Branched-chain amino acid metabolon: Interaction of glutamate dehydrogenase with the mitochondrial branched-chain aminotransferase (BCATm)

Article abstract of DOI:10.1074/jbc.M109.048777

The catabolic pathway for branched-chain amino acids includes deamination followed by oxidative decarboxylation of the deaminated product branched-chain α-keto acids, catalyzed by the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC). We found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of branched-chain α-keto acids from BCATm to E1. The protein complex also contains glutamate dehydrogenase (GDH1), 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1, pyruvate carboxylase, and BCKDC kinase. GDH1 binds to the pyridoxamine 5′-phosphate (PMP) form of BCATm (PMP-BCATm) but not to the pyridoxal 5′-phosphate-BCATm and other metabolon proteins. Leucine activates GDH1, and oxidative deamination of glutamate is increased further by addition of PMP-BCATm. Isoleucine and valine are not allosteric activators of GDH1, but in the presence of 5′-phosphate-BCATm, they convert BCATm to PMP-BCATm, stimulating GDH1 activity. Sensitivity to ADP activation of GDH1 was unaffected by PMP-BCATm; however, addition of a 3 or higher molar ratio of PMP-BCATm to GDH1 protected GDH1 from GTP inhibition by 50%. Kinetic results suggest that GDH1 facilitates regeneration of the form of BCATm that binds to E1 decarboxylase of the BCKDC, promotes metabolon formation, branched-chain amino acid oxidation, and cycling of nitrogen through glutamate.

Full text of DOI:10.1074/jbc.M109.048777

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 1, pp. 265–276, January 1, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Branched-chain Amino Acid Metabolon
INTERACTION OF GLUTAMATE DEHYDROGENASE WITH THE MITOCHONDRIAL
BRANCHED-CHAIN AMINOTRANSFERASE (BCATm)*
Received for publication, July 24, 2009, and in revised form, October 8, 2009 Published, JBC Papers in Press, October 26, 2009, DOI 10.1074/jbc.M109.048777
Mohammad Mainul Islam‡, Manisha Nautiyal§, R. Max Wynn¶, James A. Mobleyʈ, David T. Chuang¶,
and Susan M. Hutson‡1
From the ‡Department of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute, Blacksburg, Virginia 24061, the
§Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, the
¶Departments of Biochemistry and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390,
and the ʈDepartment of Surgery, University of Alabama at Birmingham, Birmingham, Alabama 35294
The catabolic pathway for branched-chain amino acids includes
deamination followed by oxidative decarboxylation of the deami-
nated product branched-chain ␣-keto acids, catalyzed by the
mitochondrial branched-chain aminotransferase (BCATm) and
branched-chain ␣-keto acid dehydrogenase enzyme complex
(BCKDC). We found that BCATm binds to the E1 decarboxylase
of BCKDC, forming a metabolon that allows channeling of
branched-chain ␣-keto acids from BCATm to E1. The protein
complex also contains glutamate dehydrogenase (GDH1), 4-ni-
trophenylphosphatase domain and non-neuronal SNAP25-like
protein homolog 1, pyruvate carboxylase, and BCKDC kinase.
GDH1 binds to the pyridoxamine 5؅-phosphate (PMP) form of
BCATm (PMP-BCATm) but not to the pyridoxal 5؅-phosphate-
BCATm and other metabolon proteins. Leucine activates
GDH1, and oxidative deamination of glutamate is increased fur-
ther by addition of PMP-BCATm. Isoleucine and valine are not
allosteric activators of GDH1, but in the presence of 5؅-phos-
phate-BCATm, they convert BCATm to PMP-BCATm, stimu-
lating GDH1 activity. Sensitivity to ADP activation of GDH1 was
unaffected by PMP-BCATm; however, addition of a 3 or higher
molar ratio of PMP-BCATm to GDH1 protected GDH1 from
GTP inhibition by 50%. Kinetic results suggest that GDH1 facil-
itates regeneration of the form of BCATm that binds to E1
decarboxylase of the BCKDC, promotes metabolon formation,
branched-chain amino acid oxidation, and cycling of nitrogen
through glutamate.
cosome”) are organized into complexes, and this assembly is
regulated by the oxidation and phosphorylation states of the
proteins. The advantages of such a supramolecular assembly
include efficient channeling of substrates between enzymes in a
pathway, regulating pathway flux by association and dissocia-
tion, and by targeting the assembly of the interacting proteins to
the appropriate intracellular structures. Previously, using liver
mitochondrial extracts and purified BCATm,2 we showed that
the first two enzymes in BCAA catabolism, mitochondrial
branched-chain aminotransferase (BCATm) and branched-
chain ␣-keto acid decarboxylase (E1) of the BCKDC, associate
in vitro (5). It is not yet known if this BCAA metabolon forms in
tissues expressing BCATm, and if other associated proteins are
functionally involved in the metabolon.
BCATs (mitochondrial and cytosolic isozymes) catalyze
reversible transamination of BCAAs (leucine, isoleucine, and
valine) to form the branched-chain ␣-keto acids (BCKAs) (6).
Transamination results in transfer of the ␣-amino group from
BCAA to ␣-ketoglutarate (␣-KG) to form glutamate (Glu) as
shown in Reactions 1 and 2.
BCAT⅐PLP ϩ R1-CH͑NH2͒-COOH ^
BCAT⅐PMP ϩ R1-CO-COOH
REACTION 1
BCAT⅐PMP ϩ R2-CO-COOH ^
BCAT⅐PLP ϩ R2-CH͑NH2͒-COOH
REACTION 2
It has been proposed that the enzymes that are responsible
for catalyzing sequential reactions in several metabolic path-
ways are highly organized in supramolecular complexes termed
metabolons (1). The concept of metabolic enzymes associating
to form a supramolecular complex was hypothesized 50 years
ago (2). Recently, Benkovic and co-workers (3) have shown the
in vivo assembly of six proteins in the purine catabolic pathway
to form a “purinosome.” The formation of the purinosome
appears to be dynamically regulated by stimulation of de novo
purine biosynthesis in response to changes in purine levels.
Another group (4) also has shown that glycolytic enzymes (“gly-
Here, BCAT⅐PLP and BCAT⅐PMP denote the pyridoxal
2 The abbreviations used are: BCATm, mitochondrial branched-chain amino-
transferase; BCAA, branched-chain amino acid; BCKA, branched-chain
␣-ketoacid; BCKDC, branched-chain ␣-ketoacid dehydrogenase complex;
BCAT, branched-chain aminotransferase; BCATc, cytosolic branched-chain
aminotransferase; DCPIP, 2,6-dichlorophenolindophenol; DTT, dithiothre-
itol; E1, branched-chain ␣-keto acid decarboxylase/dehydrogenase; E2,
dihydrolipoyltransacylase; E3, dihydrolipoamide dehydrogenase; KIC,
␣-ketoisocaproate; ␣-KG, ␣-ketoglutarate; NIPSNAP1, 4-nitrophenylphos-
phatase domain and non-neuronal SNAP25-like protein homolog 1; PLP,
pyridoxal 5Ј-phosphate; PMP, pyridoxamine 5Ј-phosphate; ThDP, thi-
amine diphosphate; BDK, BCKDC kinase; CHO, Chinese hamster ovary;
MALDI, matrix-assisted laser desorption ionization; CHAPS, 3-[(3-cholami-
dopropyl)dimethylammonio]-1-propanesulfonic acid; DSP, dithiobis(suc-
cinimidyl propionate); PC, pyruvate carboxylase.
