From the Departments of § Biochemistry
and Internal Medicine, University of Texas
Southwestern Medical Center, Dallas, Texas 75390
Received for publication, September 1, 2000, and in revised form, October 27, 2000
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ABSTRACT |
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The human mitochondrial branched-chain
The human mitochondrial branched-chain To understand the structure and function of BCKD as well as the
biochemical basis for MSUD, we have recently solved the
three-dimensional structure of the human enzyme at 2.7-Å resolution
(5). The human BCKD structure reveals the presence of two TDP-binding
pockets, with each formed at the interface between the In this report, we carried out site-directed mutagenesis on residues in
the TDP-binding and K+ ion-binding sites as well as
residues that are affected in MSUD patients. Both the wild-type and
mutant BCKD proteins were expressed in Escherichia coli in
the presence of cotransformed chaperonins GroEL and GroES. These
properly assembled heterotetramers were characterized for their kinetic
parameters and their ability to bind cofactor TDP. The results confirm
the roles of key amino acid residues in catalysis as well as cofactor
and metal ion binding as implicated by the crystal structure of
human BCKD. Moreover, the study depicts the conservation and divergence
among TDP-dependent enzymes, which catalyze the
cofactor-mediated oxidative or nonoxidative decarboxylation reaction.
Construction of Expression Plasmids for Mutant
His6-tagged BCKD--
The Altered Site in vitro
mutagenesis system (Promega, Madison, WI) was used to introduce desired
mutations into the cDNA for the Expression and Purification of Human His6-tagged
BCKD--
The recombinant His6-tagged BCKD heterotetramer
was efficiently expressed in E. coli strain CG-712
(ESts) by cotransformation of the pGroESL plasmid
overproducing chaperonins GroEL and GroES as described previously (14,
15). Wild-type and mutant His6-tagged BCKD heterotetramers
were isolated from cell lysates using a
Ni2+-NTA-derivatized Sepharose CL-6B column (Qiagen,
Chatsworth, CA) as described previously (16). BCKD proteins were
further purified on a Superdex-200 gel filtration column (2.6 × 60 cm) connected to an FPLC system from Amersham Pharmacia Biotech. The
column buffer consisted of 50 mM potassium phosphate, pH
7.5, 250 mM KCl, 10% (v/v) glycerol, 5 mM
dithioerythritol, 1 mM benzamidine, and 1 mM
phenylmethylsulfonyl fluoride. BCKD activity during purification was
assayed spectrophotometrically (see below). Protein concentrations were
determined using the Coomassie Plus protein reagent from Pierce with
absorbance read at 595 nm. Alternatively, during enzyme purification,
protein concentrations were determined by the direct measurement of
absorbance at 280 nm using a calculated molar extinction coefficient of
1.15 cm Assays for BCKD Activity and Kinetic Studies--
To determine
Km for TDP, a radiochemical assay based on activity
of the reconstituted BCKD complex was used (15). The rate of
decarboxylation of 0.2 mM
Measurements of Amounts of TDP Bound to Wild-type and Mutant
BCKD--
The purified recombinant human BCKD was essentially devoid
of TDP. The residual bound TDP was removed by exhaustive dialysis in
the presence of 0.2 mM EDTA. The wild-type or mutant
apo-BCKD (500 µg each) was incubated for 30 min with 150 µM Mg-TDP at 4 °C. The holo-BCKD was re-extracted with
Ni2+-NTA resin and eluted with 100 mM
imidazole. The BCKD-bound TDP was measured after oxidation to its
fluorescent derivative thiochrome diphosphate in the presence of
ferricyanide. The thiochrome diphosphate derivative was measured in a
PerkinElmer Life Sciences model LS50B luminescence spectrometer
(excitation wavelength, 375 nm; emission wavelength, 430 nm) as
described previously (18).
Other Methods--
Emission spectra over a range of 300-400-nm
wavelengths for bound TDP were obtained using the luminescence
spectrometer at an excitation wavelength of 280 nm as described
previously (19). The quenching of tryptophan fluorescence by Mg-TDP was
studied by adding increments of Mg-TDP to the cuvette containing
apo-BCKD. The range of final Mg-TDP concentrations studied was 5-400
µM. Cycles of Mg-TDP addition and fluorescence
measurements were repeated until the fluorescence quenched by the TDP
bound to BCKD reached a plateau. Circular dichroism measurements were
carried out on an AVIV (Lakewood, NJ) model 62 DS spectrometer.
