lactic acidosis and related diseases. Inborn errors of energy metabolism as a group
affect 1/5000. The cause of lacticacidemia represents a significant diagnosis problem. A
cell tries to maintain its ATP level at all cost and a universal consequence of failure of
mitochondria to produce adequate ATP is excessive lactic acid production. Defects of
PDHC lead to fatal neonatal lacticacidosis, psychomotor retardation with or without
neurodegeneration and a male-only syndrome of ataxia, mild mental retardation and
carbohydrate sensitivity (109). Most tissues can survive with little or no PHDC activity
when they can use other energy substrates, but in the brain cells that have PDHC defects
usually die.
Clinical improvement was achieved by oral administration of lipoic acid to an 8-monthold boy who had primary lactic acidosis due to a deficiency of E3 (125). Lipoic acid
treatment may be useful in reducing serum-mediated cytotoxicity in patients with acute or
chronic alcohol toxicity (126). PDHC abnormalities have been reported in over 50
patients; these patients had ataxia, psychomotor retardation, Leigh's disease, and/or some
had lactic acidosis (127). There can be tissue specificity in the deficiency since the
deficiency of both subunits of E1 was not expressed in some fibroblasts when the activity
of the PDHC was reduced by 70% in the kidney (128). E1 deficiency is associated with
lactic acidosis and central nervous system dysfunction (129). Several mutations account
for the molecular heterogeneity of PDHC deficiencies. For example, with 11 patients
(129), 7 had E1a and E1b proteins and mRNAs, 2 had mRNAs but no proteins, and 2 had
only E1b mRNA. The cDNA coding for E1ap was isolated from a patient with lactic
acidosis. Endo et al. (130) showed that there was a deletion of four nucleotides upstream
from the normal termination codon which made a new termination codon 33 bases
downstream. mRNA was present in this patient. This study was the first cloning of a
defective gene of PDHC.
The effects of mutations in the E1p subunit are extremely varied: lactic acidosis,
hydrocephaly, enlarged ventricles, Leigh disease, psychomotor retardation, microcephaly,
developmental delay, agenesis of the corpus callosum, dilated ventricles, cortical
thinning, ataxia, mental retardation, and comatose episodes. Robinson and colleague
analyzed these 14 patients for PDH-complex activity (ranged from 3.5 % to 100 %). The
individual with 100 % activity had a 46-bp repeat (frame-shift mutation results) and
premature termination (28 amino acids less). The activity in the brain was undoubtedly
reduced (131). Even an amino acid substitution in the mitochondrial import sequence of
the precursor protein can reduce the PDH-complex activity in fibroblasts (132). Because
the E1ap gene is located on the X chromosome the effects of mutations differ in males
and females (133). A patient with a defect in the X-lipoyl-containing component of the
PDHC can cause neonal lactic acidemia (134). Thiamine treatment of a male child who
was PDH deficient and had lactic acidosis (135) improved his condition
The A199T E1 mutation decreases the affinity for TPP and yields a complex with 25 %
of normal activity and a 10-fold increase in the Km for pyruvate (136). It is suggested that
the addition of lactic dehydrogenase inhibitors would be useful in treatment.
Lactic acidosis is a broad-anion gap metabolic acidosis caused by either lactic acid
overproduction or underutilization. Treatments of the underlying cause of the lactic
acidosis are ideal (136a).
Maple syrup urine disease (MSUD) and branched chain-related diseases. Deficiency
in the branched-chain -keto acid dehydrogenase cause hypervalinemia, hyperleucineisoleucinemia, maple syrup urine disease, isovaleric acidemia, glutaric aciduria type II,
ethylmalonic-adipic aciduria, 3-methylcrotonyl CoA carboxylase deficiency, 3-hydroxy3-methylglutaryl CoA lyase deficiency, and 3-ketothiolase deficiency (137). Danner et al.
(138) established the immunologic absence of the E2 protein in an individual with
MSUD. Another study (139) on cell lines derived from patients with MSUD showed a
marked decrease in E1b and faint immunostaining of E1a. Fisher et al. (140) found five
types of defects: type I, reduced E1 activity but normal amounts of E1a and E1b; type II,
reduced amounts of E1a and E1b; type III, E1a mRNA reduced; type IV, E2 mRNA
reduced, and type V, E2 reduced or absent. Thus, MSUD is a complex disease at the
molecular level. A history of MSUD from 1954 when it was recognized to 1993 has
appeared (141). The incidence is 1:200,000 live births. Numerous mutations in the
BCKD have been noted. Retroviral gene transfer has been used to correct the disease in
lymphoblasts from a Mennonite MSUD patient where Tyr 393 has been converted to Asn
in E1b.
There are gender differences in the regulation of the branched-chain 2-oxo acid
dehydrogeanses (142). In male and female rats there were differences in response to
light-dark cycles. The females showed a profound diurnal rhythm while the males did
not.
Danner and Doering at Emory have an on-line review of human mutations that affect
BCKD (143). His group (144) also the transport of the three enzyme components from
the cytoplasm to the mitochondria. E1b transport is rate limiting.
There are three groups of MSUD mutations (50): 1) mutations that affect cofactor
binding (R114W, T166M, R220W, and N222S; 2) mutations that affect the
hydrophobic core (M64T, G204S, A208T, T265R, and I281T; and 3) mutations
that affect subunit association (N126Y, Q134K, H156R, A209D, A240P,
G245R, R252H, F364C, Y368C, and Y393N.
In Turkish patients 3 missense disease specific mutations Q80E, C213Y, and T106M in the E1 gene and a
polymorphism F280F were new mutations that produced MSUD (144a). Also there was a splice site
mutation in the E2 gene. Some 36 children with MSUD were treated with a protocol that 1) inhibits
endogenous protein catabolism, 2) sustains protein synthesis, 3) prevents deficiencies of essential amiono
acids and 4) maintains normarl serum osmolarity (144b). All survived but infection or injuries can lead to
deteroriation with life-threating neurological function at any time.
Leigh's necrotizing encephalomyelopathy. In the case of an infant with lactic acidosis
and developmental delay with neuropathological changes consistent with Leigh's
necrotizing encephalomyelopathy, there was systemic deficiency of both subunits of E1
(145). A patient with well-documented clinical and biochemical PDHC deficiency was
found on postmortem examination to have the specific CNS pathology of Leigh's disease
(146). The PDHC activity was about 20% of that of normal individuals. Therefore, a
subgroup of Leigh's disease is due to a PDHC deficiency. 20% of that of normal
individuals. Therefore, a subgroup of Leigh's disease is due to a PDHC deficiency. Blass
and coworkers (147) restored PDHC activity to extracts from skin fibroblast cells from
two patients by adding E3. This shows that Leigh's disease can result from abnormalities
in either the E1 or E3 components of the complex. A defect of X has been identified in
two patients with encephalomyelopathy (148).
Alzheimer's disease. The activity of 2-ketoglutarate dehydrogenase complex is reduced
by more than 75% in patients with Alzheimer's disease (149). A 50% deficiency in
PDHC activity has also been shown (150). In 20 patients with early-onset dementia of the
Alzheimer type there was a 44% reduction in the cerebral metabolic rate for glucose
(151). This breakdown in glucose metabolism appeared to involve the PDHC, but it is not
clear if it is the primary defect. Both PDHC and KGDHC are reduced in Alzheimer
diseased brains (152). Brains that are aging or have Alzheimer disease switch from
glucose as their fuel source to ketone bodies. Heininger (152a) postulates that a soluble
factor Abeta or deprivin is the metabolic switch and inhibits pyruvate dehydrogenase. In
the older or Alzheimer brain the ketone bodies are an insufficient energy source.