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grants DK34738 (to S. M. H.) and DK26758 (to D. T. C.) from the USPHS.
1 To whom correspondence should be addressed. Tel.: 540-231-8766; Fax:
540-231-3916; E-mail: susanh5@vt.edu.
JANUARY 1, 2010•VOLUME 285•NUMBER 1
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Protein Complex in Branched-chain Amino Acid Metabolism
5Ј-phosphate (PLP) and pyridoxamine 5Ј-phosphate (PMP) BL21(DE3)pLys-competent cells). The anti-His monoclonal
forms of BCAT, respectively. R1 is the side chain of BCAAs or antibody and anti-FLAG M2 monoclonal antibodies were pur-
BCKAs, and R2 is the side chain of ␣-KG. The ␣-amino group chased from Sigma. The anti-rat BCATm antibody has been
temporarily resides on the coenzyme PLP forming PMP (6–9). described previously (20), and the antisera raised against com-
BCKA transamination products are ␣-ketoisocaproate (KIC), ponents of the BCKDC complex were the kind gift of Dr. Yoshi
␣-keto-␤-methylvalerate, and ␣-ketoisovalerate. In the second Shimomura (Nagoya, Japan). Restriction endonucleases and
half-reaction, the PMP cofactor donates its nitrogen to ␣-KG to other DNA modification enzymes were purchased from Pro-
form Glu (10). Once BCAA nitrogen enters the large glutamate mega. DNA sequencing was done at the DNA sequencing lab-
pool, it is available for synthesis of alanine and glutamine. The oratory of the Comprehensive Cancer Center at Wake Forest
forward nitrogen transfer is favored by oxidation of the BCKA University School of Medicine. The medium used for bacterial
products by BCKDC. Net nitrogen transfer from BCAAs to growth contained 0.5% yeast extract, 1% polypeptone, and 0.5%
glutamate occurs when the carbon skeleton of the BCAA is NaCl and was from Fisher. Nickel-nitrilotriacetic acid resin was
irreversibly lost via the BCKDC step (oxidation) (11).
purchased from Qiagen (Chatsworth, CA). Sequencing grade-
The second enzyme in BCAA metabolism is BCKDC, which modified trypsin was purchased from Promega (Madison, WI).
is a member of a family of highly conserved macromolecular Bovine GDH1 is available commercially and was purchased
enzyme complexes. The other two protein complexes in this from Sigma. All other chemicals were of the highest grade com-
family are pyruvate dehydrogenase and ␣-ketoglutarate dehy- mercially available.
drogenase (12–14). The catalytic reaction of all these three pro-
Animals—Male Sprague-Dawley rats (200–300 g) were
tein complexes is similar and leads to the oxidative decarbox- housed three to four per cage and maintained with standard
ylation of ␣-ketoacids (BCKAs, pyruvate, and ␣-KG) giving rise chow food diets and water in a temperature and light controlled
to branched-chain acyl-CoAs, acetyl-CoA, or succinyl-CoA, environment. The animals were sacrificed under a mixture of
respectively (Reaction 3).
ketamine/xylazine anesthesia, and the tissues were collected in
MSE buffer (225 mM D-mannitol, 75 mM sucrose, 1 mM EDTA,
pH 7.0).
R1-CO-COOH ϩ CoA-SH ϩ NADϩ
3
Preparation of Mitochondria and Mitoplasts—The mito-
chondria from different rat tissues were prepared according to
methods described previously (5, 21). Mitochondria from rat
pancreas were prepared as described in MacDonald et al. (22).
To prepare mitoplasts from liver mitochondria, the mitochon-
drial pellets were diluted to 100 mg/ml with H medium (75 mM
sucrose, 225 mM D-mannitol, 50 mM HEPES, and 1 mM bovine
serum albumin) (23). An equal volume of extraction mixture
(12 mg/ml digitonin and 0.5 mg/ml bovine serum albumin) was
added and diluted by a 3-fold volume of H medium and centri-
fuged at 12,500 rpm for 10 min. The pellets were then resus-
pended in H medium, and centrifugation was then repeated.
The pellets were then resuspended in a minimal volume of H
medium. The respiratory control ratio was determined sepa-
rately in the presence of magnesium by measuring the ratio of
the respiratory rate in the presence of ADP (0.5 mM) to the rate
measured after cessation of ADP phosphorylation with 20 mM
glutamate and 1 mM malate as substrates (24). No mitochon-
drial preparation with a ratio less than 6 was used. Protein was
determined using the Biuret assay. The mitochondria and mito-
plasts (100 mg) were allocated into separate tubes and centri-
fuged, and the pellets were either used immediately or stored at
Ϫ80 °C. Pellets could be stored frozen for up to 4 weeks. Mito-
chondrial proteins were extracted using solubilizing buffer (SB
buffer: 50 mM potassium phosphate, 0.2 mM EDTA, 0.75%
CHAPS, 25 mM KCl, 1 mM DTT, pH 7.4, and protease inhibitor
mixture set III (Calbiochem)) as described previously (5).
Purification of Recombinant BCAT Proteins—The overex-
pression and purification of the wild type and mutant BCAT
proteins were performed according to the method of Conway et
R1-CO-S-CoA ϩ CO21ϩ NADH ϩ Hϩ
REACTION 3
We have shown that the deaminated BCKA product of
BCATm is directly transferred to the E1 decarboxylase of
BCKDC through complex formation between the two
enzymes and substrate channeling (15). The association
between BCATm and E1 is specific for the PLP form of the
BCATm isozyme, requires a reduced CXXC center, is inhib-
ited by phosphorylation of E1, and is dissociated by the BCKDC
reaction product NADH (5). The E1 enzyme catalyzes the
ThDP-mediated decarboxylation of BCKAs (12). The product
of the reaction, carbanion-ThDP, undergoes the E1-catalyzed
reductive transfer of carbanion to the lipoyl moiety, which is
covalently bound to dihydrolipoyl transacylase (E2) (16). The
dihydrolipoyl residue is reoxidized by the FAD moiety of dihy-
ϩ
drolipoamide dehydrogenase (E3), and NAD is the ultimate
electron acceptor. This reaction is the committed step in BCAA
oxidation.