Catalytic Residues for the TDP-mediated Decarboxylation--
In
human BCKD, residues that form each Mg-TDP cofactor binding pocket are
derived from two separate subunits. Fig.
1 shows that residues from both the
As shown in Fig. 1, Glu-76-
His-146-
Ser-292-
The side chain of Tyr-102-
The amounts of TDP bound to His6-tagged wild-type and
mutant BCKD were measured by incubating the enzymes with 150 µM of Mg-TDP. Following Ni2+-NTA extraction,
the bound TDP was determined in the form of thiochrome pyrophosphate
(18). This method allowed for end point measurements of bound TDP in
BCKD incubated at saturating concentrations of the cofactor. The
results showed that each mole of wild-type BCKD binds ~2 mol of TDP
(Fig. 2A). The data confirm
the presence of two cofactor-binding sites in the BCKD heterotetramer.
It is noteworthy that the active site residues involved in catalysis or
substrate binding have little effect on TDP binding. This notion is
supported by the slight decrease in TDP binding as observed with the
E76A- Active Site Residues That Coordinate to the Mg2+
Ion--
As shown in Fig. 1, the Mg2+ ion is octahedrally
coordinated between the cofactor phosphates, the carbonyl of
Tyr-224- Residues That Interact with Diphosphate Oxygens of
TDP--
Gln-112-
His-291- Residues in the Novel K+ Ion-binding Site of the Identification of the Trp Residue Quenched by Bound TDP--
One
of the characteristics associated with TDP-dependent
enzymes involves quenching of tryptophan fluorescence upon binding of
the cofactor (19). This property has been used as a means to determine
the level of the cofactor binding by TDP-dependent enzymes
(22, 27). To identify the Trp residue quenched by BCKD-bound TDP, we
replaced Trp-136- Conclusion--
The present study defines the functional roles of
active site residues in human BCKD, which catalyzes the oxidative
decarboxylation of branched-chain
The human BCKD is tightly regulated by reversible
phosphorylation/dephosphorylation (4). Residue Ser-292-
The utilization of the octahedrally coordinated K+ ion site
to stabilize an essential loop structure in the TDP binding fold has
not been described in other TDP-dependent enzymes. Both
BCKD and pyruvate dehydrogenase require high concentrations of the K+ ion to stabilize enzyme activity (16, 31, 34). It is
very likely that the novel K+-binding sites are conserved
in the decarboxylase/dehydrogenase components of -ketoacid decarboxylase/dehydrogenase (BCKD) is a heterotetrameric
(
2
2) thiamine diphosphate
(TDP)-dependent enzyme. The recently solved human BCKD
structure at 2.7 Å showed that the two TDP-binding pockets are located
at the interfaces between
and
' subunits and between
' and
subunits. In the present study, we show that the E76A-
' mutation
results in complete inactivation of BCKD. The result supports the
catalytic role of the invariant Glu-76-
' residue in increasing
basicity of the N-4' amino group during the proton abstraction from the
C-2 atom on the thiazolium ring. A substitution of His-146-
'
with Ala also renders the enzyme completely inactive. The data are
consistent with binding of the
-ketoacid substrate by this residue
based on the Pseudomonas BCKD structure. Alterations in
Asn-222-
, Tyr-224-
, or Glu-193-
, which coordinates to the Mg2+ ion, result in an inactive enzyme (E193A-
) or a
mutant BCKD with markedly higher Km for TDP and a
reduced level of the bound cofactor (Y224A-
and N222S-
).
Arg-114-
, Arg-220-
, and His-291-
interact with TDP by directly
binding to phosphate oxygens of the cofactor. We show that natural
mutations of these residues in maple syrup urine disease (MSUD)
patients (R114W-
and R220W-
) or site-directed mutagenesis
(H291A-
) also result in an inactive or partially active enzyme,
respectively. Another MSUD mutation (T166M-
), which affects one of
the residues that coordinate to the K+ ion on the
subunit, also causes inactivation of the enzyme and an attenuated
ability to bind TDP. In addition, fluorescence measurements establish
that Trp-136-
in human BCKD is the residue quenched by TDP binding.