Therefore, outer regions of the brain are shut down to conserve fuel for survival of the
inner core.
Primary biliary cirrhosis. The autoantibodies of primary biliary cirrhosis recognize E2
and inhibit its function (153). In 10/10 patients there was 30-100 % inhibition of PDHC
activity and the inhibition was directly proportional to the ELISA value (151). Another
group of investigators has identified the 70-kD M2 autoantigen as the E2 of
mitochondrial PDHC. In this study 38/40 patients' sera reacted positively with E2 (154)
A synthetic peptide containing the lipoic acid binding site, KATIGF, absorbed the
reactivity of sera from patients having primary biliary cirrhosis (155). The target for the
autoantibodies corresponds to a functional site of dihydrolipoamide acetyltransferase.
Would these sera also block lipoic acid activation since they are directed toward the same
amino acid sequence? All 40 patients with primary biliary cirrhosis have autoantibodies
directed against at least one of the E2 components of the -keto acid dehydrogenases.
Treatment of this disease accounts for about 80 % of European liver transplants (156).
The primary structure of the human M2 mitochondrial autoantigen E2 has been
determined (156a). There are two presumed lipoyl bearing sequences: ETKATVGFE and
ETKATIGFE. Avidin, the protein that avidly binds biotin, interacts with the lipoyl
domains in PDHC (157). Both lipoic acid and biotin are bound to -amino groups of
lysines and there is similarity in the amino acid sequence around these lysines. An
octadecapeptide from amino acid residue 167-184 was specifically recognized by the
anti-M2 autoantibodies from patients with primary biliary cirrhosis (158). A flow
cytometric method has been developed to detect the anti-PDH antibody in primary biliary
cirrhosis (159). The presence of lipoyl residues is crucial for recognition of human PDCE2 by antisera from patients with primary biliary cirrhosis (160). A library of 21
monoclonal antibodies to the E. coli PDH-complex and its components have been
developed (161). All the antibodies were bound to the complex; 17 were bound to the E1
subunit. The degree of inhibition of enzymatic activity varied from 0 to > 98 %. A new
assay for E1 activity based on N-acetyl-4-thiopyridine was developed. Competitive
epitope mapping revealed that there were at least six separate binding regions (162). One
antibody counteracted the GTP regulation of PDH-complex. GTP is an allosteric
inhibitor. A semiautomated PDH-complex assay based on use of microtiter plates has
been developed for the diagnosis of primary biliary cirrhosis (163). The results of assays
following cyclosporin treatment showed a decrease in antibodies that correlated with the
improvement of liver function. Halothane hepatitis has associated antibodies that are
directed against the lipoyl-containing components of the 2-oxoacid dehydrogenase
complex (164). The mechanisms of autoimmune liver disease have been reviewed (164a).
Chronic viral infections could initiate primary biliary cirrhosis (164b). There isonly one
treatment that is effective other than liver transplant and it is ursodeoxycholic acid
(164c). The diagnosis usually uses three criteria: 1) elevated alkaline phosphatase, 2)
antimitochondrial antibodies, and liver biospy.
Systemic sclerosis. Many patients with have the multisystem connective tissue disorder
of systemic sclerosis produce antibodies to the E1 (165). Systemic sclerosis is also an
autoimmune disease.
Lipoic Acid Activation and the Lipoamidase Reaction
Lipoic acid is enzymatically activated in an ATP-dependent reaction before being
covalently incorporated into the E2 and protein X (E3BP) units of the -keto acid
dehydrogenases.
The reaction is
E1 + ATP + Lipoic Acid
&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;
> E1-lipoyl-AMP + PP
E1-lipoyl-AMP + E2
&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;
&emdash;> Lipoyl-E2 + AMP + E1
Lipoyl-E2 + apo-PDHC
&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;
&emdash;&emdash;> Lipoyl-PDHC + E2
Yang and Frey (166) found that only the R-enantiomer of lipoic acid is a substrate for the
enzymes of the complexes. Morris et al (167) have identified the gene encoding the
lipoate-protein ligase A in E. coli. It codes for a 337 amino acid residue protein of 38 kD
that attaches radioactive lipoic acid to apopyruvate dehydrogenase. A lipoyltransferase
that catalyzes the second reaction above involving the lipoyladenylate was purified and
characterized by Fujiwara, et al. (168) from bovine liver mitochondria. The same group
(168a) purified the lipoic acid activating enzyme to homogeneity and showed that GTP
was 1000 times more active than ATP. The product was lipoyl-GMP. cDNA clones
encode a precusor of the enzyme. The amino acid sequence is identical to that of the
medium-chain fatty acid CoA ligase. The lipoate protein ligase from E. coli has been
purified and characterized by Guest and colleagues (169). The purified enzyme is
monomeric with a Mr of 38000. It is inactivated by forming an intramolecular disulfide.
Studies with mutants that lack this enzyme have another lipoylation system.
The lipoamide arm of the glycine decarboxylase is not freely swinging (170). Lipoic acid
is the prosthetic group for the H-component of the glycine decarboxylase that catalyzes
the oxidative decarboxylation and deamination of glycine with the formation of CO2,
NH3, and N5,N10-methylene-5, 6, 7, 8-tetrahydropteroyl-glutamate. One of the
components, L-protein, is dihydrolipoamide dehydrogenase. These observations are
based on the X-ray crystallographic analysis of two forms of the H-protein. The Hprotein has been crystallized and its structure determine to a 2.6 Å resolution (171). The
131-amino acid residues form seven -strands arranged into two antiparallel -sheets
forming a sandwich structure. One -helix is observed at the C-terminal end. The lipoyl
moiety points toward the H34 and D128 resiudes.
Guest and colleagues (172) have engineered an E. coli that has PDH-complexes with
lower numbers of lipoyl domains. They show that the organism has a reduced growth rate
and yield. While not explaining why three lipoyl domains are in the E2 of E. coli, these
results show that more than one is required for maximum growth efficiency. Properties of
the complexes, the growth under limiting glucose conditions is a more drastic condition
that shows a functional debilitation. Additional study (173) has revealed that although
there are three lipoyls on E2p only one lipoyl domain is sufficient to give full catalytic
activity in vitro. Only the outermost lipoyl domain must be lipoylated for activity. The
presence of the other lipoyl domain provides a structural extension rather than extra
catalytically active cofactors. Constructs with three lipoyl domains give maximum
growth rate while only one domain is required to give the greatest in vitro specific
activity.
The E. coli lipoyl-ligase can lipoylate the H protein of the glycine decarboxylase complex
when exogenous lipoic acid is supplied (174). Selenolipoic acid, a lipoic acid analog,
where both S atoms are replaced by Se, can be attached to the H protein. The enzyme is
26 % as active as the S-containing one for glycine decarboxylation (175). In reactions
where redox is required it is poorly active. The changed redox potential is responsible.
