Tài liệu Báo cáo khoa học: Site-directed mutagenesis of a loop at the active site of E1 (a2b2) of the pyruvate dehydrogenase complex A possible common sequence motif docx

Site-directed mutagenesis of a loop at the active site of E1 (a
2
b
2
)
of the pyruvate dehydrogenase complex
A possible common sequence motif
Markus Fries*, Hitesh J. Chauhan
†
, Gonzalo J. Domingo
‡
, Hyo-Il Jung
§
and Richard N. Perham
Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, UK
Limited proteolysis of the pyruvate decarboxylase (E1, a
2
b
2
)
component of the pyruvate dehydrogenase (PDH) multi-
enzyme complex of Bacillus stearothermophilus has indicated
the importance for catalysis of a site (Tyr281-Arg282) in the
E1a subunit (Chauhan, H.J., Domingo, G.J., Jung, H I. &
Perham, R.N. (2000) Eur. J. Biochem. 267, 7158–7169). This
site appears to be conserved in the a-subunit of hetero-
tetrameric E1s and multiple sequence alignments suggest
that there are additional conserved amino-acid residues in
this region, part of a common pattern with the consensus
sequence -YR-H-D-YR-DE This region lies about 50
amino acids on the C-terminal side of a 30-residue motif

previously recognized as involved in binding thiamin
diphosphate (ThDP) in all ThDP-dependent enzymes. The
role of individual residues in this set of conserved amino
acids in the E1a chain was investigated by means of site-
directed mutagenesis. We propose that particular residues
are involved in: (a) binding the 2-oxo acid substrate,
(b) decarboxylation of the 2-oxo acid and reductive acety-
lation of the tethered lipoyl domain in the PDH complex,
(c) an Ôopen–closeÕ mechanism of the active site, and
(d) phosphorylation by the E1-specific kinase (in eukaryotic
PDH and branched chain 2-oxo acid dehydrogenase com-
plexes).
Keywords: pyruvate dehydrogenase; multienzyme complex;
thiamin diphosphate; limited proteolysis; enzyme mecha-
nism.
The family of 2-oxo acid dehydrogenase (2-OADH) multi-
enzyme complexes contains three members: the pyruvate
dehydrogenase (PDH), the 2-oxoglutarate dehydrogenase
(OGDH) and the branched-chain 2-oxo acid dehydrogenase
(BCDH) complexes. These are responsible for the oxidative
decarboxylation of pyruvate, 2-oxoglutarate and branched-
chain 2-oxo acids, respectively, in each case generating the
corresponding acyl-CoA and NADH. The complexes all
occupy important positions in the metabolism of the cell
and generally comprise three component enzymes: a 2-oxo
acid decarboxylase (E1, EC 1.2.4), a dihydrolipoyl acyl-
transferase (E2, EC 2.3.1), and dihydrolipoyl dehydroge-
nase (E3, EC 1.8.1.4). E1 catalyses the first and irreversible
step of the overall reaction, the thiamin-diphosphate
(ThDP)-dependent oxidative decarboxylation of the 2-oxo

acid, followed by the reductive acylation of a lipoyl
prosthetic group covalently bound to a lysine residue in
the lipoyl domain of the E2 chain. The reaction catalysed by
E1 is rate-limiting for the overall activity of the complex
[1,2], probably at the reductive acylation step [2,3]. The E2
component catalyses the transfer of the acyl group from the
lipoyl-lysine group to CoA, and the cycle is completed by
reoxidation of the resulting dihydrolipoyl group catalysed
by E3, a flavoprotein, generating NADH and H
+
from
NAD
+
. For recent reviews, see de Kok et al.andPerham
[4,5].
In eukaryotic PDH and BCDH complexes, modulation
of catalytic activity is achieved by phosphorylation-
dephosphorylation of E1; an E1-specific kinase inactivates
E1 by phosphorylating certain serine residues in the E1a
component and activity is restored by the action of a specific
phosphatase [6–8]. Depending on the organism and the type
of 2-OADH complex, the E1 component exists either as a
heterotetramer (a
2
b
2
) or a homodimer (a
2
). In the PDH
complexes of Gram-negative bacteria and in all OGDH