Thus, our results define the functional roles of key amino acid
residues in human BCKD and provide a structural basis for MSUD.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-ketoacid
decarboxylase/dehydrogenase
(BCKD)1 is a thiamine
diphosphate (TDP)-dependent enzyme, which catalyzes the
oxidative decarboxylation of branched-chain
-ketoacids derived from
leucine, isoleucine, and valine (1, 2). BCKD is the E1 component of the
macromolecular BCKD complex (Mr = 4 × 106), which comprises multiple copies of BCKD or E1,
dihydrolipoyl acyltransferase (E2), dihydrolipoamide dehydrogenase
(E3), a specific kinase, and a specific phosphatase (3). Human BCKD is
a heterotetrameric protein consisting of two
(Mr = 45,500) and two
(Mr = 37,500) subunits. In the oxidative
decarboxylation reaction, BCKD or the E1 component catalyzes the
TDP-mediated decarboxylation of a branched-chain
-ketoacid to
produce an enamine-TDP, with concomitant reduction of the lipoamide
moiety covalently attached to E2. This is followed by the reaction of
enamine-TDP with the reduced dihydrolipoamide, which results in an
S-acyldihydrolipoamide. E2 catalyzes the transfer of the
acyl group from the tetrahedral intermediate to CoA to give rise to a
branched-chain acyl-CoA. The nonconjugated dihydrolipoamide is
subsequently reoxidized by E3, which is a flavoprotein, with NAD+ as the ultimate electron acceptor. The last step
resets the cycle for oxidative decarboxylation of the branched-chain
-ketoacid. Mammalian BCKD is regulated by a reversible
phosphorylation (inactivation)/dephosphorylation (activation) cycle
under different hormonal and dietary stimuli (4). In patients with
heritable maple syrup urine disease (MSUD), activity of the BCKD
complex is deficient. This results in the accumulation of
-ketoacids
with severe clinical consequences including often fatal ketoacidosis,
neurological derangement and mental retardation (2).
and
'
subunits or between the
' and
subunits (5). This topology is
similar to the BCKD from Pseudomonas putida (6) and is
equivalent to the homodimeric transketolase from Saccharomyces
cerevisiae, where its two TDP-binding sites are formed at the
head-to-tail interfaces between the two identical subunits (7). In
contrast, crystal structures of pyruvate decarboxylases from
Saccharomyces uvarum (8), S. cerevisiae (9) and
Zymomonas mobilis (10), pyruvate oxidase from
Lactobacillus plantarum (11), and benzoylformate decarboxylase from P. putida (12) indicate the presence of
four TDP-binding sites in each homotetramer. The cofactor binding fold in these TDP-dependent enzymes is located at the interface
between the
domain of one subunit and the
domain of the other.
The physiological significance in doubling of the TDP-binding sites compared with transketolase and BCKD is not apparent at present. In
addition, two novel K+ ion-binding sites in human BCKD, one
on the
subunit and one on the
subunit, were identified. MSUD
mutations that affect residues in the TDP-binding and the
K+ ion-binding sites or residues involved in subunit
assembly of BCKD have been described. These findings have provided a
basis for understanding structure and function of human BCKD as well as
how these properties are affected by naturally occurring MSUD mutations.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
or the
subunit. Detailed
protocols for the mutant vector construction and subsequent mutagenesis
were described previously (13). Briefly, oligonucleotides for the
desired mutations and the
-lactamase repair primer were annealed to
the single-stranded form of pAlter-
or pAlter-
vector. After the
second strand synthesis and two rounds of ampicillin selection, clones
harboring the correct mutations were isolated for plasmid preparation.
DNA segments containing the mutations were used for cassette
replacements of the expression vector pHis-TEV-E1 for wild-type BCKD
(13).
1 mg
1 ml for
the
2
2 heterotetramer.
-keto[1-14C]isovalerate ([1-14C]KIV) by
BCKD in the presence of an excess of E2 and E3 was measured at varying
concentrations of TDP. Double reciprocal plots were used to determine
Km and Vmax values for
cofactor TDP. For the determination of kinetic parameters for substrate
KIV, a spectrophotometric assay, also according to the reconstituted BCKD complex activity, was employed. The assay mixture contained 50 mM potassium phosphate, pH 7.5, 100 mM NaCl, 3 mM NAD+, 0.4 mM CoA, 2 mM MgCl2, 2 mM dithiothreitol,
0.1% Triton X-100, 400 µM TDP, 7 nM
lipoylated recombinant bovine E2, and 0.4 µM recombinant
human E3 (17). The reduction of NAD+ absorbance at 340 nm
at different substrate concentrations was used to derive the
kobs value. The plots observed the pseudo-first order decay. The double-reciprocal plots of kobs
values versus KIV concentrations were used to determine
Km and kcat values for
KIV.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and
' subunits are involved in binding to TDP, and the
Mg2+ ion is held in an octahedral coordination. This
topology maintains TDP in a strained "V-shaped" conformation having
torsion angles
T = 100°, and
P =
71° to facilitate the proton extraction from the C-2 carbon (5).
