(Received for publication, November 18, 1994; and in revised form, December 12, 1994)
From the
The pyruvate dehydrogenase (E1) component of the mammalian
pyruvate dehydrogenase complex catalyzes the oxidative decarboxylation
of pyruvate with the formation of an acetyl residue and reducing
equivalents, which are transferred sequentially to the dihydrolipoyl
acetyltransferase and dihydrolipoamide dehydrogenase components. To
examine the role of tryptophanyl residue(s) in the active site of E1,
the enzyme was modified with the tryptophan-specific reagent N-bromosuccinimide. Modification of 2 tryptophan residues/mol
of bovine E1 (out of 12 in a tetramer
) resulted in complete inactivation
of the enzyme. The inactivation was prevented by preincubation with
thiamin pyrophosphate (TPP), indicating that the modified tryptophan
residue(s) is a part of the active site of this enzyme. Fluorescence
studies showed that thiamin pyrophosphate interacts with tryptophan
residue(s) of E1. The magnetic circular dichroism (MCD) spectral
intensity at
292 nm was decreased by
15% for E1 + TPP
relative to the intensity for E1 alone. Because this MCD band is
uniquely sensitive to and quantitative for tryptophan, the simplest
interpretation is that 1 out of 6 tryptophan residues present in E1
(
dimer) interacts with TPP. The natural circular dichroism
(CD) spectrum of E1 is dramatically altered upon binding TPP, with
concomitant induction of optical activity at
263 nm for the
nonchiral TPP macrocyle. From CD studies, it is also inferred that loss
of activity following N-bromosuccinimide treatment occurred
without significant changes in the overall secondary structure of the
protein. A single peptide was isolated by differential peptide mapping
in the presence and absence of thiamin pyrophosphate following
modification with N-bromosuccinimide. This peptide generated
from human E1 was found to correspond to amino acid residues
116-143 in the deduced sequence of human E1
, suggesting that
the tryptophan residue 135 in the
subunit of human E1 functions
in the active site of E1. The amino acid sequences surrounding this
tryptophan residue are conserved in E1
from several species,
suggesting that this region may constitute a structurally and/or
functionally essential part of the enzyme.
The multienzyme pyruvate dehydrogenase complex (PDC) ()plays a pivotal role in energy metabolism by regulating
the oxidation of pyruvate. The mammalian complex consists of several
catalytic as well as regulatory components namely, pyruvate
dehydrogenase (E1), dihydrolipoyl acetyltransferase, dihydrolipoyl
dehydrogenase, protein X, E1
-kinase, and phospho-
E1
-phosphatase(1, 2) . The E1 tetramer
(
) has two active centers with equal
catalytic efficiency but different affinity for thiamin pyrophosphate
(TPP)(3) . Considering the importance of this component in the
overall catalytic reaction in the complex, little is known about the
structure of the active site. Studies involving chemical modification
of E1 have implicated the amino acid residues lysine(4) ,
histidine, arginine, tryptophan, and cysteine (5) as being
critical for enzyme function. The involvement of tryptophan in TPP
binding was suggested by chemical modification of pigeon breast E1
using N-bromosuccinimide (NBS)(6) .
The primary
structures of both and
subunits of E1 from several species
have been deduced from their cDNA
sequences(7, 8, 9, 10, 11) .
The possible functional significance of several motifs in these deduced
sequences have also been proposed based on sequence
homology(12, 13) . Hawkins et al. (12) have identified a sequence motif of approximately 30
residues in length in several TPP-dependent enzymes; this motif begins
with the sequence GDG and ends with NN. Aspargine has also been
suggested as a possible residue for TPP binding(12) . Dahl et al. (14) have speculated that tryptophan residue
214 of the
subunit of human E1 may be a possible site of TPP
binding. In a number of TPP-dependent enzymes like Escherichia coli pyruvate oxidase(15) , Zymomonas mobilis pyruvate
decarboxylase(16) , and Saccharomyces cerevisiae transketolase(17) , tryptophan has been implicated in TPP
binding from spectral analysis and chemical modification studies. Thus,
clarification of the involvement of tryptophan or aspargine residue(s)
in TPP binding in these enzymes is of fundamental importance.
To
investigate the possible role of tryptophan residue(s) in the
structure-function relationship and to explore its specific
localization in the E1 component of mammalian PDC, we have treated both
highly purified bovine E1 and recombinant human E1 with NBS, a
tryptophan-specific chemical reagent. We have identified by
differential peptide mapping and reverse phase high performance liquid
chromatography (HPLC) followed by sequence analysis that the tryptophan
residue of E1, which is involved in TPP binding, corresponds to the
tryptophan residue at position 135 in the human E1 subunit.
All samples were in
pH 7.2 potassium phosphate buffer containing 1 mM Mg-EDTA.