complexes, E1 is a homodimer; in PDH complexes of
Correspondence to R. N. Perham, Department of Biochemistry,
University of Cambridge, Sanger Building, Old Addenbrooke’s Site,
80 Tennis Court Road, Cambridge CB2 1GA, UK.
Fax: + 44 1223 333667, Tel.: + 44 1223 333663,
E-mail:
Abbreviations: BCDH, branched-chain 2-oxo acid dehydrogenase;
DCPIP, 2,6-dichlorophenolindophenol; E1, 2-oxo acid decarboxylase;
E1a,alphasubunitofE1;E1b, beta subunit of E1; E1p, pyruvate
decarboxylase of PDH complex; E2p, dihydrolipoyl acetyltransferase;
E3, dihydrolipoyl dehydrogenase; IPTG, isopropyl thio-b-
D
-galacto-
side; OGDH, 2-oxoglutarate dehydrogenase; PSBD, peripheral
subunit-binding domain; SPR, surface plasmon resonance;
ThDP, thiamin diphosphate.
Enzymes: pyruvate decarboxylase (EC 1.2.4.1); dihydrolipoyl acetyl-
transferase (EC 2.3.1.12); dihydrolipoyl dehydrogenase (EC 1.8.1.4).
*Present address: Cambridge Institute for Medical Research,
Wellcome Trust/MRC Building, Box139 Addenbrooke’s Hospital,
Hills Road, Cambridge CB2 2XY, UK.
Present address: Adprotech Ltd, Chesterford Research Park, Little
Chesterford, Saffron Walden, Essex CB10 1XL, UK.
àPresent address: Seattle Biomedical Research Institute, 4 Nickerson
Street, Seattle, WA 98109, USA.
§Present address: Wolfson Institute for Biomedical Research, The
Cruciform Building, Gower Street, London WC1E 6BT, UK.
(Received 19 September 2002, revised 5 December 2002,
accepted 20 December 2002)
Eur. J. Biochem. 270, 861–870 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03444.x

Gram-positive bacteria and eukaryotes and in BCDH
complexes, E1 is a heterotetramer [9]. In all cases, E1 acts as
a functional dimer with two active sites of equal catalytic
efficiency [10,11] and two ThDP per E1, albeit bound with
different affinities [11–13]. There is some evidence that the
two active sites of pigeon breast muscle E1p work in an
Ôalternating site mechanismÕ and that intersubunit inter-
actions may play an essential part in the catalysis by, and
regulation of, the enzyme [11]. This is consistent with the
finding that phosphorylation of only one of two active sites
in mammalian E1p leads to complete inactivation [7].
Crystal structures of the heterotetrameric E1s from the
Pseudomonas putida [14] and human [15] BCDH complexes,
both E1(a
2
b
2
) heterotetramers, are available (Fig. 1). The
E1a chain houses a conserved ThDP-binding site [16], the
phosphate groups of ThDP being anchored by residues of
this sequence motif through a bound Mg
2+
ion, and the
aminopyrimidine end bound by the b-subunit [17]. Thus,
both subunits are needed for a catalytically active enzyme.
Limited proteolysis of E1 (a
2
b
2
) from the PDH com-

plexes of Bacillus subtilis [18] and pig heart [19] leads to
cleavage of the E1a subunit, whereas the E1b subunit
remains intact. A detailed study of the limited proteolysis of
the E1 component of B. stearothermophilus PDH complex
[20] identified Tyr281 (for chymotrypsin) and Arg282 (for
trypsin) of the E1a subunit, two conserved residues in a loop
at the entrance to the active site (Fig. 1), as the main
cleavage sites. Cleavage of the E1a-subunit activated the
enzyme, as determined by a decarboxylation assay of the
free E1 in the presence of an artificial electron acceptor, but
the catalytic activity of a reconstructed PDH complex was
found to be substantially lowered. Multiple sequence
alignments reveal additional conserved residues in the
region of Tyr281 and Arg282. We describe here a study of
the role of these conserved residues and compare the results
with those of limited proteolysis in ascribing possible
functions to them.
Materials and methods
Materials
Restriction endonucleases were obtained from Pharmacia
Biotech (St Albans, UK) or New England Biolabs (Hitchin,
UK), Pfu DNA polymerase was from Stratagene (Cam-
bridge, UK) and T4 DNA ligase from Promega
Fig. 1. Crystal structure of heterotetrameric
(a
2
b
2
)E1.(A) P. putida E1b [14]. (B) Human
E1b [15]. The two a-subunits are shown in

orange and red, the two b-subunits in light
blue and dark blue. The cofactor ThDP-
Mg
2+
, located at the interface between an
a- and b-subunit, is indicated in spacefill.
Residues 301–314 are not included in the
human E1b structure, so that not all residues
of the sequence motif could be highlighted.
(C) Conserved active site motif derived
from the crystal structure of P. putida E1b.
(D) Conserved active site motif derived from
the crystal structure of human E1b.
862 M. Fries et al. (Eur. J. Biochem. 270) Ó FEBS 2003
(Southampton, UK). Isopropyl thio-b-
D
-galactoside
(IPTG) and phenylmethanesulfonyl fluoride were purchased
from Melford Laboratories (Chelsworth, UK), bacterio-
logical media from Beta Laboratory (West Molesely, UK)
and Duchefa (Haarlem, the Netherlands), and ampicillin
from Beecham Research Laboratories (Brentford, UK).
Pyruvate, NAD
+
, ThDP, 2,6-dichlorophenolindophenol
(DCPIP), coenzyme A and FAD were from Sigma (Poole,
UK). Solid [2-
14
C]pyruvic acid, sodium salt (specific activity,
15.9 mCiÆmmol