Most of these residues are conserved among TDP-dependent
enzymes including Glu-193-
, Gly-194-
(not shown), and
Asn-222-
in the TDP-binding motif
GDGX26-28NN (20) and the invariant
catalytic residue Glu-76-
'. To establish the functional role of
these active site residues, site-directed mutagenesis was carried out.
His6-tagged wild-type and mutant BCKD carrying a single
amino acid substitution were expressed in E. coli by cotransformation with chaperonins GroEL and GroES. The proteins were
purified by Ni2+-NTA extraction followed by FPLC gel
filtration on a HiLoad Superdex 200 column. The mutant BCKD used for
this study uniformly formed heterotetramers, and no gross
conformational changes were detected by circular dichroism spectroscopy
(data not shown). The highly purified wild-type and mutant BCKD were
used to determine their kinetic parameters and abilities to bind
cofactor TDP.
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Fig. 1.
Residues in the cofactor TDP binding fold of
human BCKD. The inverted V-shaped conformation of cofactor TDP is
stabilized by stacking of the aminopyrimidine ring against the side
chain of Tyr-102- ' from the
' subunit (in greenish
yellow) and the side chain of Leu-164-
from the
subunit (in magenta). The invariant Glu-76-
' important
for cofactor activation coordinates to the N-1' atom of the
aminopyrimidine ring (3.4 Å apart). A ketoacid substrate analog (in
gray) labeled isocaproate is covalently modeled
into the side chain of His-146-
', based on the crystal structure of
BCKD from Pseudomonas putida (6). The carboxylate group of
the inhibitor interacts with the N-4' amino group of TDP (separated by
a distance of 4.3 Å). The side chain of Ser-162-
also coordinates
to the N-4' amino group (3.0 Å apart) to position the cofactor in the
correct conformation. Residue Ser-292-
is phosphorylation site 1 of
human BCKD. The diphosphate moiety of TDP is stabilized, in part, by an
octahedral coordination of the Mg2+ ion. Two of the amino
acid ligands Glu-193-
and Asn-222-
in this coordination are
shown. Side chains of Arg-114-
, Arg-220-
, and His-291-
, are,
in turn, in direct contact with the distal phosphate oxygens, whereas
the side chains of Gln-112-
and Tyr-113-
(not shown) interact
with the proximal phosphate oxygens of the diphosphate moiety of
TDP.
' is directly bound to the N-1' atom of
the aminopyrimidine ring at a distance of 3.4 Å, whereas the
main-chain carbonyl group of Ser-162-
coordinates to the N-4' group
on the opposite side of the ring, with the two entities being 3.0 Å apart. The electron withdrawal caused by the interaction between
Glu-76-
' and the N-1' atom increases the basicity of the N-4' amino
group through tautomerization. This interaction is essential for
efficient proton abstraction from the C-2 atom of the thiazolium ring
of TDP, giving rise to the reactive ylide (21). The nucleophilic attack
of the
-ketoacid substrate at the reactive C-2 carbon of the ylide
results in a tetrahedral adduct. Decarboxylation of this intermediate
produces a 2-
-carbanion or enamine-TDP, which is stabilized by a
neutral resonance. This mechanism for TDP-mediated decarboxylation is
entirely conserved (22). In the case of human BCKD, the decarboxylated
acyl moiety on the enamine-TDP is transferred to the E2 dithiolane ring
to produce the tetrahedral adduct S-acyldihydrolipoamide. In
the present study, substitution of Glu-76-
' with Ala (E76A-
,
Table I) renders BCKD completely
inactive. The data establish Glu-76-
' as an essential catalytic
residue. On the other hand, the interaction between the carbonyl group
of Ser-162-
and the N-4' amino group is required to orient the
latter for proton abstraction from the C-2 atom of the thiazolium ring.