In this study, we have identified other proteins found in the
complex in tissues expressing BCATm and have explored
potential functions of GDH1 on BCAA metabolism. Leucine
has been reported to allosterically activate GDH1, which is
important for the stimulation of insulin secretion by this amino
acid (17–19). We demonstrate that GDH1 is the protein part-
ner for PMP-BCATm. Facilitating the recycling of BCATm to
form metabolon, GDH1 acts as a catalytic machine.
EXPERIMENTAL PROCEDURES
Chemicals—Plasmid pCR 2.1 TA cloning vector and pcDNA3. al. (25, 26). Mutant (C315A, C318A, and C315A/C318A) and
1/Zeo(ϩ) were purchased from Invitrogen, and pET28-a(ϩ) oxidized BCATm were prepared according to the method of
plasmid was purchased from Novagen (Madison, WI). Bac- Yennawar et al. (27). The recombinant human E1, E2, and E3
terial strains were purchased from Novagen (Escherichia coli proteins were expressed as described previously (28, 29).
266 JOURNAL OF BIOLOGICAL CHEMISTRY
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Protein Complex in Branched-chain Amino Acid Metabolism
Preparation of Protein and Antibody Affinity Columns—To manufacturer’s instructions. Proteins were alkylated with
prepare the protein columns, recombinant BCAT (1 mg/ml in 2 iodoacetamide, and enzymatic digestion was performed using
ml) was dialyzed against bicarbonate buffer (0.2 M NaHCO3 sequencing grade trypsin gold (Promega, Madison, WI). The
and 0.5 M NaCl, pH 8.0) overnight. Two g of activated CH- extent of digestion was determined by SDS-PAGE, and then 20
Sepharose௡ 4B (Sigma) were suspended in 200 ml of cold 1 mM ␮l of the digested sample was purified on a C18 peptide trap
HCl for 10–15 min. The rest of the protocol for this ligand- (Michrom Bioresources, Auburn, CA) and concentrated in a
resin coupling was performed according to the manufacturer’s SpeedVac apparatus followed by resuspension in 20 ␮l of 0.1%
instructions. The columns were stored at 4 °C in phosphate- MS grade formic acid. One-half of the sample (10 ␮l) was
buffered saline with 0.02% sodium azide, and 5 mM DTT was diluted 1:20, and ϳ4 ␮l (ϳ200 ng of peptide mixture) was
added to the BCATm column. To prepare antibody columns, injected onto a Thermo Surveyor high pressure liquid chroma-
affinity-purified human BCATm antibody was coupled to tography tied to a Thermo LTQ ion trap mass spectrometer
recombinant protein A beads and cross-linked with disuccin- with a nano-electrospray source (Thermo Electron, San Jose,
imidyl suberate (Pierce) following the protocol provided by the CA). The liquid chromatography conditions and search param-
manufacturer.
eters were run using conditions published previously (30). Data
Isolation of BCATm-bound Proteins—Isolation of BCATm- were searched against the NCBInr mammalian data base using
bound proteins from the protein columns was performed as SEQUEST with common filtering cutoff values, i.e. cross-cor-
described previously with some modification (5). The BCAT relation (Xcorr) values Ͼ1.8 for doubly charged ions and Ͼ2.5
affinity columns were equilibrated with the SB buffer (contain- for triply charged ions. In addition, ranking of preliminary score
ing 1 mM DTT for BCATm), and the filtered mitochondrial values of Ͻ5 and preliminary score values of Ͼ350 also were
extract was passed through the column. The column was then required for positive peptide identifications. A number of sig-
washed with SB buffer with addition of 100 mM KCl. Bound nificant hits were observed for each protein with Xcorr above 5
proteins were eluted with 10 or 5 mM NADH in SB buffer with- as reported under “Results.”
out DTT. The eluted proteins were precipitated using acetone
Spectrophotometric Kinetic Analysis—GDH1 enzymatic
at Ϫ20 °C overnight, and the protein pellet was washed with assays were performed using a Beckman Coulter DU-800 spec-
10% trichloroacetic acid followed by ether/ethanol (1:1 v/v). trophotometer at 30 °C equipped with a Beckman Coulter tem-
The protein pellets were dried under nitrogen, resuspended in perature controller, and the reaction was monitored using the
SDS loading buffer, and analyzed by SDS-PAGE. For the anti- coenzyme absorbance changes at 340 nm. The reactions were
body columns, the mitochondrial extract in SB buffer was performed in 1 ml of 0.1 M sodium phosphate buffer, pH 8.0, for
applied to the column. The bound proteins were eluted using oxidative deamination in the presence of 50 mM glutamate and
ϩ
the RC buffer (4 M urea, 0.5 M NaCl in 0.1 M sodium acetate, pH 0.2 mM NAD and at pH 7.0 for reductive amination in the
4.0). The rest of the steps were exactly as described above.
presence of 0.1 mM NADH, 50 mM NH4Cl, and 5 mM ␣-keto-
Cell Lines and Dithiobis(succinimidyl Propionate) (DSP) glutarate (31).
Cross-linking—CHO cell lines were grown in Dulbecco’s mod-
Kinetics of the E1 decarboxylase reaction in presence of GDH1
ified Eagle’s medium supplemented with 10% fetal calf serum. and BCATm were determined in the presence of an artificial elec-
When the cells reached confluency (80–90%), they were trans- tron acceptor 2,6-dichlorophenolindophenol (DCPIP) as in Ref.
fected with pcDNA3.1/Zeo(ϩ) containing BCATm cDNA, and 5. The assay mixture contained 100 mM potassium phosphate,
expression of the protein was evaluated using an established pH 7.5, 2.0 mM MgCl2, 0.2 mM ThDP, and 0.1 mM DCPIP. E1
assay (26). To cross-link protein complexes, the cells were was reconstituted with BCATm at a 1:1 molar ratio, and GDH1
washed with phosphate-buffered saline and then incubated was added at varied molar ratios to BCATm. The rate of decar-
with the thiol-cleavable cross-linker DSP at 1 mM concentra- boxylation at 30 °C was measured by monitoring the reduction
tion in dimethyl sulfoxide (DMSO) and with occasional shaking of the dye at 600 nm (15, 33).