)1
) was from New England Nuclear (Bos-
ton, MA, USA). Centrifugal filter devices were purchased
from Millipore.
Bacterial strains and plasmids
E. coli strain TG1recO and plasmids pKBstE1a and
pKBstE1b, expressing genes encoding the B. stearother-
mophilus E1a and E1b subunits, respectively, have been
described previously [21]. E. coli strain BL21 (DE3) [B,
F
–
, ompT, hsdS
B
(r
B
-,m
B
-), gal, dcm (DE3)] from Novagen
(Madison, USA) was employed to express the B. stearo-
thermophilus E3 gene (from pBSTNAV/E3) and E2p gene
(from pETBstE2) [21] and a subgene encoding a di-domain
that comprises the lipoyl domain, the peripheral subunit-
binding domain and the linker between them (from
pET11d2D and pET11ThDD). The subgene in plasmid
pET11d2D encodes residues 1–171 of the wild-type B. ste-
arothermophilus E2p chain [22]; plasmid pET11ThDD
encodes the same di-domain but with a thrombin-cleavage
site inserted in the linker region between the lipoyl and
binding domains [23]. Lipoate protein ligase A was purified
from E. coli BL21 (DE3) cells transformed with the plasmid

pTM202 [24].
Recombinant DNA techniques and mutagenesis
Recombinant DNA techniques were carried out as described
elsewhere [25]. DNA fragments were isolated from gels using
the QIAquick gel extraction kit, and plasmids were prepared
by means of the Qiagen plasmid kit, both from Qiagen
(Hilden, Germany). Site-specific mutations were introduced
into the plasmid pKBstE1a using splicing-by-overlap exten-
sion PCR [26]. The fidelity of the amplified DNA fragments
was established by DNA sequence analysis after subcloning
into the vector. All the sequences differed slightly from the
original pKBstE1a plasmid [21] in the noncoding region
following the stop codon. The differences (four C instead of
three C at positions 1405–1407, and two additional G after
position 1435) were the same in all instances, suggesting that
there may have been errors in this part of the original
pKBstE1a sequence.
Protein purification
Purification of wild-type and mutant E1s was carried out
as described previously [21,27]. The E1aS283C mutant
was purified with 1 m
M
dithiothreitol added to all
buffers to prevent formation of disulfide bonds. E2
and E3 were purified as described elsewhere [28]. The
E2p di-domain [29] and the thrombin-cleavable di-
domain and the lipoyl domain subsequently released by
thrombin treatment [23] were purified essentially as
described elsewhere. The apo-forms of the lipoyl domain,
di-domain and the E2 component were lipoylated by

means of lipoate protein ligase A, also essentially as
described elsewhere [22,30].
Enzyme assays
The E1 component was assayed for catalytic activity by
means of three different assays.
DCPIP assay. This measures the rate of reduction of the
artificial electron acceptor, DCPIP, by the E1 component
[31]. The decrease in A
600
was monitored at a temperature of
30 °C. The assay mixture contained 0.2 m
M
ThDP, 2 m
M
MgCl
2
,50l
M
DCPIP, 100 m
M
potassium phosphate,
pH 7.0 and 20 lg of E1. The reaction was started by
adding pyruvate (final concentration 400 l
M
) after the assay
mix had been incubated for 10 min at 30 °C.
Reductive acetylation assay. This measures the reductive
acetylation of the free lipoyl domain with [2-
14
C]pyruvate at

25 °C [30,32]. The assay mixture (350 lL) contained
0.2 m
M
ThDP, 1 m
M
MgCl
2
,0.3mgÆmL
)1
BSA, 20 m
M
potassium phosphate, pH 7.0, plus 3 nmol lipoyl domain
and 5 lg E1. The reaction was started by addition of
14
C-
labelled pyruvate (1 lCi, about 60 nmol) after the assay mix
had been incubated for 10 min at 25 °C.
PDH assay. This measures the rate of formation of
NADH at 340 nm and 30 °C after the complex has been
reconstituted from its individual components [1,33,34]. The
assay contained 0.2 m
M
ThDP, 1 m
M
MgCl
2
,2.6m
M
cysteine HCl, 2 m
M

pyruvate, 0.13 m
M
CoA, 50 m
M
potassium phosphate, pH 7.0. E1, E2 and E3 were added
in molar ratios of E1(a
2
b
2
)/E2(a)/E3(a
2
)of3:1:3;the
amount of E2 used was 0.5 lg, 1.0 lg, 1.5 lgand2.0lg.
The reaction was started by adding the pyruvate and CoA
after the PDH complex had been allowed to assemble in
the assay mixture for at least 3 min at 30 °C. Specific
activities for the reconstituted PDH complex are expressed
as U per mg E2. All assays were normally carried out
at least three times and the results are expressed as the
mean ± SD.
Determination of kinetic parameters
K
m
values for pyruvate and ThDP were determined using
the DCPIP assay by varying the concentrations of pyruvate
from 0.2 l
M
to 4000 l
M
and the ThDP concentrations from