Experimental conditions for data collection were: wavelength range,
250-350 nm; 9-16 scan accumulation; spectral step
resolution, 0.1 nm; scan rate, 20 nm/min; slit width, 1 nm; response,
0.25 s. Data manipulation and calculations utilized the Jasco J-720
software. CD data are presented as (M cm
). Far ultraviolet (protein region CD spectra
of both native and NBS-modified E1 preparations were recorded between
190 and 260 nm at 25 °C using a J-600 Spectropolarimeter (Jasco,
Tokyo).
Figure 1: A, effect of TPP on the tryptophanyl fluorescence emission spectrum of bovine E1. Varying concentrations of TPP were added to E1 (0.1 mg/ml), and emission spectra were collected from 300-400 nm after excitation at 295 nm. B, plot of relative fluorescence intensity of E1 at the emission maximum (335 nm) as a function of concentration of TPP.
Fig. 2presents the MCD spectra of
bovine E1 + TPP (solidline), E1 (dashedline), and the (E1 + TPP) - E1 difference
spectrum (dotted-dashed line). The MCD maximum is, as
expected, at 292 nm for both native and the (E1 + TPP)
samples. A 15-17% decrease in intensity, concomitant with a red
shift of
0.3 nm, occurred upon binding of TPP to bovine E1
(compare the solid and dashedspectra). This
is perhaps most clearly shown by the dotted-dashed line, the
difference spectrum of (E1 + TPP) - E1. These data strongly
suggest that 1 out of 6 tryptophan residues of E1 interacts with TPP
leading to a major loss in its MCD signal. We cannot eliminate partial
reduction of the signal of more than one Trp, but the reduction in
signal must result from an interaction changing the electronic
character of Trp, and this is most easily explained with a change in
single Trp.
Figure 2: MCD spectra of bovine E1. Dashedline, E1 (45.5 µM) in potassium phosphate buffer (pH 7.2) and 1 mM Mg-EDTA; solidline, E1 (43.7 µM) plus TPP (87.4 µM) in same buffer; dotted-dashed line, difference spectrum ((E1 + TPP) - E1). Data were obtained and calculated using the J-720 software.
One possibility for the loss in signal is that binding
of TPP to E1 involves a ``stacking'' interaction with the
tryptophan residue(5) . It is interesting, if this model is
accurate, that stacking should cause a loss of MCD intensity (to our
knowledge, such a perturbation of tryptophan MCD by stacking has not
been previously addressed). The MCD bands of tryptophan were identified
by comparision with their absorption bands (assigned by Strickland et al.(27) ). The 292-nm MCD band of indole
chromophores, i.e. tryptophan, was assigned by Holmquist and
Vallee (26) to a
-
*, L
(0-0)
transition. Albinsson et al. (28) showed that the UV
transition moment was at + 45 ± 5
relative to
the pseudo symmetry long axis. Thus, our data suggest that the nature
of the tryptophan-thiamin interaction results in loss of this
transition. This is of particular interest given the normal
insensitivity of this feature to perturbants such as solvent, pH, or
protein conformation.
Fig. 3presents the natural CD spectra
of bovine E1 + TPP (solidline), E1 (dashedline), and TPP alone (parallelingbaseline). As evident from the spectrum, TPP alone has no
optical activity. However, upon binding to bovine E1, a dramatic
spectral change occurred (compare solid and dashedspectra). The entire spectrum becomes shifted upwards,
and there is a new, intense feature at 263 nm, coincident with the
electronic absorption maximum of TPP. Thus, these data represent a
classic example of ``induced optical activity,'' with the
optically inactive TPP coupling to the optically active protein
chromophore, resulting in the observed spectral pattern. There is also
a dramatic shift at higher wavelengths, as previously reported by
Khailova and co-workers(3, 5) .
Figure 3:
CD spectra of bovine E1. Dashedline, E1 (45.5 µM) in potassium phosphate
buffer (pH 7.2) and 1 mM Mg-EDTA (pH 7.2); solidline, E1 (43.7 µM) + TPP (87.4
µM) in the same buffer; and lineparallelingbase line, TPP (200 µM). CD spectra are
presented as (M cm
), where
= 3300
.
Figure 4:
A, inactivation of E1 with NBS. E1 (6.4
µM) in 20 mM potassium phosphate buffer (pH 6.5)
was preincubated in presence and absence of TPP and/or pyruvate and
then treated with varying concentrations of NBS for 1 min and expressed
as NBS/E1 molar ratio. Treatments were E1 (), E1 preincubated
with 0.4 mM TPP (
) and with 1 mM TPP (
),
E1 preincubated with 0.05 mM TPP plus 0.5 mM pyruvate
(
), and E1 preincubated with 0.5 mM pyruvate (
). B, correlation between the oxidation of tryptophan residue(s)
(
) in E1 and absorbance (
) with the NBS/E1 molar ratio. The
number of tryptophan residues oxidized was calculated from the decrease
in absorbance at 280 nm.