for 30 min at room temperature. The reaction was quenched for
For the overall assay of the BCAA metabolon complex (Reac-
15 min with 10 mM Tris, pH 8.6. After washing with phosphate- tion 3) in the presence of GDH1, the enzymes were exchanged
buffered saline, the cells were lysed in HEPES buffer (50 mM into phosphate buffer (30 mM potassium phosphate, pH 7.5)
HEPES, pH 8.0) containing 0.2 mM NaCl, 1 mM EDTA, 1% Tri- containing 5 mM DTT using a PD-10 column, and the enzyme
ton X-100, and protease inhibitor mixture. The lysate was concentrations were calculated from the absorption maxima at
passed through a BCATm antibody column, and the bound 280 nm (27). The protein complex was reconstituted with
proteins were eluted RC buffer containing a high concentration BCATm, E1, and lipoylated E2 and E3 at a molar ratio of 12:12:
of urea. The eluted proteins were identified using nonreducing 1:55, in which lipoylated E2 exist as a 24-mer. NIPSNAP1 and
SDS-PAGE and Western blotting (20 ␮g of proteins were GDH1 were added in different molar ratios to BCATm. The
loaded in ϪBCATm and ϩBCATm lanes, and 4 ␮g of proteins assay mixture contained 30 mM potassium phosphate, pH 7.5,
ϩ
were loaded in complex lanes) and MALDI-mass spectrometry. 100 mM NaCl, 3 mM NAD , 0.4 mM CoA, 2 mM MgCl2, 2 mM
Protein Identification by Tandem Mass Spectrometry and DTT, 0.1% Triton X-100, and 2 mM ThDP. The overall reaction
N-terminal Edman Degradation—To identify the proteins in was monitored by formation of NADH at 340 nm. Replacement
the affinity chromatographic eluate, the IP pellet was resus- of NaCl salt with the more physiological relevant KCl salt did
pended in 50 ␮l of the reaction mixture (6 M guanidine HCl, 50 not show any difference in the kinetic rate constants for
mM Tris-HCl, pH 8.0, and 4 mM DTT), which was followed by a BCKDC. However, KCl inhibited the activity of GDH1 (34).
standard in-solution trypsin digestion protocol following the Therefore, we always used NaCl in the overall reaction mixture.
JANUARY 1, 2010•VOLUME 285•NUMBER 1
JOURNAL OF BIOLOGICAL CHEMISTRY 267

Protein Complex in Branched-chain Amino Acid Metabolism
constants of GTP, and M1 is the
factor associated with maximum
extent of GTP inhibition. All cal-
culations and analyses were per-
formed using IGOR Pro software
(WaveMetrics Inc.).
E1 Phosphorylation Assay—The
phosphorylation of E1 was carried
out in the phosphorylation reaction
mixture (30 mM HEPES, pH 7.4, 2
mM DTT, 1.5 mM MgCl2, and 0.2
mM EGTA). BCATm, E1, E2, and E3
proteins were mixed at 12:12:1:55
molar ratio in 0.1 ml of reaction
mixture, and 0.1 ␮g of maltose-
binding protein-tagged rat BCKDC
kinase (BDK) was added. The mix-
ture was preincubated at room tem-
perature for 15 min. The phosphor-
ylation reaction was started after
addition of 0.4 mM ATP to the reac-
tion mixture, and the reaction was
terminated at different time points
by addition of higher salt concentra-
tion (15). Overall BCKDC activity
FIGURE 1. Human BCATm-associated proteins in rat liver mitochondria. Rat liver mitochondria were
extracted in SB Buffer. The extract was applied to a BCATm-Sepharose affinity column, and the column was
washed with SB Buffer until no more UV-absorbing material was eluted, followed by washing with SB Buffer
plus100mM KCl. TheBCATmboundproteinswereelutedwithSBBuffercontaining10mM NADHandidentified
by tandem mass spectrometry as described under “Experimental Procedures.” Lower band immediately under
PC is a degraded fragment of PC.
was measured as described above.
TABLE 1
CD Spectroscopic Analysis—Allosteric changes of GDH1
were analyzed by CD spectroscopic measurements using a
Jasco J-720 spectropolarimeter equipped with a variable tem-
perature accessory. The spectra (350 to 190 nm) were acquired
using a protein concentration of 20 ␮M in 10 mM potassium
phosphate buffer, pH 7.0, and a 0.01-cm path length. CD spec-
tra of BCATm were used as the blank to obtain the GDH1
spectra. Values were measured as ⌬⑀ ϭ ⌬A⅐c⅐l (where c is molar
concentration and l is path length in centimeters). Differences
in CD spectra were calculated by ⌬AЈ ϭ ⌬Asolution Ϫ ⌬Areference
and were obtained by numerical subtraction. Allosteric changes
in GDH1 were detected at 260 nm, and the binding constant
was determined from the curve fitting of ⌬A2Ј 60 versus
[BCATm]. The final spectra were the average of three different
accumulations.
Identification of metabolon proteins in extrahepatic tissues
SEQUEST was used for liquid chromatography/tandem mass spectrometry analy-
sis, and MASCOT was used for matrix-assisted laser desorption ionization time-of-
flight/time-of-flight analysis, although in most cases both were used. Data were
searched against the NCBInr mammalian data base using SEQUEST using common
filtering cutoff values, i.e. cross-correlation (Xcorr) values Ͼ1.8 for doubly charged
ions and Ͼ2.5 for triply charged ions. In addition, ranking of preliminary score
values of Ͻ5 and preliminary score values of Ͼ350 also were required for positive
peptide identifications. Replicate experiments were carried out with similar results.
ϩ, present; ϩ/Ϫ, lower content; Ϫ, absent; NC, not confirmed.
Mitochondrial source
MASCOT
Skeletal Brown adipose
ID
Pancreas
Heart Kidney
muscle
tissue
PC
E3
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
Ϫ
ϩ
ϩ/Ϫ
ϩ
ϩ
Ϫ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
Ϫ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
Ϫ
GDH1
ϩ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
NC
E2
ϩ
BCKD-E1␣
BCKD-E1␤
NIPSNAP1
BDK
ϩ
ϩ
Ϫ
NC
RESULTS
Tissue Distribution of BCAA Metabolon Protein Partners—
Using rat liver mitochondrial extracts, we have shown previ-
ously the potential for a BCAA supramolecular complex con-
sisting of BCATm and BCKDC proteins (5). As shown in Fig. 1,
other proteins were also found in the liver mitochondrial com-
plex. These non-BCKDC liver mitochondrial proteins, which
bound to the BCATm affinity column, were GDH1, pyruvate
carboxylase (PC), BDK, and NIPSNAP1 (Fig. 1). To determine
the potential metabolon composition in tissues that express
BCATm, BCATm-specific antibodies were used to isolate asso-
ciating proteins from mitochondria prepared from the pan-
creas, kidney, heart, skeletal muscle, and brown adipose tissue.
The proteins found in these tissues are shown in Table 1. There
were tissue-specific differences in metabolon composition that
The apparent rate constants (kapp) at different substrate con-
centrations for all of the above assays were determined from the
absorption changes at the individual wavelength maximum.
The kapp rate constants were fit using the following Equation 1,
k
app ϭ kcat͓S͔/͑Km ϩ ͓S͔͒
(Eq. 1)
The kcat and Km values were obtained from the fitted curves and
are shown in Tables 3–5.