0.04 l
M
to 200 l
M
. When determining the kinetic param-
eters for ThDP, the pyruvate concentration was kept at
1m
M
. Data were analysed and fitted to a Michaelis–
Menten curve using the
SIGMA PLOT
software.
Temperature-dependence of catalytic activity
The dependence of the catalytic activity of E1 on tempera-
ture was investigated using the DCPIP assay over a
temperature range of 15 °Cto85°C. Assay mixtures were
incubated for exactly 10 min at the relevant temperature
before the reaction was started by the addition of pyruvate
and then monitoring the decrease in A
600
.
Ó FEBS 2003 Active site of E1 component of PDH complex (Eur. J. Biochem. 270) 863
Interaction of E1 with the peripheral subunit-binding
domain
Mixtures of E1 and di-domain were submitted to non-
denaturing PAGE using the Pharmacia Phast System. The
interaction of di-domain with E1 was also investigated using
surface plasmon resonance detection (BIAcore, Pharmacia
Biosensor AB), immobilizing the lipoyl domain by means of
its lipoyl group. Both sorts of experiment were carried out as

described in detail elsewhere [35].
Results
Multiple sequence alignment of E1 components
of 2-OADH complexes
No obvious sequence homology can be detected between
homodimeric forms of E1p and E1o. Likewise, amino-acid
sequences of heterotetrameric (a
2
b
2
) E1s do not readily
align with sequences of homodimeric E1s. However, a
sequence motif of about 30 amino acids, beginning with a
highly conserved amino-acid triplet of GDG and ending
withNN,hasbeenidentifiedascommontoallThDP-
dependent enzymes [16], and confirmed as a ThDP-binding
motif in the crystal structures of several ThDP-utilizing
enzymes [17]. Aside from this motif, no active site residues
other than those directly in contact with ThDP have been
reported as conserved [36].
However, inspection of the available E1 sequences
suggests that there may be another set of amino-acid
residues common to the E1 (a
2
b
2
) components of 2-OADH
complexes. This consensus set, -Y/F/WR-H-D-YR-D/EE-
in the E1a chains (Fig. 2), includes the Y281R282 site of
limited proteolysis [20] and lies about 50 amino-acid

residues to the C-terminal side of the ThDP-binding motif.
The crystal structures of P. putida [14] and human [15] E1b
show them as located on a loop region in Ela close in space
to the cofactor ThDP-Mg
2+
(Fig. 1). Also included in this
region are two serine residues in the Ela chain of mamma-
lian PDH and BCDH complexes that serve as phosphory-
lation sites 1 and 2, responsible for inhibiting E1 when they
become phosphorylated. Other consistently conserved resi-
dues in the aligned sequences appear to mark the beginning
and end of the loop region: notably -PXXXE- (where X is
generally a large aliphatic residue) at the N-terminal end,
and -DP- (or -DHP-) at the C-terminal end (Fig. 2).
Choice of mutations
The importance of the loop region in the a-subunit of the
B. stearothermophilus E1p component was highlighted by its
susceptibility to limited proteolysis with trypsin and chymo-
trypsin and the effects of the cleavages on catalytic activity
[20]. In the present study, residues Phe266, Arg267 and
Asp276 in the loop region of B. stearothermophilus E1a were
replaced with alanine,as were Tyr281 and Arg282, the sites of
cleavage with chymotrypsin and trypsin, respectively. Like-
wise the neighbouring residue, Ser283, corresponding to a
phosphorylation site in eukaryotic BCDH complexes, was
also replaced with alanine. The Y281S/R282S mutation was
constructed to test the effect of preserving a hydrophilic but
uncharged character in the loop region, and the S283C
mutant was a replacement of the serine hydroxyl group with
the more nucleophilic thiol group (also potentially capable of

subsequent chemical modification, if required).
Effects of mutations on catalytic activity
The catalytic activity of E1 mutants was investigated using
three different assays: the DCPIP assay, which measures E1
activity bymonitoring the reduction ofDCPIP as an artificial
electron acceptor instead of lipoamide; the reductive acety-
lation assay, which measures the rate of reductive acetylation
of the lipoyl group on a free lipoyl domain; and the PDH
assay, which measures the overall activity of a PDH complex
reconstituted from E2, E3 and the relevant mutant E1.
In all assays, the E1aS283C and E1aF266A mutants
behaved essentially the same as wild-type E1. In the DCPIP
assay, the E1aY281A and E1aR282A mutants displayed a
catalytic activity about twice that of wild-type E1. The single
mutants E1aR267A and E1aD276A and the multiple
mutants E1aY281A/R282A/S283A and E1aY281S/R282S
displayed catalytic activities 2.5 and 3.5 times, respectively,
that of wild-type E1. In contrast, the reductive acetylation
assay showed no significant changes for the mutants
compared with wild-type E1. However, in the PDH assay,
the catalytic activity fell to about 50% of the wild-type value
for the E1aD276A and E1aR282A mutants and to about
25% for the E1aY281A mutant. The double mutants
showed even lower (< 20%) activity, but the most
Fig. 2. Active site sequence alignment of the E1a chains of 2-OADH
complexes. The numbering refers to the sequence of the E1a chain
from B. stearothermophilus (ODPA_BACST). The sequences were
taken from the SwissProt and NCBI databases and aligned using
CLUSTALW
1.8. (*), identical residues in sequences in the alignment by