There are redundant pathways for lipoyl ligation in E. coli (176). lipA encodes a ligase
that attaches exogenously supplied lipoic acid. lipB is required for the functioning of a
second ligase which uses the lipoyl group from the biosynthetic pathway. The
lipoyltransferase has been cloned from bovine liver (177). It contains 373 amino acids, a
mitochondrial leader and has a MW of 39,137 Da. The sequence is 35 % identical with
that of E. coli lipoate-protein ligase A. Lipoyltransferases I and II from bovine liver
mitochondria can lipoylate E2p and E2o effectively but E2b had a low rate of lipoylation
(178). By in vitro mutagensis studies it was found that G-54 of E2o was important for the
ligation reaction.
The lipB gene, lipoyl-protein ligase, from yeast has 27 % sequence identity with the E.
coli protein (179). The yeast has also been cloned by Marvin, Williams, and Cashmore
(179a). The biotinylating and lipoylating enzymes are part of an evolutionarily related
protein family that contains a homologous catalytic module (180). There is a single
conserved lysine that is expected to contribute to the binding of lipoic acid in the LplA
and LipB enzymes. There is no three-dimensional structure available for any of the
liopoylating enzymes. The modeling has been done using the biotinylating enzyme BirA
from E. coli.
The human lipoyltransferase gene is located on chromosome band 2q11.2. It is 88 %
identical to the bovine enzyme and 31 % identical to the E. coli one (180a).
The biotinylating and lipoylating enzymes look at different keys in determining what is a
proper substrate for posttranslational modification. The lipoylating enzyme, LplA,
recognized the MKM sequence and responds to structural cues in the flanking -strand of
the substrate protein (181).
The surface antigentic protein P64K fromthe pathogenic Neisseria meningitidis is a
chimera of the lipoyl domains of E2, the linker regions of E2, and E3. Lipoyl protein
ligase attached a lipoyl group (181a).
The enzyme lipoamidase hydrolytically removes the bound lipoic acid from the E2 and
protein X units of the -keto acid dehydrogenases. The reaction is:
Lipoyl-PDHC + H2O
&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;
> apo-PDHC + Lipoic acid
These two reactions may be another means of regulating the activity of the  -activating
system has been little studied since its original characterization in the 1950s and 1960s.
There have been more studies on lipoamidase because of its usefulness in studying the
complexes, but many questions remain. The molecular biology (base sequence, etc.) of
the complexes is being elucidated. However, there have been no such studies on the
lipoic acid-activating system and lipoamidase. Oizumi and Hayakawa (182) have purified
lipoamidase 295-fold to near homogeneity from guinea pig liver. Lipoamidase was found
in the microsomal membrane fraction and was labile. The subunit molecular mass was 68
kDa; under nondenaturing conditions the molecular mass was 120 kDa, suggesting a
homodimer. Lipoamidase has a pI of 5.7, a pH optimum of 8.0, and is not inhibited by
PCMB. It is inhibited by DFP and is thus a serine peptidase.
Recent Results on the Regulatory Enzymes
The catalytic subunit of bovine pyruvate dehydrogenase phosphatase has been cloned and
sequenced (183). The protein has 467 amino acid residues with a Mr of 52,625. The
activity of enzyme expressed in E. coli was near that of the native bovine PDP. The
primary structure of the pyruvate dehydrogenase kinase has been determined and it
establishes a new family of eukaryotic protein kinases (184). The cDNA codes for 434
amino-acid protein. T he protein lacks the motifs usually associated with ser/thr-protein
kinases. There is homology with the prokaryotic histidine kinase. The lipoyl group is
involved in the interaction with the kinase (185). Reed's group (186) has developed a
one-step purification for the catalytic subunit of the recombinant bovine pyruvate
dehydrogenase phosphatase. The PDPc binds to the inner lipoyl domain of the E2 of
mammalian pyruvate dehydrogenase.
To conserve carbohydrate reserves, the reaction of PDH-complex is down-regulated
when the citric acid cycle is provided with sufficient acetyl~CoA. The E1 kinase activity
is increase by increased NADH/NAD+ and acetyl~CoA/CoA ratios. Roche and associates
(187) showed that the lipoyl moities on E2 were required for the control by the
coenzymes. The control of the kinase is changed according to the molecular state of the
inward lipoyl group: oxidized, reduced, or acetylated.
There are at least three isoenzymatic forms of PDpK (PDpK1 and PDpK2) (188). cDNAs
for these enzymes were cloned from human liver. The PDpK3 is almost exclusively
expressed in heart and skeletal muscle suggesting a muscle-specialized function. PDpK2
has the highest expression in most tissues and is probably the one responsible for the
majority of regulation. Although the rat branched-chain -ketoacid dehydrogenase kinase
has sequence similarity to histidine-protein kinase, autophosphorylated kinase is not
active (189).
Using site-directed mutagenesis Korotchkina and Patel the three phosphorylation sites
were changed from ser to ala to allow analysis of the phosphorylation reaction (190). The
rates of phosphorylation and inactivation were site specific. Phosphorylation of each site
resulted in complete inactivation of the E1p. The rates of dephosphorylation of the
various sites were similar and there is a random dephosphorylation mechanism. The same
authors ((190a) have reviewed the phosphorylation by four kinases on the three sites.
There appears to be site specificity and independence. The roles of amino acids residues
surrounding the phosphorylation site of BCKDH have been analyzed (191). Changing of
arg-288, his-292, or asp-291 resulted in inactive enzymes. The his-292 and a S-293
mutation prevented TPP binding. The arg-288 was not phosphorylated, but all others
were. Roche's group (192) has implicated tryptophan-135 in the TPP binding site of E1p.
Two metal ions, Ca2+ and Mg2+, independently modulate -ketoglutarate dehydrogenase
complex activity (193).
There are tertiary and quaternary interactions that regulate the yeast 2-oxo acid
decarboxylases using the substrate pyruvate and the cofactor TPP. These studies have
been reviewed by Guest and colleagues (194).
Roche and colleagues (195) have reviewed the regulation by the four pyruvate
dehydrogenase kinases and two pyruvate dehydrogenase phosphatases. Tissue-specific
and metabolic state-specific control is acheived by the selective expression and distinct
regulatory properties of the enzymes. One or more of the lipoyl domains in E2 selectively
bind each PDK and PDP.
Web connections
1. Pyruvate dehydrogenase complex

Complex
 http://www.biochemtech.unihalle.de/PPS2/course/section11/complexes.html
Structure
http://w3pharm.u-shizuokaken.ac.jp/~bioorg/macromol/pdh/pdh_complex.html
Deficiency
http://www.nlm.nih.gov/mesh/jablonski/syndromes/syndrome548.html
Architecture
 http://www.bmsc.washington.edu/people/hol/WimFigs5.html
&
word description
but must navigate back to section 5 from above.