GTP inhibition curves in the absence or presence of PMP-
BCATm were analyzed using the following mathematical Equa-
tion 2,
% activity ϭ 100⅐͑M1͓͑GTP͔͒/͑Ki ϩ ͓GTP͔͒͒
(Eq. 2)
where [GTP] is the concentration of GTP; Ki is the inhibition were consistent with differences in expression of the individual
268 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 1•JANUARY 1, 2010

Protein Complex in Branched-chain Amino Acid Metabolism
proteins in these tissues. For example, PC is a binding partner in
all tissues except heart. It is the prominent band in the liver
complex (Fig. 1). Proteomic studies of tissue mitochondria also
show that PC is abundant in other tissues like brown adipose
tissue and kidney, whereas there are low levels expressed in
skeletal muscle, and it is absent in heart (35). BCKDC proteins
were found in the metabolon in all tissues except skeletal mus-
cle. All five subunits of BCKDC are found in skeletal muscle;
however, the high phosphorylation state of E1 may prevent
metabolon formation (see below). We used chow-fed animals
where liver E1 is highly dephosphorylated and the skeletal mus-
cle E1 is highly phosphorylated (36, 37). NIPSNAP1 was found
only in heart and kidney tissues. BDK was associated with
metabolon proteins in pancreas and kidney mitochondria and
was not observed in brown adipose tissue mitochondria. Asso-
ciation of BDK with the metabolon was variable. When BDK
was present, GDH1 was not found in the associated proteins.
Published mitochondrial protein array data show that the tis-
sue-specific expression of GDH1 protein is consistent with its
presence in the BCAA metabolon in the tissues shown in Table
1 (38).
Previously (5), we demonstrated channeling of the BCATm
transamination product to the E1 component of BCKDC that
enhanced E1-catalyzed decarboxylation of the BCKAs and
overall BCKDC activity. Addition of ␣-KG stimulated activity
further by regenerating the PLP form of BCATm that binds to
E1, i.e. cycling versus single turnover reaction. If GDH1 is a
metabolon protein, it could potentially supply ␣-KG for
BCATm through oxidative deamination of glutamate. In this
study, we focused on potential functions of GDH1 in the BCAA
metabolon and identified its binding partner.
FIGURE 2. DSP cross-linking of the complex proteins (A) and identifica-
tion of binding partner of GDH1 (B). A, CHO cell lines were transfected with
pcDNA3.1/Zeo(ϩ) containing BCATm gene DNA and incubated with DSP.
The BCATm-associated proteins were isolated using a BCATm antibody col-
umn, and the complex proteins were identified by mass spectrometry or
Western blotting using anti-BCATm antibody. Lanes are as follows: ϪBCATm,
cell extract from untransfected cell lines; ϩBCATm, cell extract of BCATm
transfected cell lines; complex, BCATm-associated proteins eluted using the
BCATm antibody column. All experiments were performed under nonreduc-
ing conditions. B, GDH1 was coupled to Profound co-immunoprecipitation
beads and incubated with PLP-BCATm, PMP-BCATm, E1, E2, pyruvate dehy-
drogenase-E2, E3, PC, or NIPSNAP1. Flow-through (F) and bound proteins
elutedwithNADH(E)wereanalyzedbySDS-PAGE. PMP-BCATmwasboundto
GDH1. Although a small amount of PLP-BCATm appeared to bind with GDH1,
it could represent PMP-BCATm contamination of the purified PLP-BCATm
preparation.
Identification of the Protein Complex Using Chemical Cross-
linking and Affinity Chromatography—To identify the BCAA
metabolon proteins in in situ, we used CHO cell lines. The
freshly prepared cell lines (Fig. 2A, ϪBCATm) were transfected
with pcDNA vector containing the BCATm gene DNA (Fig.
2A, ϩBCATm), and the BCATm overexpressed cell lines were
harvested in the presence of the cell-permeable chemical cross-
linker DSP. BCATm-bound proteins were captured using a
BCATm antibody column and eluted from the column with RC
buffer (Fig. 2A, complex), and the complex proteins were iden-
tified using MALDI-mass spectrometry. Analysis of the pro-
teins in the nonreducing SDS-PAGE demonstrates that all
BCAA metabolon proteins (BCATm, BCKDC, GDH1, PC, and
NIPSNAP1) were present in this DSP-cross-linked complex.
Western blotting of the gel with anti-BCATm antibody
demonstrated that there was some endogenous BCAT in CHO
cell lines (Fig. 2A, light band, ϪBCATm), and CHO cell lines
showed overexpressed the BCATm after transfection with
BCATm cDNA (Fig. 2A, ϩBCATm). The thick band at 200 kDa
(Fig. 2A, complex) indicated that BCATm is associated with
other proteins in a supramolecular complex.
PMP-BCATm Induces Allosteric Changes in GDH1—Mam-
malian GDH1 is a homohexamer. Each subunit is characterized
by an N-terminal glutamate-binding domain, NAD-binding
domain, and a special antenna domain. The residues in the
antenna region are characteristic for mammalian GDH1 and
are involved in allosteric regulation of this enzyme (39, 40).
GDH1 forms abortive complexes with NAD(P)H⅐Glu and
NAD(P)⅐␣KG. Mammalian GDH1 exhibits complex allosteric
regulation by a large number of allosteric effectors. The main
allosteric activators are leucine and ADP, which act by destabi-
lizing the abortive complex of GDH1. GTP is a potent inhibitor
that acts through stabilization of the abortive complexes (41–
44). Changes in the allosteric conformation of GDH1 can be
analyzed using CD spectroscopy (45, 46). To determine
To identify the binding partner(s) of GDH1, PMP-BCATm,
PLP-BCATm, E1, E2, E3, and PC were used as bait proteins.
Affinity chromatography was performed using NADH contain-
ing SB buffer to elute bound proteins. As shown in Fig. 2B, only
the PMP form of BCATm clearly bound to GDH1.
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Protein Complex in Branched-chain Amino Acid Metabolism
FIGURE 4. Oxidative deamination of glutamate by GDH1 is stimulated by
PMP-BCATm. Activity was measured with (closed circles) and without (open
circles) PLP-BCATm. A, leucine (Leu) alone stimulates GDH1 activity, which is
enhanced by further addition of PLP-BCATm. Isoleucine (B) and valine (C) do
not affect activity in the absence of BCATm.
FIGURE 3. PMP-BCATm induces allosteric changes in GDH1. A, CD changes
at 260 nm show a conformational change in GDH1 on addition of increasing
concentrations of PMP-BCATm. B, GDH1 and BCATm interact at a molar ratio
of 1:3 which corresponds to a 1:1 active site ratio of the GDH1 hexamer and
BCATm dimers. Titration of GDH1 in the presence of C315A/C318A BCATm (C)
or oxidized BCATm (D) showed no significant allosteric changes in GDH1,
indicating there is no interaction between GDH1 and the mutant or oxidized
BCATm.