The oxidation of bovine E1 with NBS produced an
instantaneous decrease in absorbance at 280 nm with the transformation
of the indole to oxindole chromophore (Fig. 4B). The
maximal change in absorbance during the NBS titration can be used to
determine the number of tryptophan residues oxidized(22) . Fig. 4B shows the concentration dependence for NBS
modification of tryptophan residues in E1 and supports a stoichiometric
reaction. At relatively low NBS/E1 molar ratio (40:1), 2 tryptophan
residues/mol of E1 () were oxidized.
Based on the amino acid sequences of human E1
and E1
subunits, there are a total of 12 tryptophan residues in the tetrameric
E1 (2 in
and 4 in
). Only 2 of the 12 tryptophan residues
were modified by NBS under our experimental condition. Thus, a single
tryptophan residue per subunit of bovine E1 appeared to be at or near
the catalytic site. The number of essential tryptophan residues
oxidized also correlates with the number of active centers of the
enzyme(3) . Table 1provides results showing the extent
of tryptophan modification in the absence and presence of different
ligands. TPP (1 mM) alone caused almost complete protection of
two tryptophan residues, but other combinations were ineffective in
protecting the tryptophan residues from NBS modification (Table 1). The CD spectrum in the far ultraviolet region of the
modified enzyme was essentially identical with that of the untreated
enzyme (results not shown), indicating little or no significant change
in secondary structure of the protein upon modification.
Figure 5: Reverse phase HPLC elution profile of tryptic digest of human E1. Absorbance was monitored at 280 nm for the detection of tryptophanyl peptides. The profiles correspond to E1 modified with NBS (0.25 mM) in the presence of TPP (1 mM) (A) and E1 modified with NBS (0.25 mM) in the absence of TPP (B). The plussign indicates the peak of interest as described under ``Results and Discussion.'' Experimental details were as described under ``Experimental Procedures.''
The peptide was expected to be very long, as judged from
an HPLC elution profile and from the predicted trypsin cleavage sites.
The peptide was sequenced for the first 15 amino acid residues. The
sequence GPNGASAGVAAQHSQ matched completely with the sequence from 116
to 130 amino acid residues in human E1, indicating tryptic
cleavage occurred after Arg-115 in this protein. The next predicted
trypsin cleavage site would be at Lys-143, giving rise to a peptide of
28 amino acid residues with only 1 tryptophan at 135. By a similar
differential procedure, a peptide with the sequence AWYG was isolated
from bovine E1 following V8 protease digestion. The sequence agrees
with that around the same Trp in the human E1
subunit (cf. Fig. 6, discussed below). Although we are unable
to explain the production of this peptide by V8 protease, this further
supports the conclusion that it is this specific Trp of the E1
subunit, which is protected from modification by TPP. These results
strongly support the contention that Trp-135 of E1
is involved in
TPP binding.
Figure 6:
Comparison of the amino acid sequences
surrounding the specific tryptophan residue in the subunit of
PDC-E1 from recombinant human E1 (H*, present study), human (H)(8) , rat (R)(9) , pig (P)(10) , nematode (N)(11) , and Bacillus stearothermophillus (B)(29) .
Amino acid sequences of the E1 subunit of PDC have
been deduced from cDNAs isolated from human(8) ,
rat(9) , pig(10) , nematode(11) , and Bacillus stearothermophillus(29) . Comparison of the
aligned sequences of these E1
subunits indicates that Trp-135 in
human E1
is conserved among these proteins; amino acid residues
surrounding the modified tryptophan are highly conserved (Fig. 6). Thus, this region may constitute a part of the enzyme
active site.
Because it has not been possible to isolate
functionally active E1 and E1
separately, it is not known
whether TPP binds to the
or the
subunit. A putative TPP
binding motif as reported by Hawkins et al. (12) for
TPP-dependent enzymes is found only in the E1
subunits of the
-keto acid dehydrogenase complexes. Based on the sequence
similarities between E1
subunits of branched chain keto acid
dehydrogenase and transketolase from several species, Zhao et
al. (30) have recently proposed that the E1
subunit
may participate in TPP binding. X-ray crystallography studies of
transketolase from S. cerevisiae have demonstrated that the
TPP binding site is located in a deep cleft between the two subunits
(homodimer), and residues(187-198) from both subunits interact
with TPP(31) . X-ray crystallographic studies also revealed
that the TPP binding site in transketolase is lined with several
conserved amino acids such as His, Asp, Arg, Leu, and Ile(32) .
However, the identification and exact role of specific amino acid
residue(s) in TPP binding of TPP-dependent enzymes is still unclear.
This paper is the first evidence for the localization of a specific
tryptophan residue in the E1
subunit at or near the TPP binding
site in the TPP-dependent mammalian E1.