2. NMR structure of the lipoyl domain
 http://coli.polytechnique.fr/NMR.html
3. Reactions of the complex
 http://fig.cox.miami.edu/Faculty/Tom/bil255/pyruvate.gif
mechanism
http://www.chem.umd.edu/biochem/jollie/462/enzymes/glycol/pyrdhm.htm
4. Pyruvate dehydrogenase E1 polypeptide 1, small deletions
 http://www.uwcm.ac.uk/uwcm/mg/ns/4/118895.html
5. Crystallography, sequence, and comparisons

E1
http://heme.gsu.edu/glactone/PDB/Proteins/Krebs/1pyd.html

E1
http://web1.ebc.uu.se/molev/publications/cfg2000/descrip
tions/PDA1.html
E1
http://web1.ebc.uu.se/molev/publications/cfg2000/de
scriptions/PDB1.html
E1o
http://web1.ebc.uu.se/molev/publications/cfg2000/de
scriptions/KGD1.html
X

http://web1.ebc.uu.se/molev/publications/cfg2000/de
scriptions/PDX1.html
E2
http://chemistry.gsu.edu/glactone/PDB/Proteins/Krebs/1iyu.html

Various
http://web1.ebc.uu.se/molev/publications/cfg2000/de
scriptions/LAT1.html
E2o
http://web1.ebc.uu.se/molev/publications/cfg2000/de
scriptions/KGD2.html
E3
http://www.scripps.edu/pub/goodsell/interface/interface_images/1ebd.html

Various listed
http://web1.ebc.uu.se/molev/publications/cfg2000/de
scriptions/LPD1.html
6. PDB files

search for variations
http://www.rcsb.org
7. Lipoic acid synthase
http://web1.ebc.uu.se/molev/publications/cfg2000/descriptions/LIP5.html
Literature Cited
1. Roche, T. E., &. Patel, M.S., (Eds.) (1989) -Keto Acid Dehydrogenase Complexes: Organization,
Regulation, and Biomedical Aspects, Ann. NY Acad. Sci. 573, 474 pp.
2. Patel, M.S., & Roche, T.E. (1990) FASEB J., 4, 3224-3233.
3. Bisswanger, H., & Ullrich, J. (Eds.) (1991) Biochemistry & Physiology of Thiamin Diphosphate
Enzymes, VCH, Weinheim, 453 pp.
4.Yeaman, S.J. (1989) Biochem. J., 257, 625-632.
5. Perham, R.N., Packman, L.C., & Radford, S.E. (1987) Biochem. Soc. Symp., 54, 67-81.
6. Reed, L.J., & Hackert, M.D. (1990) J. Biol. Chem., 265, 8971-8974.
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Appendix
Lester James Reed
A Tribute to Lester J. Reed
From Ref (1).
This conference and volume [Roche, T.E. & Patel, M.S. (eds) (1989) -Keto Acid
Dehydrogenase Complexes: Organization, Regulation, and Biomedical Aspects, Ann. NY
Acad. Sci. 573, 474 pp.] are a tribute to Dr. Lester J. Reed for his many outstanding
contributions to the field of -keto acid dehydrogenase complexes. Lester gouged out of
the forest of biochemical distractions a highway that runs from lipoic acid chemistry to
exquisite information on the structure, function, and regulation of -keto acid
dehydrogenase complexes. We wish to pay tribute to Lester for his dedication in this
brilliant effort and for his integrity and quiet leadership, which have made lasting
impressions on those who have had the privilege to be associated with him.
A Brief Biography
Lester J. Reed was born on January 3, 1925, in New Orleans, Louisiana. He received his
B.S. degree at Tulane University in 1943 and completed his Ph.D. under Reynold C.
Fuson at the University of Illinois in 1946. Having achieved the latter degree at the ripe
old age of 21, he took a position as a postdoctoral research associate with Vincent
duVigneaud at Cornell University Medical College from 1946 to 1948.
In 1948, he joined the University of Texas at Austin, where he has been a professor since
1958, director of the Clayton Foundation Biochemical Institute since 1963, and Ashbel
Smith Professor since 1984.
Affiliations and Honors
Professor Reed is a member of several professional societies, has served on many
advisory councils and editorial boards, and has been the recipient of several honors.
Memberships. Phi Beta Kappa, Sigma Xi, American Chemical Society, American Society
for Biochemistry and Molecular Biology, the Protein Society, American Association for
the Advancement of Science (Fellow), National Academy of Sciences, American
Academy of Arts and Sciences.
Advisory Councils and Editorial Boards. Biochemistry Study Section of the National
Institutes of Health; Editorial Board, Archives of Biochemistry and Biophysics;
Nominating Committee and Executive Committee, Division of Biological Chemistry,
American Chemical Society; Membership Committee and Nominating Committee,
American Society of Biological Chemists; Editorial Board, Journal of Biological
Chemistry; Editorial Board, Biofactors; U.S. National Committee for the International
Union of Biochemistry.
Honors. Eli Lilly and Co. Award in Biological Chemistry (American Chemical Society),
1958; election to the National Academy of Sciences, U.S.A., 1973; Honorary Doctor of
Science Degree, Tulane University, 1977; election to the American Academy of Arts and
Sciences, 1981; Ashbel Smith Professor, University of Texas, 1984.
Major Contributions to Research on the Structure, Function, and Regulation of KetoAcid Dehydrogenase Complexes
Lester joined the University of Texas at Austin, following his productive studies in
synthetic organic chemistry with Fuson and in intermediary metabolism with
duVigneaud. In 1949, the pioneering studies of Roger Williams in characterizing B
vitamins were being extended in Williams's laboratory to characterizing the "acetatereplacing factor." The latter studies were initiated by Esmond Snell at the University of
Wisconsin and continued in Texas after Snell moved to Austin. Dr. Williams invited
Lester to undertake the characterization of this factor.
The following is a selected list of landmark contributions of Lester Reed to the field of keto acid dehydrogenase complexes:
Isolation and characterization of lipoic acid
Identification of the functional form of lipoic acid
Resolution of Escherichia coli pyruvate and -ketoglutarate dehydrogenase complexes and characterization
of the components
Utilization of electron microscopy to analyze the structural organization of the bacterial and mammalian
complexes
Regulation of the mammalian pyruvate dehydrogenase complex by phosphorylation dephosphorylation
Isolation and characterization of the pyruvate dehydrogenase kinase and pyruvate dehydrogenase
phosphatase
Identification of inner and outer domain structure of the transacylase components
Isolation and characterization of the branched-chain -keto acid dehydrogenase phosphatase and its
inhibitor protein
While establishing the fundamental organization of these quintessential multienzyme
complexes, Lester also had a seminal role in formulating concepts concerning the unique
properties attendant to the organization of enzymes in a clustered state. The following
listing of selected major contributions (arbitrarily divided as convenient) gives a rough
time-frame for the construction of the Lester J. Reed "highway," as well as a road map
for traveling the route. To be honest, my intention was to make a shorter list, but I could
not delete from, but only expand on, these significant discoveries. Here, then, is a
selected list of Lester's major contributions.
1949-l954
Isolation, characterization, and synthesis of -lipoic acid: structural and physical
characterization, broad distribution and enrichment in mitochondria, high-yield synthesis,
35
S-labeled cofactor, analogs.
19S4-l9S8
Functional form of lipoic acid: attachment to -amino group of lysine, ATP requiring
(lipoyl-adenylate intermediate) reaction of lipoic acid-activating enzyme, lipoyl-X
hydrolase reaction.
1957-1963
Purification of E. coli pyruvate and -ketoglutarate dehydrogenase complexes,
flavoprotein nature of the dihydrolipoyl dehydrogenase component, resolution and
reconstitution of the E. coli pyruvate debydrogenase complex, characterization of
component enzymes, sequence around the -amino lipoyllysine group, purification of
lipoamidase. model reactions, formation of 2-acetyl-thiamin pyrophosphate (AcTPP),
other aspects of reaction mechanism of components.