alone. However, addition of PLP-BCATm to the reaction mix-
ture accelerated GDH1-catalyzed deamination by 75–80% and
70–75% (Fig. 4, B and C). These results suggest that addition of
PLP-BCATm plus a BCAA destabilizes the abortive complex of
whether addition of BCATm affects the conformation of GDH1 by converting PLP-BCATm to the PMP form, releasing
GDH1, GDH1 (20 ␮M) was incubated with increasing concen- the BCKA product ␣-ketoacid. The PMP-BCATm then binds
trations of BCATm (10–200 ␮M), and CD spectra were GDH1 facilitating catalysis. The association of PMP-BCATm
recorded (see Fig. 3A). The intense CD spectral change associ- with GDH1 results in an allosteric conformational change that
ated with addition of PMP-BCATm is clearly visible at 260 nm. enhances the rate of oxidative deamination of glutamate and
Neither PLP-BCATm (data not shown), the CXXC mutant (Fig. suggests substrate channeling of the ␣-KG product to PMP-
3C) or oxidized BCATm (Fig. 3D), nor cytosolic BCATc (data BCATm, reforming glutamate and PLP-BCATm. We have per-
not shown) affected the CD spectra. Plotting ⌬A260 versus formed similar experiments using CXXC center oxidized and
[PMP-BCATm] suggested a 1:3 complex with a calculated Kd of mutated BCATm and cytosolic BCATc (data not shown). No
2 ␮M (Fig. 3B), which is similar to the calculated Kd value for the apparent changes in GDH1 catalysis were found with these
BCATm-E1 interaction (5).
proteins suggesting that the interaction between GDH1 and
PMP-BCATm Accelerates the Oxidative Deamination Reac- PMP-BCATm is enzyme-specific and requires reduced active
tion of GDH1—GDH1 catalyzes the oxidative deamination of BCATm.
ϩ
ϩ
L-glutamate using NAD or NADP as coenzyme. It can also
Reductive Amination Reaction of GDH1 Is Not Affected by
catalyze reductive amination of ␣-KG. However, in vivo reduc- PMP-BCATm—The effect of BCATm on reductive amination
tive amination is not the favored direction, because the Km was also evaluated. Addition of leucine to the GDH1 reaction
value for ammonium (50 mM) is considerably higher than phys- mixture increased the reductive amination of ␣-KG by ϳ10–
iological concentrations of ammonia (34). To evaluate the 15% (data not shown). Addition of PLP-BCATm did not result
kinetic properties of GDH1 in the presence of BCATm, we have in a significant increase in the rate of reductive amination (18–
determined the kapp of GDH1-catalyzed glutamate deamina- 22%) compared with leucine alone. Neither isoleucine nor
tion. Leucine alone enhanced the rate of GDH1-catalyzed valine had any effect on reductive amination catalyzed by
deamination of GDH1 ϳ60–65% (Fig. 4A). On addition of GDH1 (data not shown). Addition of PLP-BCATm to the reac-
PLP-BCATm, the rate of oxidation of GDH1 was enhanced an tion mixture containing isoleucine or valine did not change
additional ϳ30–35% in the presence of leucine. Isoleucine or the rate of reductive amination. Similarly, addition of PMP-
valine did not have any effect on GDH1 catalysis when added BCATm to CXXC center oxidized or mutated BCATm or cyto-
270 JOURNAL OF BIOLOGICAL CHEMISTRY
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Protein Complex in Branched-chain Amino Acid Metabolism
FIGURE 5. Protection of BCKD (E1) kinase-induced inhibition of BCKDC by
BCATm. Overall activity of BCKDC was carried out in a 0.1-ml volume in the
presence of BCATm, E1, E2, and E3 components of BCKDC in a 12:12:1:53
molarratio. PhosphorylationofE1wasinitiatedbytheadditionof2mM MgCl2
and 1 mM ATP. Aliquots were taken at different time intervals, and the activity
of the whole complex was assayed as described under “Experimental Proce-
dures.” Phosphorylation of wild type E1 was performed in the absence of
BCATm(*), inpresenceof BCATm (ࡗ), CXXCcenteroxidized BCATm (⌬), CXXC
center double mutant BCATm (#), and cytosolic BCATc (ϩ). In all cases BCKDC
activity in the absence of BDK was used as control (ϫ).
solic BCATc had no effect on the GDH1 reductive amination.
These results suggest that allosteric changes in GDH1 are spe-
cific for the deamination reaction.
FIGURE 6. Effect of BCATm on the allosteric regulation of GDH1 activity in
the oxidative deamination reaction. A, ADP activation of GDH1 activity (E).
Presence of PMP-BCATm (1:6 molar ratios) had no effect on the ADP-induced
activation of GDH1. ƒ, 1 molar ratio; 224 , 2 molar ratio; ⌬, 4 molar ratio; ⅐, 6
molar ratio. B, GTP is an allosteric inhibitor of GDH1 activity (E). Addition of
PMP-BCATm at (ƒ), 1 molar ratio; (224), 2 molar ratio; ⌬, 4 molar ratio; (F), 6
molar ratio antagonizes the GTP induced allosteric inhibition of GDH1. C, rib-
bon diagram of the two subunits of hexameric mammalian GDH1 (bovine,
Protein Data Bank code 1NR7). BCATm likely competes with GTP and
enhances the opening of the catalytic cleft.
Effects of BCATm on Phosphorylation of E1␣ by BDK—Hu-
man BCATm binds directly to E1 and promotes channeling of
substrate from BCATm to E1. The structural integrity of the
phosphorylation loop of E1 (Tyr-␣286 to Glu-␣312) is required
for the binding of E1 with BCATm (5). Mutation or phosphor-
ylation of this loop results in a disordered conformation of the
loop inactivating E1 and interfering with binding to BCATm.
To determine whether BCATm affects the rate of E1␣ phos-
phorylation, we incubated BDK and BCKDC with ATP in the
GTP is a strong allosteric inhibitor of GDH1 (Fig. 6B). At 1
presence or absence of BCATm and monitored the rate of mM GTP, GDH1 lost ϳ90% of its activity. Addition of increas-
BCKDC activity. When the E1␣ subunit is fully phosphorylated, ing concentrations of PMP-BCATm to the reaction mixture
overall BCKDC activity is zero. Addition of BCATm to E1 at a resulted in increasing protection from GTP inhibition. At a 3 or
1:1 molar ratio inhibited the BDK catalyzed inhibition of higher molar ratio of PMP-BCATm to GDH1, no further pro-
BCKDC activity by 45% (Fig. 5). The data suggest that in the tection was observed. GDH1 is a homohexameric protein con-
presence of an equal molar ratio of E1 and BCATm, BDK can- taining three active sites formed from each homodimer. Each
not fully phosphorylate and inactivate E1␣. However, when E1 dimer contains one GTP-binding site, one ADP-binding site,
is not associated with BCATm (such as in the presence of and one NADH-binding site. One GDH1 could potentially bind
CXXC center oxidized or mutated BCATm or cytosolic three BCATm. The Ki (inhibition constant) value for GTP was
BCATc), BDK was able to fully phosphorylate the E1 compo- 52 nM in the absence of PMP-BCATm (Table 2). Increasing
nent and inhibit total BCKDC activity greater than 90% (Fig. 5). the molar ratio of PMP-BCATm to GDH1 in the reaction mix-
Effects of PMP-BCATm on the Allosteric Regulation of GDH1 ture resulted in increased Ki values for GTP with a maximum
Activity—Kinetic analysis shows that ADP induces an allosteric value of 8 ␮M. The data suggest that binding of PMP-BCATm
conformational change in GDH1 (39). At 100 ␮M ADP, oxida- changes the conformation of GDH1, and this abrogates the
tive deamination was enhanced by 2-fold. Addition of PMP- GDH1 abortive complex.