CLUSTALW
; (:), conserved substitutions; (.), semiconserved substitu-
tions. Residues conserved in all aligned sequences are printed in bold,
the conserved sequence motif at the active site is underlined. P1 and P2
mark phosphorylation sites 1 and 2 (serine residues) in the E1a chains
of eukaryotic PDH and BCDH complexes. ODPA and ODPT, E1a
chain of PDH complexes with heterotetrameric E1(a
2
b
2
); ODBA, E1a
chain of heterotetrameric E1(a
2
b
2
)ofBCDHcomplexes.
864 M. Fries et al. (Eur. J. Biochem. 270) Ó FEBS 2003
significant drop in PDH complex activity (to < 10%) was
experienced by the E1aR267A mutant (Table 1).
Determination of kinetic parameters
Kinetic parameters for the mutant E1s were determined for
the substrate pyruvate and the cofactor ThDP using the
DCPIP assay (Table 2). All activities recorded were good
fits to Michaelis–Menten kinetics. A big increase in the K
m
for pyruvate was observed for the E1aR267A mutant, to
nearly 300 l
M
compared with about 1 l
M

for wild-type E1.
The K
m
for pyruvate was also found to be substantially
increased to more than 50 l
M
for E1s carrying mutations at
E1aY281. This was reflected in a major drop in the value of
k
cat
/K
m
, in spite of the k
cat
being more than twice that for
wild-type E1. In contrast, the E1aD276A and E1aR282A
mutants more closely resembled wild-type E1, with a K
m
for
pyruvate only about 10 times higher. The E1aF266A and
E1aS283C mutants were almost identical to wild-type E1
(K
m
for pyruvate 1.2 l
M
and 2.1 l
M
, respectively).
Wild-type E1was found to have an apparent K
m

for ThDP
of 23 l
M
. Interestingly, the mutants with a significantly
increased K
m
for pyruvate displayed markedly (5- to 20-fold)
lower apparent K
m
s for ThDP than did wild-type E1. The
E1aR267A mutant was found to have the lowest apparent
K
m
for ThDP (< 1 l
M
), followed by the E1aY281S/R282S,
E1aY281A/R282A/S283A and E1aY281A/R282A mutants
( 3.0 l
M
). The single mutations E1aR282A (K
m
¼
5.2 l
M
), E1aY281A (K
m
¼ 5.5 l
M
)andE1aS283C
(K

m
¼ 13 l
M
) were less severe in their effects. Replacement
of E1aF266 and E1aD276 with alanine had no significant
influence on the K
m
for ThDP (K
m
¼ 21 l
M
).
Dependence of E1 activity on temperature
The temperature-dependence of the catalytic activity of E1
was measured using the DCPIP assay over a range of 25 to
85 °C (Fig. 3). The reaction mixture with E1 was incubated
for exactly 10 min at the relevant temperature and the
catalytic activity at that temperature was then determined.
The inactivation temperatures for all the E1 mutants were
found to be 5–10 °C below that of wild-type E1; mutants
with a replacement of E1aY281 tended to lose catalytic
activity at a slightly lower temperature than mutants
retaining that residue. The E1aR267A mutant displayed a
distinctly lower specific activity at higher temperatures than
the other E1a mutants or than wild-type E1. The specific
activity of the E1aD276A mutant increased steadily to
reach twice that of wild-type E1 at 65 °C, after which the
enzyme was inactivated.
Binding of E1 to the peripheral subunit-binding
domain of E2

The E1 component of the PDH complex of B. stearother-
mophilus is bound to the E2 core mainly by interaction of its
Table 2. Kinetic parameters of wild-type and mutant E1s determined by the DCPIP assay.
Pyruvate ThDP
k
cat
(s
)1
) K
m
(l
M
)
k
cat
/K
m
(
M
)1
Æs
)1
· 10
3
) k
cat
(s
)1
) K
m

(l
M
)
k
cat
/K
m
(
M
)1
Æs
)1
· 10
3
)
Wild-type 0.48 ± 0.01 1.1 ± 0.1 427 0.46 ± 0.01 23 ± 2 20
E1aF266A 0.30 ± 0.01 1.2 ± 0.1 246 0.35 ± 0.01 21 ± 2 17
E1aR267A 1.62 ± 0.02 291 ± 14 6 1.25 ± 0.01 0.56 ± 0.04 2232
E1aD276A 0.71 ± 0.01 11.1 ± 0.5 64 0.91 ± 0.02 21 ± 2 42
E1aY281A 0.82 ± 0.01 63 ± 3 13 0.97 ± 0.01 5.5 ± 0.3 177
E1aR282A 0.55 ± 0.01 8.8 ± 0.6 63 0.62 ± 0.01 5.2 ± 0.4 121
E1aS283C 0.38 ± 0.01 2.1 ± 0.2 186 0.45 ± 0.01 13 ± 1 33
E1aY281A/R282A/S283A 1.23 ± 0.02 49 ± 4 25 1.39 ± 0.02 2.9 ± 0.2 481
E1aY281A/R282A 0.96 ± 0.01 56 ± 3 17 0.98 ± 0.01 3.0 ± 0.1 326
E1aY281S/R282S 1.21 ± 0.02 66 ± 4 19 1.30 ± 0.01 2.7 ± 0.1 475
Table 1. Specific activities of wild-type and mutant E1s in the various assays.
DCPIP assay Reductive acetylation assay PDH assay
UÆmg
)1
%10