1964-1968
Electron microscopic characterization of complexes, lipoyllysine swinging arm activesite coupling mechanism, transacylase cores composed of 24 E2 subunits arranged with
432 symmetry in cube-like particle, location of the El component on the edges and E3
component on the faces of the cubic core, resolution and reconstitution of the E. coli ketoglutarate dehydrogenase complex, presence of the same E3 component in the
pyruvate and the -ketoglutarate dehydrogenase complexes, purification and molecular
organization of the pyruvate and -ketoglutarate dehydrogenase complexes from bovine
kidney, development of concepts concerning unique properties of enzymes that are
organized into complexes.
1968-1970
Regulation of the E. coli pyruvate dehydrogenase complex by phosphoenolpyruvate,
acetyl-CoA, and guanine nucleotides; regulation of bovine kidney and heart and porcine
liver pyruvate dehydrogenase complex by phosphorylation and dephosphorylation, ATPMg2+ dependent PDH kinase tightly associated with the complex and inhibited by ADP
and pyruvate, Mg2+-dependent PDH phosphatase weakly associated with the complex.
1971-1974
X-ray crystallography of inner core of E. coli dihydrolipoyl transsuccinylase establishing
octahedral (432) symmetry; preparation of the component enzymes of the pyruvate
dehydrogenase complexes from bovine kidney and heart, and characterization of their
physical and chemical properties; stoichiometry of subunits in the mammalian
complexes; subunit ratios based on sedimentation equilibrium molecular weights of
purified components of E. coli pyruvate and -ketoglutarate dehydrogenase complexes;
multiple phosphorylation sites in mammalian pyruvate dehydrogenase (PDH) and
sequence around phosphorylation sites; characterization of PDH kinase: separation of the
kinase from the transaeetylase, direct pyruvate inhibition, thiamin PP inhibition,
transaeetylase effect on Vmax and Km for PDHa, and monovalent cation effects on ADP
inhibition; role of Ca2+ in activating PDH phosphatase by increasing its association with
the transacetylase and lowering its Km for PDH; first studies on modulation of steadystate phosphorylation and dephosphorylation; kinetic data supporting multisite ping-pong
mechanism of kidney pyruvate dehydrogenase complex.
1975-1979
Kinetic mechanism of bovine kidney transacetylase, data supporting acetyl and electronpair relay system between lipoyl moieties, purification and properties of bovine kidney
branched-chain -keto acid dehydrogenase complex, acetyl-CoA/CoA and NADH/NAD+
effects on PDH kinase and PDH phosphatase activities, kinetic properties determined
with peptide substrates of PDH kinase
1982-1987
Two-domain structure of the transacetylase from E. coli and mammalian sources,
transacetylation and E2-subunit binding role of inner domain; extended lipoyl-bearing
outer domain, capacity of transacetylase domains to bind other components, contribution
of structure in the lipoyl domain to the reductive acetylation reaction catalyzed by the E I
component; X-ray crystallography of the inner domain of the E. coli transacetylase,
establishing 432 symmetry; regulatory properties of the kinase and the phosphatase,
utilizing peptide substrates.
1982-1987
Purification and properties of: the pyruvate dehydrogenase phosphatase (FAD-containing
90-kDa regulatory subunit, stimulation by polyamines); the pyruvate dehydrogenase
kinase; the branched-chain -keto acid dehydrogenase phosphatase and a potent protein
inhibitor of this phosphatase; and a distinct cation-independent, spermine-stimulated
mitochondrial phosphatase. Computer model analysis supporting a multiple random
coupling mechanism for active-site coupling through the lipoyl. domains of the E. coli
pyruvate and -ketoglutarate dehydrogenase complexes.
1986-1988
Purification of bakers' yeast pyruvate dehydrogenase complex and phosphorylationdephosphorylation by mammalian kinase and phosphatase; cloning of genes of
components of the yeast pyruvate dehydrogenase complex.
It should be readily apparent from these latest contributions that Lester is still building
the highway and opening new frontiers for further research. During preparations for this
conference, Lester gave me a list of 60 students and collaborators who have contributed
to the above work. For readers wanting more details about any of this work, I would note
that, in addition to his numerous research papers, Lester has written 38 review articles
which describe with great clarity the above work and related studies by other researchers.
Thomas E. Roche
Kansas State University
Manhattan, Kansas
October 1988
RECOLLECTIONS
From lipoic acid to multi-enzyme complexes
Lester J. Reed 1, 2
1 Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin,
Texas 78712
Adapted from Protein Science (1998) 7: 220-224
I shall retrace the high points of a trail of research that I have had the pleasure of
establishing in association with many collaborators. This trail has led from the isolation
and identification of a microbial growth factor to the structure, function, and regulation of
-keto acid dehydrogenase complexes. The high points of this trail in the 1950s were the
isolation, characterization, and synthesis of lipoic acid and identification of its functional
form. In the late 1950s and into the 1960s the trail led to the isolation, resolution, and
reconstitution of the Escherichia coli pyruvate and -ketoglutarate dehydrogenase
complexes, characterization of their component enzymes, and elucidation of their
macromolecular organization. In the late 1960s part of our research effort was directed
toward isolating and characterizing the bovine pyruvate dehydrogenase (PDH) complex.
In the early stage of this investigation we found that the complex is regulated by
phosphorylation and dephosphorylation. Resolution of the mammalian PDH complex and
characterization of its component enzymes, including the kinase and the phosphatase,
continued in the 1970s and early 1980s. We also obtained evidence that the
dihydrolipoamide acetyltransferase components of the E. coli and bovine PDH
complexes possess a multi-domain structure. In the 1980s we isolated and characterized
the bovine branched-chain -keto acid dehydrogenase complex and the phosphatase that
regulates its activity. In the late 1980s and early 1990s we cloned and disrupted the genes
encoding the components of the Saccharomyces cerevisiae PDH complex and used
protein engineering techniques to study structure-function relationships. In the mid-1990s
we cloned, sequenced, and expressed cDNAs encoding the two subunits comprising
bovine PDH phosphatase and gained a deeper understanding of their nature and
regulation (Fig. 1).
This trail of discovery started in the spring of 1949, about six months after I joined the
faculty of the Department of Chemistry at The University of Texas. At that time I started
working on the isolation of a factor that replaced acetate in the growth medium for
certain lactic acid bacteria. Research on the acetate-replacing factor was initiated by
Esmond Snell at the University of Wisconsin, continued with a graduate student, Beverly
Guirard, after Esmond moved to The University of Texas, and then pursued by Milton
Getzendaner, a graduate student under Roger Williams' supervision. I inherited this
project in the spring of 1949. We established that this factor is widely distributed in
animal, plant, and microbial cells and that animal liver is a rich source. The factor is
tightly bound to liver protein and is released only after hydrolysis in acid or base. At that
time pharmaceutical companies were processing large amounts of pork and beef liver to
obtain extracts suitable for treatment of pernicious anemia. Fresh liver was extracted with
warm water, and the residual liver proteins and fatty material were dried and sold as an
animal feed supplement. Arrangements were made with Eli Lilly and Company to obtain
liver residue, and we developed procedures for extracting and purifying the acetatereplacing factor.
In the late 1940s and early 1950s several other groups were trying to isolate factors that
were similar to, if not identical with, the acetate-replacing factor. These factors included
the "pyruvate oxidation factor, POF" of O'Kane and Gunsalus that was necessary for
oxidation of pyruvate to acetate and carbon dioxide by Streptococcus faecalis;
"protogen," an unidentified growth factor for a protozoan, Tetrahymena geleii, that was
being purified by Stokstad, Jukes, and associates at Lederle Laboratories; and the "B. R.
factor" of Kline and Barker required for growth of Butyribacterium rettgeri with lactate
as the fermentable carbon source.