BCATm to this reaction mixture (1:6 molar ratio with GDH1,
Interaction between BCATm and GDH1 Markedly Stimu-
equal number of active sites) did not produce any further lates the Transamination Reaction of PMP-BCATm—Human
enhancement of oxidative deamination (Fig. 6A). The data sug- BCATm is a PLP-dependent enzyme, and the PLP cofactor is
gest that in presence of ADP, GDH1 is probably already in the covalently attached to the Lys-202 in the active site of the
maximum open state, and PMP-BCATm has no further effect enzyme. After transamination with BCAAs (the first half-reac-
on active site opening and/or suggests that BCATm does not tion shown in Reaction 1), the PLP form of the enzyme is con-
bind in the presence of ADP.
verted to the PMP form of the enzyme. The PLP-BCATm form
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Protein Complex in Branched-chain Amino Acid Metabolism
TABLE 2
TABLE 4
Effect of PMP-BCATm on the GTP inhibition constant (Ki) of GDH1
Addition of GDH1 increases the kcat of E1-catalyzed decarboxylation
reaction
Kinetics of GTP inhibition of bovine GDH1 activity were performed in the direction
of oxidative deamination reaction. At each fixed molar ratio of PMP-BCATm and
GDH1 (left column), GTP concentrations were increased gradually up to saturation.
The Ki values for GTP were calculated using the nonlinear kinetic equation, %
activity ϭ 100⅐(M1([GTP])/(Ki ϩ [GTP])), where [GTP] is the concentration of
GTP; Ki is the inhibition constant of GTP, and M1 is the factor associated with
maximum extent of GTP inhibition (39).
The DCPIP assays were carried out with 1 mM ThDP and varying concentrations of
amino acids (5 ␮M to 5 mM leucine) or ␣-keto acids (KIC, 0.5 mM to 1 mM; ␣-KG, 0.5
mM to 20 mM). The pH of the DCPIP buffers was 8.0, and the reactions were
ϩ
performed at 30 °C. GDH1 was added in the reaction mixture with 0.2 mM NAD .
All values are the average of three independent experiments.
Variable
Additions
k
cat values
K
m values
PMP-BCATm/GDH1 molar ratio
Ki
substrates
Ϫ1
␮M
min
␮M
0
1
2
4
6
0.05 Ϯ 0.00
1.56 Ϯ 0.15
3.12 Ϯ 0.40
8.06 Ϯ 0.86
7.95 Ϯ 1.00
DCPIP assays
None
KIC
7.7 Ϯ 1
NDa
40 Ϯ 2
ND
None
␣-KG
␣-KG
KIC
7.6 Ϯ 1
90 Ϯ 4
40 Ϯ 3
␣-KG ϩ PLP-BCATm
␣-KG ϩ PLP-BCATm ϩ GDH1
␣-KG ϩ PMP-BCATm
␣-KG ϩ PMP-BCATm ϩ GDH1
Leucine
Leucine
Leucine
Leucine
259 Ϯ 15
240 Ϯ 7
720 Ϯ 13
70 Ϯ 3
280 Ϯ 18
275 Ϯ 15
TABLE 3
Pre-steady state kinetics constants of PMP-BCAT
566 Ϯ 14
a ND indicates not detectable.
Conditions were 50 mM NaHEPES buffer at pH 8.0, 0.1 M KCl, and 1 mM EDTA.
ϩ
GDH1 was added in the reaction mixture with 0.2 mM NAD . The fast reaction
increase of calculated kcat values for E1-catalyzed decarboxyla-
tion, when compared with E1 plus KIC. However, addition of
GDH1 further enhanced the kcat value by 8-fold compared with
the absence of GDH1 in the reaction mixture. When PMP-
BCATm was used instead of PLP-BCATm, the decarboxylation
activity was still higher but was lower than in the presence
of PLP-BCATm. Similarly, if GDH1 was added to the PMP-
BCATm reaction mixture, kcat was accelerated, although
lower than in the presence of PLP-BCATm. PLP-BCATm rap-
idly converts the leucine to KIC so that it rapidly enters in
E1-catalyzed decarboxylation reaction. However, PMP-
kinetics of BCATs were performed using a stopped-flow spectrophotometer
(Applied Photophysics, Leatherhead, UK). Enzymes (80 ␮M) and ligands were
placed in different syringes, and the reactions were initiated by injecting the
enzymes and ligands in the reaction chamber. The apparent rate constants at 330 nM
were performed using the software provided with the instrument.
Substrate GDH1
Enzymes
kcat
s
Km
mM
kcat/Km
Ϫ1
M
Ϫ1 sϪ1 ϫ 104
␣-KG
␣-KG
␣-KG
␣-KG
Ϫ
ϩ
Ϫ
ϩ
PMP-BCATm 375 Ϯ 20 7.9 Ϯ 1.2
4.7
3110
3.8
PMP-BCATm 5600 Ϯ 89 1.8 Ϯ 0.4
PMP-BCATc
PMP-BCATc
430 Ϯ 15 3.8 Ϯ 0.2
444 Ϯ 13 3.5 Ϯ 0.3
3.5
can be regenerated from PMP-BCATm by direct interaction BCATm should convert to PLP-BCATm before transaminating
with GDH1 and channeling of ␣-KG produced from GDH1- leucine, and this extra step reaction slowed down the reaction
catalyzed oxidative deamination or by free ␣-KG. To character- in the PMP-BCATm reaction mixture. These results suggest
ize this interaction, the fast reaction kinetics of PMP-BCATm that GDH1 binds and facilitates regeneration of PLP-BCATm,
in the presence of ␣-KG were performed in either the presence which ultimately enhanced the overall reactions of BCATm
or absence of GDH1. Results are shown in Table 3. Addition of and E1.