Since it involves a main-chain carbonyl group, a replacement with Ala
is without effect on the catalytic efficiency for the TDP, although the
same parameter for substrate KIV is significantly reduced.
Kinetic parameters of BCKD mutants affecting residues involved in
TDP-mediated decarboxylation
' aligns with His-131-
' in BCKD from P. putida. The latter residue was shown to form an adduct with a
substrate analog,
-chloroisocaproate, in the crystal structure of
the Pseudomonas enzyme (6). It has been proposed that
binding of the substrate analog to His-131-
' of the bacterial BCKD
mimics a natural
-ketoacid substrate. The complete absence of enzyme
activity in H146A-
' (Table I) is consistent with the role of this
residue in binding substrate KIV in human BCKD. The "isocaproate"
moiety modeled into the His-146-
' residue of human BCKD shows that
the carboxylate oxygen is 4.3 Å away from the N-4' amino group of TDP
(Fig. 1). The data support the possible role of His-146-
' in
positioning a native ketoacid substrate (e.g. KIV) for the
TDP-mediated decarboxylation. This point needs to be confirmed by
co-crystallization of human BCKD with
-chloroisocaproate, which has
not been successful to date. Recently, the structure of the
Desulfovibrio africanus pyruvate:ferredoxin oxidoreductase
in complex with pyruvate (23) demonstrates that a carboxylate oxygen
from pyruvate interacts directly with the N-4' amino group of TDP.
Together, the data strongly imply that the polarization of the
substrate carboxylate moiety by the N-4' amino group of the cofactor is
necessary for activation of the substrate.
and Ser-302-
are site 1 and site 2 for phosphorylation
of human BCKD by the specific kinase. Phosphorylation at site 1 results
in inactivation of BCKD, whereas phosphorylation at site 2 is silent.
Introduction of a negatively charged Asp residue in the S292D-
mutant produces the same inactivation effect as phosphorylation at this
residue (Table I). This result confirms an earlier study in which
replacement of site 1 Ser with Glu in rat BCKD also results in an
inactive enzyme (24). As shown in Fig. 1, Ser-292-
is positioned
directly above the C-2 atoms of the thiazolium ring of TDP at an 8-Å
distance. The introduction of a negatively charged phosphate group or
amino acid residue is likely to interfere with the proton abstraction
from the C-2 atom by the basic amino group at N-4' position of the
aminopyrimidine ring. Moreover, Ser-292-
is 9 Å away from
His-146-
' (Fig. 1). The presence of negative charges by
phosphorylation or site-directed mutagenesis may also disrupt binding
of the
-ketoacid substrate as suggested in earlier studies (24,
25).
' is packed against one side of the
aminopyrimidine ring of the cofactor with the side chain of Leu-164-
approaching the other side of the ring, wedging in between the two
rings of the cofactor (Fig. 1). The flanking of both sides of the
aminopyrimidine ring by these two hydrophobic residues is important to
orient the cofactor in the strained V conformation. Substitution of
either residue with an Ala has adverse effects in the catalytic
efficiency of the mutant enzymes (Table I). However, L164A-
is
markedly more severely affected than Y102A-
'. The data suggest that
Leu-164-
is more important in providing the hydrophobic environment
for the aminopyrimidine and possibly the thiazolium ring of the cofactor.
', S162A-
, H146A-
', and S292D-
mutants (Fig.
2A). In the S162A-
mutant, the near wild-type
Km for TDP agrees well with a TDP-binding
stoichiometry that is also similar to wild-type. In contrast, the
L164A-
mutant binds only a trace amount of TDP, which is consistent
with an over 400-fold increase in Km for TDP
relative to the wild-type. The data indicate that Leu-164-
that
wedges in between the two aromatic rings of the cofactor is important
for TDP binding. It is of interest that TDP binding in the Y102A-
'
mutant is not as severely affected as the L164A-
. The results
confirm that Tyr-102-
', which is across the aminopyrimidine ring
planar from Leu-164-
, is less critical for the cofactor binding.
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Fig. 2.