In the fall of 1950, a collaboration with Gunsalus and the Lilly Research Laboratories
was undertaken to isolate the acetate-replacing/pyruvate oxidation factor. The Lilly group
adapted and scaled up isolation procedures developed by us, and concentrates of the
growth factor that were 0.1 to1% pure were sent to us for further processing. One of the
most exciting times in my life occurred on or about March 15, 1951, when I obtained the
first pale-yellow crystals of the factor. The amount was minute, only about 3 mg. It was
partially characterized and given the trivial name alpha-lipoic acid (Reed et al., 1951).
The isolation procedure involved a 300,000-fold purification. A total of approximately 30
mg of crystalline lipoic acid was eventually isolated. We estimated that approximately 10
tons of liver residue were processed to obtain this small amount of the pure substance.
NMR and mass spectrometers were not available in those days, but it was possible to
establish that lipoic acid is either 6,8-, 5,8-, or 4,8-dithiooctanoic acid. That the correct
structure is 6,8-dithiooctanoic acid (1,2-dithiolane-3-valeric acid) was established by
synthesis of DL-lipoic acid, first achieved by the Lederle group (Reed, 1957). I was
intrigued by this simple, yet unique substance and wanted to know more about its
biological function, i.e., with what and how did it function in living cells. We, therefore,
set about establishing this part of the trail, which turned out to be even more exciting than
the isolation and characterization of lipoic acid. Elucidation of the mechanism of
oxidative decarboxylation of -keto acids is a fascinating chapter in modern
biochemistry. I shall review briefly the major developments in this story.
The equation shown below represents the coenzyme A and NAD+-linked oxidative
decarboxylation of -keto acids. In addition to CoA and NAD+, thiamin diphosphate, a
divalent metal ion, protein-bound lipoic acid, and FAD are required.
TPP, Lipoic acid, FAD
RCOCO2H + NAD+ +CoASH
&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;&emdash;&emdash
;&emdash;> RCO-S-CoA + CO2 + NADH +H+
With a few notable exceptions, prior to 1950 pyruvate and -keto-glutarate oxidation had
been studied with particulate preparations from animal tissues and micro-organisms that
were unsuitable for detailed analysis. However, these studies, notably those of Peters and
his associates at Oxford, including Ochoa, had shown that thiamin diphosphate is
required by enzymes that catalyze a decarboxylation of -keto acids. Other important
developments in the late 1940s and early 1950s were Lipmann's discovery of coenzyme
A, Stadtman's discovery of phosphotransacetylase and elucidation of the reaction
catalyzed by this enzyme, and Lynen's demonstration of the thioester linkage in acetylCoA.
Solubilization of bacterial and animal -keto acid oxidation systems in the early 1950s in
the laboratories of Ochoa and Green was a significant advance. Korkes, Gunsalus, and
Ochoa demonstrated that dismutation of pyruvate by enzyme preparations from E. coli
and S. faecalis required a divalent metal ion, thiamin diphosphate, CoA, and NAD+. They
succeeded in separating the pyruvate oxidation system of E. coli into two components,
designated Fraction A and Fraction B. Jaganathan and Schweet isolated a pyruvate
oxidation system from pigeon breast muscle in a highly purified state, with an apparent
molecular weight of about 4 million. These preparations were shown subsequently to
reduce NAD+ and to acetylate CoA. I remember Dick Schweet telling me about the
skepticism expressed by some well-known enzymologists concerning the nature and
purity of his "pyruvic oxidase" preparations. One prominent enzymologist suggested that
Schweet had isolated a membrane fragment, and that if he continued with the purification
he would eventually obtain a soluble enzyme with a respectable molecular weight.
Seymour Kaufman showed that dismutation of -ketoglutarate by soluble preparations
from pig heart required NAD+ and CoA and that one of the products was succinyl CoA.
Sanadi and Littlefield isolated the -ketoglutarate oxidation system from pig heart as a
highly purified preparation with an apparent molecular weight of 2 million and showed
that NAD+ and CoA were the natural electron and acyl acceptors.
The next important development was the isolation and characterization of lipoic acid
described above. The presence of a disulfide linkage in lipoic acid recalled the interesting
results of Peters and co-workers, who had observed a rather specific inhibition of the
pigeon brain pyruvate oxidation system by trivalent arsenicals, particularly Lewisite, and
a reversal of this toxic action by the dithiol 2,3-dimercaptopropanol (British antiLewisite, BAL), but not by monothiols. They postulated the existence of a dithiol
structure as part of the pyruvate oxidation system. These results were duplicated by
Gunsalus and associates with S. faecalis cells, and interpreted as indicating the
involvement of dihydrolipoic acid in pyruvate oxidation. Gunsalus proposed that lipoic
acid underwent a cycle of reactions in -keto oxidation comprising reductive acylation,
acyl transfer, and electron transfer. Lipoic acid was visualized as functioning after TPP
and before CoA and NAD+. Gunsalus, Hager, and associates obtained evidence for this
proposal using lipoic acid and derivatives thereof in substrate amounts. They
demonstrated that E. coli Fraction A contained a lipoyl transacetylase and that Fraction B
contained a lipoyl dehydrogenase. In the late 1950s, Vince Massey showed that the lipoyl
dehydrogenase component of the pig heart -ketoglutarate dehydrogenase complex is
identical with Straub diaphorase, a flavoprotein described in 1939. [Franklin Leach
observed that he could ascertain when the pyruvate complex was precipitated by its
yellow color which should have been a clue to the flavin involvement]. Mechanistic
studies by Massey and later by Charles Williams elucidated the catalytic mechanism.
Model experiments conducted by Ronald Breslow with thiamin and analogs thereof led
him to propose a mechanism of thiamin diphosphate action. 2-(1-Hydroxyethyl)thiamin
diphosphate was proposed to be "active acetaldehyde." This hypothesis was confirmed
and extended by enzymic studies carried out by Lester Krampitz and by Helmut Holzer
and their associates.
In my laboratory, we developed mild procedures for purification of the pyruvate and
alpha-ketoglutarate oxidation systems from E. coli By the late 1950s, Masahiko Koike
succeeded in isolating these enzyme systems as highly purified functional units with
molecular weights in the millions (Koike et al., 1960). It was very exciting to see in the
analytical ultracentrifuge of my friend and collaborator at NIH, Bill Carroll, a major
symmetrical peak for each of the two highly purified preparations, and that the boundary
of the yellow color of the flavoprotein was associated with the main peak. The molecular
weights of these multi-enzyme units were determined to be 4.8 and 2.4 million,
respectively. By careful, and persistent work over a period of several years, we dissected
the pyruvate and alpha-ketoglutarate dehydrogenase complexes into their component
enzymes and reassembled the large functional units from the isolated enzymes (Koike et
al., 1963). We demonstrated that each of these functional units is composed of multiple
copies of three enzymes, a pyruvate or alpha-ketoglutarate decarboxylase-dehydrogenase
(E1), a dihydrolipoyl acetyltransferase or succinyltransferase (E2), and the flavoprotein,
dihydrolipoyl dehydrogenase (E3). T hese three enzymes, acting in sequence, catalyze the
reactions shown below.