)3
UÆmg
)1
%UÆmg
)1
E2 %
Wild-type 0.128 ± 0.004 100 2.6 ± 0.3 100 12.4 ± 0.3 100
E1aF266A 0.133 ± 0.001 104 – 9.6 ± 0.2 77
E1aR267A 0.325 ± 0.002 254 – 0.93 ± 0.03 8
E1aD276A 0.319 ± 0.005 249 – 5.67 ± 0.03 46
E1aY281A 0.268 ± 0.003 210 2.8 ± 0.3 108 3.30 ± 0.02 27
E1aR282A 0.230 ± 0.007 180 2.6 ± 0.3 100 6.10 ± 0.07 49
E1aS283C 0.156 ± 0.004 122 2.3 ± 0.1 90 10.5 ± 0.3 85
E1aY281A/R282A/S283A 0.452 ± 0.003 354 3.0 ± 0.1 115 1.75 ± 0.06 14
E1aY281A/R282A 0.279 ± 0.004 218 2.2 ± 0.1 85 1.74 ± 0.04 14
E1aY281S/R282S 0.41 ± 0.01 326 2.6 ± 0.1 100 1.83 ± 0.05 15
Ó FEBS 2003 Active site of E1 component of PDH complex (Eur. J. Biochem. 270) 865
E1b chains with the PSBD of the E2 chain. To investigate
whether this interaction was impaired by the mutations in
the E1a subunit, non-denaturing PAGE and surface
plasmon resonance (SPR) detection were employed.
To assess the binding, mutant E1s were mixed with a
molar excess of di-domain (lipoyl domain and PSBD, joined
by the natural linker region) and submitted to non-dena-
turing PAGE. Binding of E1 to the di-domain will create an
E1-di-domain band shifted towards a higher molecular
mass than that of free E1. No difference in the ability of the
mutant E1s to bind to the PSBD was observed (Fig. 4A),
though it should be noted that small differences in affinity
would not be detected by this Ôband-shiftÕ assay. SPR

detection was used to determine the kinetic parameters of
the interaction of the mutant E1s with the PSBD (Fig. 4B).
For this purpose, lipoylated di-domain was immobilized on
a BIAcore sensor chip by means of the lipoyl group on the
lipoyl domain (see Materials and methods). E1 was then
injected and allowed to interact with the exposed PSBD of
the di-domain retained on the sensor surface. The k
on
(association rate constant), k
off
(dissociation rate constant)
and K
d
(dissociation constant) of the E1–PSBD interaction
were determined. The kinetic parameters for the interaction
of wild-type E1 with PSBD reported earlier [35] are: k
on
,
3.27 · 10
)6
M
)1
Æs
)1
; k
off
,1.06· 10
)3
s
)1

; K
d
,3.24·
10
)10
M
)1
. The kinetic constants and K
d
values for mutant
and wild-type E1s were found to be essentially identical
(normally within ± 10%).
Discussion
Function of the mutated amino acids
The E1aY281 and E1aR282 mutants of B. stearothermo-
philus E1 show a higher V
max
in the DCPIP assay, no change
in activity in the reductive acetylation assay and a significant
drop in overall activity in the PDH assay when compared
with wild-type E1. This is similar to the effects on E1 catalysis
of limited proteolysis at Tyr281 and Arg282 [20]. Therefore,
Fig. 4. Gels and SPR. (A) Non-denaturing polyacrylamide gel elec-
trophoresis of wild-type and mutant E1s with di-domain at a 16-fold
molarexcessofdi-domainoverE1.Lane1:E1wild-type;lane2:E1
wild-type + di-domain; lane 3: E1aF266A mutant; lane 4:
E1aF266A mutant + di-domain; lane 5: E1aR267A mutant; lane 6:
E1aR267A mutant + di-domain; lane 7: E1aD276A mutant; lane 8:
E1aD276A mutant + di-domain. The gels for the other mutants were
virtually identical (data not shown). (B) SPR sensorgrams for the