Stoichiometry of bound TDP in wild-type and
mutant BCKD. Wild-type or mutant apo-BCKD
(His6-tagged) at 500 µg each was incubated with 150 µM Mg-TDP for 30 min at 4 °C. The holoenzymes were
extracted with Ni2+-NTA resin, and eluted proteins were
oxidized with ferricyanide. The thiochrome-TDP released from the
enzymes was measured by fluorescence emission at 430 nm following an
excitation at 375 nm. The results are expressed as mol of TDP bound per
mol of BCKD heterotetramers. A, mutants affecting catalytic
residues required for the TDP-mediated decarboxylation. B,
mutants affecting residues that coordinate to the Mg2+ ion
of TDP. C, mutants affecting residues that interact with
diphosphate oxygens of TDP. D, mutants affecting residues in
the K+ ion-binding site on the subunit.
(not shown) and the side chains of Asn-222-
and
Glu-193-
. A substitution of Glu-193-
with Ala results in complete
loss of BCKD activity (Table II). An
E193K-
mutation recently identified in an MSUD patient also renders
the enzyme inactive (data not shown). The N222S-
mutation, which was
identified in an MSUD patient, results in marked increases in
Km for KIV and TDP, whereas the kcat values are one half of the wild-type. These
data, taken together, indicate that the carbonyl groups in the side
chains of Glu-193-
and Asn-222-
are critical for Mg2+
ion binding. The hydroxyl group of the Ser side chain in the N222S-
mutant is probably a poorer ligand than Asn for the Mg2+
ion. The O
of a Ser residue would not be able to extend
as far toward the Mg2+ ion as the O
1 of the
Asn residue; this is likely to decrease affinity for the Mg2+ ion at this site. The less attenuated effect in
affinity for KIV and TDP in the Y224A-
mutant, compared with the
above two mutants is consistent with the fact that the main-chain
carbonyl group of Tyr-224-
coordinates to the metal ion. The
critical roles of Glu-193-
and Asn-222-
side chains in the metal
ion binding is supported by the markedly diminished levels of bound TDP
in the E193A-
, N222A-
, and Y224A-
mutants (Fig.
2B). The Y224A-
mutant also shows a markedly decreased
ability to bind TDP despite an only moderate increase in
Km for the cofactor.
Kinetic constants of BCKD mutants affecting residues involved in
Mg2+ ion binding
and Tyr-113-
are hydrogen-bonded to the
proximal and the terminal phosphate oxygens of TDP, respectively (5). Substitution of Gln-112-
with an Ala residue has marginal effects in
catalytic efficiency of BCKD as well as Km values for KIV and TDP (Table III). The data
suggest that either the side chain of the Gln-112-
is not an
essential ligand to the Mg2+ ion or that a neighboring
residue(s) can fill in this function. The introduction of an Ala
residue into the Tyr-113-
position results in more than 50- and
100-fold increases in Km for KIV and TDP, indicating
that Tyr-113-
is an important ligand to the terminal phosphate
oxygen. Remarkably, a replacement of Arg-114-
, which also
coordinates to the same terminal phosphate oxygen (Fig. 1), with an Ala
residue results in a completely inactive BCKD. The R114W-
mutation,
which occurs in MSUD patients, also renders the enzyme completely
inactive. As for Arg-220-
, which coordinates to another distal
phosphate oxygen, when changed to Ala, Lys or Trp (an MSUD mutation)
also results in complete loss in enzyme activity. The combined results
strongly suggest that the ionic interactions between positively charged
Arg-114-
or Arg-220-
and the negatively charged phosphate oxygens
are critical in maintaining the conformational integrity of the
diphosphate group of TDP (Fig. 1). In the case of R220K-
, the
positively charged Lys residue is pointing away from the distal
phosphate oxygen in the BCKD structure. Therefore, the ionic
interactions between the Lys side chain and the phosphate oxygen cannot
occur.