CH3COCO2- + E1TPP + H+ <==> CO2 + E1 CH3C(OH)=TPP (1)
E1 CH3C(OH)=TPP + E2_LipS2 <==> E1TPP + E2-Lip(SH)-S-COCH3 (2)
E2-Lip(SH)-S-COCH3 + CoASH <===> E2_Lip(SH2) + CH3COSCoA (3)
E2-Lip(SH2) + E3FAD <===> E2-LipS2 + Dihydro-E3FAD (4)
Dihydro-E3FAD + NAD+ <===> E3FAD + NADH + H+ (5)
Fig. 2 Reaction sequence in -keto acid oxidation. Abbreviations: TPP, thiamin
diphosphate; LipS2 and Lip(SH)2, lipoyl moiety and its reduced form.
E1 catalyzes both the decarboxylation of the -keto acid (reaction 1) and the subsequent
reductive acylation of the lipoyl moiety that is covalently bound to E2 (reaction 2). E2
catalyzes the acyl transfer to CoA (reaction 3), and E3 catalyzes the re-oxidation of the
dihydrolipoyl moiety with NAD+ as the ultimate electron acceptor (reactions 4 and 5).
Hayao Nawa showed in the late 1950s that the lipoyl moiety in the E. coli pyruvate and
-ketoglutarate dehydrogenase complexes is attached in amide linkage to the epsilonamino group of a lysine residue (Nawa et al., 1960). An enzyme that hydrolyzes the
lipoyllysyl linkage, lipoamidase, as well as an ATP-dependent enzyme that
reincorporates the lipoyl moiety, i.e., a lipoate-protein ligase, were detected in S. faecalis
extracts and partially purified. [This was the lipooic acid activating story which has been
told above and which was the subject of Franklin Leach's thesis]. We proposed that this
linkage provides a flexible arm, about 14 Å in length, for the reactive 1,2-dithiolane ring,
permitting the lipoyl moiety to rotate among the catalytic sites of the three component
enzymes of each complex. This is the so-called "swinging-arm" active-site coupling
mechanism. Some 15 years later, Richard Perham and Cees Veeger and their associates
attached a spin label to the protein-bound lipoyl moieties and showed by ESR
spectroscopy that the lipoyl moieties exhibited considerable rotational mobility.
These were exciting times for us in the late 1950s and early 1960s. We visualized the E.
coli PDH complex as an organized mosaic of enzymes. To obtain evidence for this
hypothesis, we turned to electron microscopy. I contacted Humberto Fernandez-Moran,
who was then at the Massachusetts General Hospital, and arranged to bring a sample of
the PDH complex to his laboratory. This was in January of 1962, and was indeed a
memorable occasion. When our sample was negatively stained with phosphotungstate to
provide contrast and then examined in the electron microscope, we saw a beautifully
organized structure. The particles seen in the electron microscope had a diameter of about
300 Å, and there was a definite indication of subunits arranged in a regular manner
(Fernandez-Moran et al., 1964). Within about two years, we set up an electron
microscopy laboratory in the Biochemical Institute at The University of Texas. Electron
microscopy studies were carried out by my associate Robert Oliver, X-ray
crystallographic studies by collaborators David DeRosier and Marvin Hackert, and
sedimentation equilibrium molecular weight determinations by Petr Munk. The results
demonstrated that both the acetyltransferase and the succinyltransferase (E2) consist of
24 apparently identical polypeptidechains arranged as eight trimers in a cube-like particle
exhibiting octahedral (432) symmetry. Multiple copies of E1 and E3 are attached to E2
by noncovalent bonds. In the PDH complex, 12 E1 dimers and 6 E3 dimers are
apparently arranged, respectively, on the 12 edges and in the six faces of E2 (Reed,
1974).
In the late 1960s part of our research effort was directed toward isolation and
characterization of the mammalian pyruvate and -ketoglutarate dehydrogenase
complexes, which are localized to mitochondria, within the inner membrane-matrix
compartment. Procedures were developed for preparation of mitochondria on a large
scale from bovine kidney and heart (with the advice and assistance of my friend and
colleague, Dan Ziegler), and relatively mild procedures were developed to isolate the
pyruvate and -ketoglutarate dehydrogenase complexes from the mitochondrial extracts.
In the course of attempts to stabilize these complexes in crude extracts of bovine kidney
mitochondria, Tracy Linn observed that the PDH complex, but not the alphaketoglutarate dehydrogenase complex, underwent a time-dependent inactivation in the
presence of ATP. A systematic investigation revealed that the bovine kidney and heart
PDH complexes are regulated by a phosphorylation-dephosphorylation cycle (Linn et al.,
1969).
Phosphorylation and concomitant inactivation of the complex is catalyzed by an ATPdependent kinase, which is tightly bound to the complex, and dephosphorylation and
concomitant reactivation is catalyzed by a Mg2+-dependent phosphatase, which is loosely
attached to the complex. It seemed curious at the time (1968) that inactivation of the PDH
complex by phosphorylation had not been detected earlier. The explanation may lie in a
remark by Henry Lardy after receiving a preprint of our paper on the phosphorylation and
inactivation of the PDH complex. (This finding) "explains why we have never been able
to get pyruvate to be oxidized in submitochondrial particles, because we invariably add
ATP to keep things in the 'optimum' state." This control mechanism was subsequently
confirmed in the laboratories of Otto Wieland, Philip Randle, S.E. Severin, and other
investigators with preparations of the PDH complex from other mammalian tissues and
from pigeon breast muscle, plant tissue, and Neurospora crassa.
Over a period of several years our group separated the bovine kidney and heart PDH
complexes into their component enzymes, including the kinase and the phosphatase, and
characterized the individual enzymes (Linn et al., 1972). The bovine heart PDH complex
has a molecular weight of about 9.5 million. Its subunit composition is now known to be
60 E2 subunits, 30 E1 tetramers (alpha2beta2), and 12 E3 dimers, which are positioned
on the E2 core by 12 E3-binding protein (protein X) monomers. The E1alpha subunit
undergoes phosphorylation and dephosphorylation. The appearance of E2 in the electron
microscope is that of a pentagonal dodecahedron, and its design is based on icosahedral
(532) symmetry. We proposed that the E1 tetramers are located on the 30 edges and the
E3 dimers in the 12 faces of the pentagonal dodecahedron.
A novel architectural feature of dihydrolipoamide acyltransferases was revealed initially
in our laboratory by limited proteolysis of the E. coli acetyltransferase and by electron
microscopy. Dennis Bleile found in the late 1970s that trypsin cleaved the
acetyltransferase, which contained radioactive lipoyl moieties, into two large fragments
(Bleile et al., 1979). One fragment, designated the lipoyl domain, contained the
covalently bound lipoyl moieties and exhibited an extended structure. The other tryptic
fragment exhibited a compact structure and contained the active site, the intersubunit
binding sites, and the binding sites for E1 and E3. The assemblage of compact catalytic
domains constitutes the inner core of E2, conferring the cube-like appearance in the
electron microscope. The two domains are connected by a trypsin-sensitive hinge region.
We suggested that movement of lipoyl domains and not simply rotation of lipoyllysyl
moieties provides the means to span the physical gaps between catalytic sites on the
complex. These early findings on the domain structure of dihydrolipoamide
acyltransferases were confirmed and extended by studies involving molecular genetics,
limited proteolysis, and proton NMR spectroscopy in the laboratories of John Guest and
Richard Perham. Briefly, the amino-terminal segment possesses one, two, or three lipoyl
domains, followed by a domain that is involved in binding E3 and/or E1, and then by a
catalytic domain that contains the active site as well as additional subunit binding sites.