interaction of wild-type and mutant E1s with the PSBD. Shown are the
sensorgrams for wild-type E1, the E1aF266A, E1aR267A and
E1aD276A mutants. The sensorgrams of the other mutants were
essentially identical (data not shown).
Fig. 3. Temperature-dependence of the activity in the DCPIP assay of
wild-type and mutant E1s.
866 M. Fries et al. (Eur. J. Biochem. 270) Ó FEBS 2003
it is clear that those effects were not due simply to cleavage of
the Ela chain backbone at these positions.
In comparing the results from the different assays, one
should be aware of the differences in reaction conditions.
Thus, the reductive acetylation assay is performed with the
lipoyl domain at a concentration of 8.5 l
M
and E1 at
95 n
M
, whereas in the PDH assay the local concentrations
of E1 and lipoyl domains in the assembled complex are in
the millimolar range and closer to equimolar. This may be
of particular importance as the interaction between E1 and
the lipoyl domain for the E. coli PDH complex is estimated
to have a dissociation constant in the millimolar range or
higher despite the fact that the K
m
is c. 20 l
M
[37,38].
Another difference in the assays is the concentration of the
substrate, pyruvate. The different types of assay are reflected

in the different rates; the PDH assay has a turnover number
100-fold higher than the DCPIP assay and 4800-fold higher
than the reductive acetylation assay (referring to wild-type
E1, Table 1). Thus, subtle changes in the kinetics of the E1
reaction may not be detected by the reductive acetylation
assay, but will be detected by the PDH assay.
The interaction of E1 (a
2
b
2
)withthePSBDwas
unaffected by any of the mutations in E1a (Fig. 4).
Therefore, any effects of the mutations on the catalytic
activities of the PDH complex must be due to direct effects
on the reactions catalysed by E1. The falls in PDH complex
activity were most marked for the R267A mutant and all the
Y281A mutants (Table 1). Given that there were no
detectable effects on the catalytic activities in the reductive
acetylationassaymeasuredwiththefreelipoyldomainas
substrate (Table 1) and that the PDH complex activity was
measured with pyruvate at a concentration of 2 m
M
,well
above the highest K
m
(300 l
M
) recorded for any mutant E1
(R267A), the mutations appear to be affecting the ability of
E1 to catalyse the reductive acetylation of the tethered lipoyl

domain in the assembled PDH complex. It should be noted
that Tyr281 and Arg282 are located at the mouth of the
funnel-shaped active site of E1, at the bottom of which lies
the ThDP cofactor some 20–25 A
˚
from the protein ÔsurfaceÕ
[14]. Thus it may be that these mutations are affecting the
efficacy of the recognition of the tethered lipoyl domain by
E1, an essential prelude to reductive acetylation of the
pendant lipoyl group [5,9,39]. The damaging effects of
mutations in the b-turn region of the B. stearothermophilus
lipoyl domain on its reductive acetylation by native E1 have
been reported earlier [23,40].
The E1aR267A mutant of E1 exhibited a vastly
increased K
m
for pyruvate (300 l
M
) compared with wild-
type E1 (1 l
M
). Interaction of the positively charged side
chain of Arg267 with the negatively charged carboxyl
group of the 2-oxo acid substrate thus appears to be very
likely. The crystal structures of E1 show the corresponding
arginine residues in P. putida [14] and human [15] E1s to be
in a suitable position to participate in such an electrostatic
interaction (Fig. 1). In contrast, given the K
m
(9 l

M
)for
pyruvate shown by the E1aR282A mutant and its other
modest differences from wild-type E1 (Tables 1 and 2), a
direct role for Arg282 in the E1 reaction appears improb-
able. This too is consistent with the E1 crystal structures in
that the arginine residues corresponding to E1aR282 of the
B. stearothermophilus E1 are not pointing towards the
active site, identified by the C2-carbon of the cofactor
ThDP.
Although Arg282 is even more strictly conserved in
sequence alignments than Tyr281, the latter is more
important for the reaction catalysed by B. stearothermophi-
lus E1p. Apart from the K
m
for pyruvate being markedly
increased for all the Tyr281 mutants (Table 2), these
enzymes started to lose catalytic activity at lower temper-
atures than the other mutants or wild-type E1 (Fig. 3). This
suggests that Tyr281 has some part to play in conferring
thermal stability. In the crystal structure of pyruvate
decarboxylase (EC 4.1.1.1) from Zymomonas mobilis,a
tyrosine sidechain is found in the active site and is suitably
oriented to take part in catalysis, as model building with
reaction intermediates suggests [41]. It was speculated that
the tyrosine might form a hydrogen bond with the carboxy
group of 2-(2-hydroxypropionyl)-ThDP and contribute to
the stabilization of its negative charge. The conserved
Tyr281 in the B. stearothermophilus E1a chain may play a
similar role in the E1 component of this and other 2-oxo