Kinetic constants of BCKD mutants affecting residues coordinating to
diphosphate oxygens of TDP
coordinates to another distal phosphate oxygen in the
TDP-binding pocket (Fig. 1). Substitution of this residue with Ala
results in a trace amount of BCKD activity (Table III). A previous
report showed that the same mutation in the rat BCKD is associated with
complete absence of activity (26); however, a spectrophotometric assay
was used in the latter study, which is less sensitive than the
radiochemical assay employed here. The combined results are thus
consistent with His-291-
as an essential ligand to the diphosphate
group of TDP. It is noteworthy that the Km for TDP
with the His-291-
mutant is increased by 40-fold over the wild-type
(Table III). This is consistent with about 15% of TDP bound to this
mutant compared with the wild-type (Fig. 2C). Modifications
of the three other residues Arg-114-
, Arg-220-
, and Gln-112-
that coordinate to the diphosphate oxygens invariably yield marginal
binding of TDP (Fig. 2C). The data strongly suggest that the
amino acid ligands to the diphosphate moiety of TDP are as important as
the ligands to the aminopyrimidine ring in maintaining the V
conformation of the cofactor. It should be mentioned that in addition
to being a ligand for the TDP diphosphate group, His-291-
has been
implicated to function as a catalytic residue (6). As described above,
BCKD functions as both a decarboxylase and a dehydrogenase during the
oxidative decarboxylation of
-ketoacids. His-291-
is proposed to
serve as a proton donor during the reduction of the disulfide bond of
the E2-attached lipoamide, which occurs during acyltransfer from the
enamine-TDP to E2. In the H291A-
mutant, the reduction of lipoamide
on E2 by the dehydrogenase activity of BCKD may be disrupted. The
additional role of His-291-
as a proton donor to the E2-attached
lipoamide will require further studies.
Subunit--
The crystal structure of human BCKD disclosed that the
and the
subunits each has a distinct K+ ion binding
fold that has not been described previously in any TDP-dependent enzyme (5). Fig.
3 shows that the K+ ion on
the
subunit stabilizes a loop containing residues Ser-161-
, Thr-166-
, and Gln-167-
, which are directly involved in ligating to the metal ion through their side chains. The structural integrity of
this loop structure is essential for ordering Ser-162-
and Leu-164-
for interactions of these residues with the cofactor TDP as
described above. An MSUD mutation, T166M-
, is likely to abolish the
coordination of the Thr side chain to the K+ ion, resulting
in a disorder of this loop with a concomitant disruption of TDP
binding. This accounts for the loss of both enzyme activity (Table
IV) and the inability of the mutant
enzyme to bind TDP (Fig. 2D). It is of interest that the
replacement of Thr-166-
with an Ala has little effect on the
catalytic efficiency of BCKD. It is possible that the side chain of
Ala, which is shorter than that of Met, does not aberrantly protrude
into the K+ ion binding pocket. As a result, the octahedral
coordination is not severely impaired by this substitution. This is
reflected by near wild-type catalytic efficiency of the T166A-
mutant when assayed at saturating TDP concentrations (Table IV).
However, the binding affinity of the mutant enzyme for the cofactor is still reduced as indicated by an elevated Km for TDP (Table IV) and by a significant decrease in the amount of the bound
cofactor compared with the wild-type (Fig. 2).
View larger version (30K):
[in a new window]
Fig. 3.
The K+ ion-binding site on
the subunit of human BCKD. The metal ion
is bound by two main-chain carbonyl groups and by the side
chains of Ser-161-
, Thr-166-
, and Gln-167-
. The side
chain of Leu-164-
and the main-chain carbonyl group of Ser-162-
make direct contacts with cofactor TDP. The octahedral coordination of
the metal ion stabilizes the loop structure on the
subunit
(residues 161-167) that is essential for the efficient binding of the
cofactor.
Kinetic parameters of BCKD mutants affecting residues involved in
K+ ion binding
, which is the Trp residue closest to the
aminopyrimidine ring of the cofactor at 15-18 Å, with a Phe. As a
control, a distal Trp-309-
was also converted to a Phe. The
W136F-
mutant BCKD showed Km values for KIV and
TDP, which are similar to those for the wild-type (data not shown).
Therefore, Trp-136-
appears to be nonessential for efficient binding
of both the substrate and the cofactor. The apoenzymes of the wild
type, W136F-
, and W309F-
were excited at 280 nm. The fluorescence
emission spectra of the wild-type and W309F-
apo-BCKD were similar
(Fig. 4). The maximal fluorescence emissions at 340 nm for both proteins are progressively quenched when
titrated with increasing concentrations of Mg-TDP. In contrast, the
maximal emission at 340 nm of the W136F-
mutant enzyme is not
quenched over the same concentration range of Mg-TDP. The result
established that Trp-136-
is the residue that is quenched upon TDP
binding. In pyruvate decarboxylase from Z. mobilis, the Trp-487 was shown to also be quenched by the bound TDP (28). The
crystal structure of this bacterial pyruvate decarboxylase shows that
Trp-487 is 18 Å away from the aminopyrimidine of a chemically modified
cofactor, 2-(1-hydroxyethyl)-TDP (10). Thus, the Trp residues in human
BCKD and the bacterial pyruvate decarboxylase, which are quenched upon
TDP binding, are in similar conformations relative to the cofactor. In
the related pyruvate dehydrogenase, Trp-135-
was reported to be the
residue that is quenched in response to TDP binding using chemical
modification and magnetic circular dichroism methods (29). Although the
structure of the pyruvate dehydrogenase has not yet been determined,
Trp-135-
in this enzyme aligns with the Trp-136-
in human BCKD.