The domains are connected by flexible segments or hinge regions that are rich in alanine,
proline, and charged amino acid residues. Recently, Wim Hol and associates determined
the crystal structure at 2.6 Å resolution of the cube-like inner core of the
dihydrolipoamide acetyltransferase from the Azotobacter vinelandii PDH complex.
Richard Perham and associates used multi-dimensional NMR to determine the threedimensional solution structures of the lipoyl domain and the E1/E3-binding domain of the
acetyltransferase from Bacillus stearothermophilus. Hol and associates also determined
the crystal structure of this E3-binding domain complexed with an E3 dimer. These
structures provide a deeper understanding of how the lipoyl domain can move between
the active sites of E2 and E3 in the PDH complex.
In the late 1970s Flora Pettit and Steve Yeaman purified to apparent homogeneity and
characterized the bovine branched-chain -keto acid dehydrogenase complex. In the
1980s Zahi Damuni isolated and characterized the phosphatase that participates in the
regulation of this complex, and he also isolated and characterized a potent heat-stable
inhibitor of the phosphatase.
Hormonal regulation of the mammalian PDH complex is particularly fascinating because
it involves signal transduction not only across the cell membrane but also across the inner
mitochondrial membrane to target the PDH phosphatase and, consequently, the PDH
complex, located in the mitochondrial matrix. It is now known that the major regulators
of the phosphatase activity are Ca2+ and Mg2+, which involve the hormones epinephrine
and insulin, respectively. In the early 1970s our group partially purified PDH phosphatase
from bovine heart and kidney mitochondria and showed that it requires Mg2+ or Mn2+ for
activity. Denton, Randle, and Martin subsequently reported that Ca2+ stimulated the
activity of the phosphatase in the presence of Mg2+. Flora Pettit and Tom Roche in our
group showed that Ca2+ mediates translocation of the phosphatase to the E2 component
of the PDH complex, presumably in proximity to its substrate, phosphorylated E1,
thereby increasing the rate of dephosphorylation. This Ca2+-mediated translocation
apparently is the molecular basis of the epinephrine-induced activation of PDH
phosphatase observed by Richard Hansford, Richard Denton, and other investigators.
In the early 1980s Martin Teague, Flora Pettit, and co-workers purified PDH phosphatase
to near homogeneity and showed that it consists of a Mg2+-dependent and Ca2+stimulated catalytic subunit (50 kDa; PDPc) and a flavoprotein of unknown function (100
kDa; later designated PDPr) (Teague et al., 1982). Zahi Damuni showed that polyamines,
particularly spermine, increase the sensitivity of PDH phosphatase to Mg2+. Denton and
associates subsequently showed that insulin stimulates the activity of PDH phosphatase
in adipose tissue by increasing the sensitivity of the phosphatase to Mg2+. Spermine
apparently mimics the insulin effect. The function of PDPr remained a mystery until
Janet Lawson recently cloned and expressed cDNA encoding PDPc. By comparing the
properties of recombinant PDPc and the native PDH phosphatase heterodimer (PDPc
bound to PDPr), we obtained insight into the function of PDPr. Jiangong Yan found that
PDPr decreases the sensitivity of PDPc to Mg2+ and that spermine increases the
sensitivity of PDH phosphatase but not PDPc to Mg2+, apparently by interacting with
PDPr (Yan et al., 1996). We interpret these observations to indicate that PDPr blocks or
distorts the Mg2+-binding site of PDPc and that spermine produces a conformational
change in PDPr (allosteric effect) that reverses its inhibitory effect. These observations
raise the intriguing prospect that an insulin-induced allosteric effect on PDPr may
underlie its stimulation of PDH phosphatase activity.
To gain further understanding of structure-function relationships in eukaryotic PDH
complexes, we initiated in the late 1980s molecular genetic studies of the PDH complex
in the yeast Saccharomyces cerevisiae. The genes encoding the five proteins comprising
the complex (E1 , E1, E2, E3BP, and E3) were cloned, sequenced, expressed, and
disrupted. Studies on E3-binding protein (E3BP) confirmed and extended previous
studies of Tom Roche and of Gordon Lindsay and their associates with the protein X
component of the bovine PDH complex. E3BP and E2 apparently evolved from a
common ancestor. E3BP possesses an amino-terminal lipoyl domain, followed by an E3binding domain, and then by a carboxyl-terminal domain that is involved in anchoring
E3BP to the inner core of E2. Binding studies in conjunction with cyroelectron
microscopy and three-dimensional image reconstruction in collaboration with James
Stoops and Timothy Baker and their associates revealed a unique structural organization
of the S. cerevisiae PDH complex and, by analogy, of the mammalian complex (Stoops et
al., 1977). E2 consists of 20 cone-shaped trimers at the vertices of a pentagonal
dodecahedron. There are 12 large openings that lead into a central cavity. It was generally
believed that the other components of these complexes are bound on the outside of the E2
scaffold. By contrast, our results show that E3BP binds near the tips of the E2 trimers
within the central cavity and anchors an E3 dimer inside each of the 12 pentagonal faces
of E2. Our finding that the E2 structure, with 532 molecular symmetry, can physically
accommodate only one BP-E3 complex in each of its 12 pentagonal-shaped faces
provides a satisfactory explanation of the unique polypeptide chain ratio in the S.
cerevisiae and mammalian PDH complexes (60 E1:60 E1:60 E2:12 BP:24 E3).
I hope these recollections have given some appreciation of the thrill and excitement I
have experienced in establishing this trail of research from lipoic acid to the structure,
function, and regulation of the -keto acid dehydrogenase complexes. I have been
accompanied in the various stages of this journey by excellent associates, including
undergraduate, graduate, and postdoctoral students, technicians, and members of the
senior staff of the Biochemical Institute, and by collaborators at other universities and
institutes.
Acknowledgments
I am pleased to acknowledge the Clayton Foundation for Research and the National
Institutes of Health for generous financial support.
References
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component of pyruvate dehydrogenase complex from Escherichia coli. Proc Natl Acad Sci USA 76:43854389.
Fernandez-Moran H, Reed LJ, Koike M, Willms CR. 1964. Electron microscopic and biochemical studies
of pyruvate dehydrogenase complex of Escherichia coli. Science 145:930-932.
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dephosphorylation. Proc Natl Acad Sci USA 62:234-241.
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2
Lester J. Reed was born in New Orleans, Louisiana, in 1925 and received a B.S. in chemistry from Tulane
University in 1943. He was awarded a Ph.D. in organic chemistry from the University of Illinois in 1946
under R.C. Fuson. He was a research associate in biochemistry under Vincent du Vigeaud at Cornell
University Medical College from 1946 to 1948. He joined the Department of Chemistry at The University
of Texas in 1948 as an Assistant Professor. He is currently Ashbel Smith Professor of Chemistry and
Biochemistry. He was Director of the Clayton Foundation Biochemical Institute from 1963 to 1996. Dr.
Reed is a member of the National Academy of Sciences and the American Academy of Arts and Sciences.
He received the Eli Lilly & Co. Award in Biological Chemistry of the American Chemical Society in 1958,
an honorary doctorate from Tulane University in 1977, and the Merck Award of the American Society for
Biochemistry and Molecular Biology in 1994.