acid dehydrogenase (Fig. 1) complexes.
The E1aD276A mutant displayed moderate changes in
its kinetic properties compared with wild-type E1, some-
what similar to those observed for the R282A mutant.
Adjacent to E1aD276 in the primary structure of the E1a
chain from B. stearothermophilus E1p is another aspartate
residue, E1aD277. This is replaced by proline in many E1a
chains (Fig. 2), and has not been examined here. However,
the corresponding residue, E1aD296 of rat E1b, has been
reported to be essential [42], so further experiments may be
justified. The E1aF266A and E1aS283C mutants behaved
almost identically to wild-type E1 in all aspects investigated;
a crucial role for Phe266 and Ser283 in the reaction
mechanism can thus be excluded.
A common sequence motif
The high conservation of amino-acid residues in the
sequence spanning positions 255–295 discussed above was
noted earlier [43], including the phosphorylation sites P1
and P2 (Fig. 2), but no particular role was assigned to them.
The importance of three residues in the E1a component of
rat E1b, namely, E1aR288, E1aH292 and E1aD296 of rat
E1b, was recognized by alanine mutagenesis [42]. These
residues were among 10 in the neighbourhood of phos-
phorylation site 1 (E1aS293) that were examined, and their
replacement with alanine resulted in totally inactive
enzymes. They correspond to residues Arg267, His271
and Asp276 in the proposed YR–H–D–YR–DE sequence
motif (Fig. 2). Our results indicate that Arg267 of
B. stearothermophilus E1p is involved in binding the 2-oxo
acid substrate and that E1aTyr281 and, to a lesser extent,

E1aAsp276 and E1aArg282, have some effect on the
decarboxylation of pyruvate and the reductive acetylation
of the tethered lipoyl domain in the active PDH complex
(although the replacement of none of these residues caused
complete inhibition). Other experiments (M. Fries & R. N.
Perham, unpublished work) indicate that E1aHis271 has a
crucial part to play in the decarboxylation of pyruvate,
probably by serving to stabilize the energetically unfavou-
rable dianion formed after nucleophilic attack of ThDP at
Ó FEBS 2003 Active site of E1 component of PDH complex (Eur. J. Biochem. 270) 867
the 2-oxo group of the substrate [for a detailed formulation
of the mechanism to date, see [44]).
The importance of this region is emphasized by the
reports of clinically deleterious mutations in the human E1a
chain; for example, the replacement of His263 (equivalent to
His271 in B. stearothermophilus E1a, Fig. 2) with leucine is
associated with a very low PDH complex activity, as is the
replacement of Arg273 (equivalent to Arg282) in B. stearo-
thermophilus E1a with cysteine [45,46]. It is interesting to
note that the R282A mutation in B. stearothermophilus E1a
had only a modest effect on the E1 catalytic activity in vitro
(Tables 1 and 2), perhaps because the amino-acid replacing
the arginine is different.
Two phosphorylation sites in eukaryotic E1a chains are
located within the region of the sequence motif outlined in
Fig. 2. In addition to the E1aR288A mutant of rat E1b
being inactive, it was incapable of becoming phosphory-
lated, suggesting that this residue may be involved in the
interaction with the E1 kinase [42]. Studies on synthetic
peptides as substrates for mammalian pyruvate dehydro-

genase kinase have indicated that an acidic residue to the
C-terminal side of phosphorylation site 1 (Ser293) is also an
important specificity determinant for the kinase [47]. This
might be true similarly for phosphorylation site 2 (Ser300).
There are conserved acidic residues to the C-terminal side of
both phosphorylation sites 1 and 2 in the Ela sequence
alignment (Fig. 2).
Non-equivalence of active sites has been observed in the
crystal structure of the thiamin-dependent yeast pyruvate
decarboxylase; one active site was found to be in an open
conformation, with two loop regions disordered, whereas in
the other these loop regions were well-ordered and shielded
the active site from the bulk solution [48]. The loop housing
the conserved sequence motif in B. stearothermophilus E1p
is exposed and flexible, as indicated by limited proteolysis;
moreover, it appears to adopt two different conformations,
one susceptible to proteolysis and the other not [20]. Thus,
this loop in the B. stearothermophilus E1p might take part in
a similar Ôopen-closeÕ mechanism. If in the mutant E1s the
loop region became more disordered, it might not function
properly as a lid for the active site, thereby granting easier
access for the cofactor ThDP to its binding site. The lower
thermal stability of the mutant E1s and the increased V
max
in the DCPIP assay, which could be due to easier access of
DCPIP to the active site or to a more ready release of
product, would be consistent with this idea.
We have looked at the recently determined crystal
structure of the dimeric E1p from the E. coli PDH complex
[49] but have been unable to locate a similar sequence of

amino acids in a comparable position with respect to the
active site. Some conclusions about the importance of
individual residues in the E. coli E1p have been drawn [49],
but a detailed comparison of the two types of E1 (dimeric
and heterotetrametric) will have to be the subject of further
investigation.
Acknowledgements
This work was supported by a research grant from the Biotechnology
and Biological Sciences Research Council (to R. N. P.). M. F. is
grateful to the Verband der Chemischen Industrie and the Cusanu-
swerk for a studentship and Clare Hall for additional support, H. J. C.
thanks the BBSRC for the award of an earmarked Research
Studentship, and H I. J. is grateful to the Cambridge Overseas Trust,
St John’s College, Cambridge and the Department of Biochemistry,
University of Cambridge for financial support.
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