The data show the conservation of the quenchable Trp residue in
TDP-dependent enzymes.
View larger version (21K):
[in a new window]
Fig. 4.
Identification of Tyr-136-
as the residue quenched by TDP binding. Wild-type or mutant
(W309F-
and W136F-
) apo-BCKD was contained in a cuvette housed in
a luminescence spectrometer at 23 °C. Fluorescence emission spectra
of 300-400 nm for apo-BCKD were obtained following an excitation at
280 nm. The quenching of tryptophan fluorescence was studied by adding
increments of Mg-TDP to the cuvette to the final concentrations of 0, 10, 15, 20, 30, 40, 60, 100, 200, and 400 µM (from
top to bottom spectra in each of the three
panels). The absence of fluorescence quenching in the
W136F-
mutant at saturating Mg-TDP concentrations indicates that
Tyr-136-
is the residue involved in fluorescence quenching by the
bound cofactor.
-ketoacids. The results depict a
high degree of conservation in the structure and function of the TDP
binding fold in TDP-dependent enzymes. These enzymes
include those that catalyze the TDP-mediated oxidative decarboxylation,
such as human BCKD (5), and the TDP-mediated nonoxidative
decarboxylation, such as yeast pyruvate decarboxylase (8) and
benzoylformate decarboxylate (12), as well as those that promote the
two-carbon transfer, for example, yeast transketolase (7, 30). Human BCKD is distinct among TDP-dependent enzymes in that the
enzyme is both a decarboxylase and dehydrogenase. The enamine-TDP
intermediate becomes a substrate for the acyltransfer reaction during
the BCKD-mediated reduction of lipoamide covalently attached to the E2
core of the BCKD complex. The mechanism for the BCKD-catalyzed
acyltransfer reaction remains to be elucidated at the structure level.
, which is
responsible for phosphorylation and the resultant inactivation of BCKD,
is located inside the TDP-binding pocket. From a structural standpoint, it is unclear how the specific BCKD kinase enters this TDP binding fold
to phosphorylate Ser-292-
. However, the phosphorylation efficiency
of BCKD is markedly enhanced when the enzyme is in complex with the E2
acyltransferase (31-33). It can, therefore, be speculated that binding
of BCKD to E2 may induce conformational changes that render the
TDP-binding pocket more accessible to the kinase. These questions will
be addressed by solving the structure of human BCKD that in complex
with the binding domain of E2.
-ketoacid
dehydrogenase complexes. The presence of K+ ion-binding
sites explains the dependence of high K+, but not
Na+, ion concentrations on the inhibitions of BCKD
phosphorylation by TDP (31). A survey of the literature shows that the
Pseudomonas dialkylglycine decarboxylase also uses the
octahedral K+ ion coordination to maintain the conformation
of a loop structure in the cofactor pyridoxal phosphate binding fold
(35, 36). This topology appears to be conserved among certain
cofactor-dependent decarboxylases.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant DK-26758, American Heart Association, Texas Affiliate, Grant 95G-074R, and Welch Foundation Grant I-1286.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9038. Tel.: 214-648-2457; Fax: 214-648-8856; E-mail: David.Chuang@UTSouthwestern.edu.
Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M008038200
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ABBREVIATIONS |
---|
The abbreviations used are:
BCKD or E1, branched-chain -ketoacid decarboxylase/dehydrogenase;
E2, dihydrolipoyl transacylase;
E3, dihydrolipoamide dehydrogenase;
FPLC, fast protein liquid chromatography;
NTA, nitrilotriacetic acid;
KIV,
-ketoisovalerate;
MSUD, maple syrup urine disease;
TDP, thiamine
diphosphate;
TEV, tobacco-etch virus.
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