GDH1 enhanced the kcat value of PMP-BCATm-catalyzed
GDH1 Alone Does Not Change the Rate of BCKDC Catalysis—
reamination of ␣-KG by 15-fold and reduced the Km by 4.5-fold. To see if there is an effect of GDH1 alone on BCKDC catalytic
However, there were no changes in the kinetic constants for activities, we determined the kinetic constants of E1 and
PMP-BCATc transamination in the presence of GDH1, indi- BCKDC overall catalysis in the presence of GDH1. As shown in
cating that the interaction between GDH1 and PMP-BCATm is the Table 5, addition of GDH1 (1:6 molar ratio of E1) resulted in
specific. These results are consistent with increased catalytic no apparent change in rate constants, kcat or dissociation con-
efficiency as a result of the interaction between BCATm and stants, or Km values of the E1 decarboxylation reaction. The
GDH1.
results are consistent with the absence of direct association
Decarboxylation Catalytic Activity of E1 Is Accelerated by the between E1 and GDH1 and the specificity of E1 for BCKAs
Association between PMP-BCATm and GDH1—E1 uses the (␣-KG is not a substrate for E1) (5). After the first cycle of
BCKA product of BCAA deamination reaction catalyzed by transamination and decarboxylation, PMP-BCATm was pro-
PLP-BCATm. As mentioned previously, PLP-BCATm is regen- duced which was recycled to PLP-BCATm by ␣-KG. Because
erated from PMP-BCATm by direct interaction with GDH1. GDH1 produces ␣-KG through oxidative deamination of glu-
To further understand the significance of the association, we tamate, E1 decarboxylation assays were performed in the pres-
measured the kinetics of E1 decarboxylation reaction either in ence of PLP-BCATm, BCAAs, GDH1, and glutamate (no added
the presence or absence of GDH1 and using DCPIP as an elec- ␣-KG or BCKA). As shown in Table 5, facilitating regeneration
tron acceptor. If the association between PMP-BCATm and of PLP-BCATm by GDH1 plus glutamate enhanced the calcu-
GDH1 results in more efficient regeneration of PLP-BCATm lated kcat by 10–12-fold when compared with E1 plus BCKAs
and this step can be limiting, the rate of E1-catalyzed oxidative alone. The Km values for BCAAs were also lower in the presence
decarboxylation reaction might proceed faster in the presence of GDH1 and glutamate as compared with native BCATm
of GDH1 than the absence of GDH1. The increase would result enzyme (Table 5) (27, 47). There were no detectable changes in
in a higher kcat values, Km values for BCAAs and BCKAs are not kinetic constants for E1 decarboxylation in the presence of
directly comparable, because they are specific for specific sub- CXXC mutated or oxidized BCATm and GDH1, indicating the
strates for their respective enzymes. As shown in Table 4, in the importance of the reduced redox center for BCATm binding to
presence of leucine and PLP-BCATm, there was a 12-fold E1 or GDH1.
272 JOURNAL OF BIOLOGICAL CHEMISTRY
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Protein Complex in Branched-chain Amino Acid Metabolism
TABLE 5
Effect of GDH1 on the kinetics of E1 and BCKDC catalysis
In both the DCPIP assay and the overall assay, ThDP (1 mM) concentrations were kept constant. With GDH1 addition, glutamate (20 mM) was used to produce
␣-ketoglutarate. The maximum concentration of the substrates used in the titration was always 10-fold higher than the Km values of the substrates for each individual
reaction. All values are the average of three independent experiments.
DCPIP assay
Overall assay
kcat
Km
␮M
kcat
min
Ile
Km
␮M
Ϫ1
Ϫ1
min
Ile
Addition
BCKAa
GDH1a
Leu
Val
Leu
Ile
Val
Leu
Val
Leu
Ile
Val
7.7 Ϯ 1 8.0 Ϯ 1
13 Ϯ 1
41 Ϯ 4
35 Ϯ 5
50 Ϯ 4
149 Ϯ 13 180 Ϯ 10 215 Ϯ 18
42 Ϯ 7
35 Ϯ 6
53 Ϯ 3
7.6 Ϯ 1 7.9 Ϯ 1
14 Ϯ 1
39 Ϯ 3
34 Ϯ 3
52 Ϯ 6
157 Ϯ 10 175 Ϯ 11 199 Ϯ 12
39 Ϯ 5
37 Ϯ 3
50 Ϯ 3
640 Ϯ 18
PLP-BCATmb
ϩ
89 Ϯ 5 99 Ϯ 5 165 Ϯ 5
7.0 Ϯ 1 7.2 Ϯ 1
280 Ϯ 15
255 Ϯ 13
690 Ϯ 25 250 Ϯ 9
279 Ϯ 7
390 Ϯ 17
210 Ϯ 23
189 Ϯ 20
GDH1
BCATm-Oxc ϩ
14 Ϯ 1 15,800 Ϯ 20 9060 Ϯ 25 45000 Ϯ 80 123 Ϯ 8
135 Ϯ 13 177 Ϯ 12 8600 Ϯ 14 8900 Ϯ 33 47,000 Ϯ 60
GDH1
a The kinetic values are for corresponding ␣-ketoacids of leucine, ␣-ketoisocaproate, isoleucine, ␣-keto-␤-methylvalerate, valine, and ␣-ketoisovalerate.
b The Km values for the BCAAs in the transamination reaction with PLP-BCATm alone are 1.6 mM Leu, 1.3 mM Ile, and 7.7 mM Val (27).
c The Km values for the BCAAs are 10-fold higher in oxidized BCATm than WT-BCATm, and oxidized BCATm (BCATm-Ox) does not form protein complex with other
metabolon protein partners (27).
PC, and a protein with unknown
function, NIPSNAP1, were found
in the multiprotein complex (Figs.
1 and 2). We show that GDH1 can
participate functionally by regen-
erating the form of BCATm that
binds to E1, creating a kinetically
more efficient channeling of BCATm
products to the oxidative pathway.
Not only is the metabolon kineti-
cally more efficient, but metabolon
formation results in lower Km values
for BCAAs and glutamate that are in
line with physiologically observed
concentrations of these amino acids
in cells. In cells, the concentrations
of the ␣-keto acids (BCKAs and
␣-KG) are significantly lower than
their respective amino acids. Our
results suggest that changes in flux
will occur in response to physiolog-
FIGURE 7. Proposed model of BCAA metabolon. PLP-BCATm binds to E1, promoting channeling of KIC from
BCATm to E1, leading to decarboxylation of KIC, and release of CO2. PMP-BCATm dissociates from E1. Product
isovaleryl-CoA is released from E2. E3 reoxidizes the lipoamide cofactor (data not shown), forming NADH.
GDH1 binds PMP-BCATm and regenerates PLP-BCATm through oxidative deamination of Glu by GDH1 and
reamination of ␣-KG by PMP-BCATm. Regenerated PLP-BCATm binds E1 to restart the reaction cycle. Abbrevi-
ations used are as follows: PLP-BCATm, pyridoxal form of mitochondrial branched-chain aminotransferase;
PMP-BCATm, pyridoxamine form of mitochondrial branched-chain aminotransferase; LBD, lipoyl-bearing
mutation; SBD, subunit-binding domain.
ically relevant changes in the amino
acids.
In the model shown in Fig. 7, the
PLP form of BCATm binds to the
E1 component of BCKDC, and after
the first transamination half-reac-
tion reaction, PMP-BCATm is re-
leased from the complex. It needs to be converted to PLP-
BCATm to complete the catalytic cycle (Fig. 7). GDH1 is a
binding partner for PMP-BCATm. Kinetic results provide the
evidence that the association between PMP-BCATm and
GDH1 enhanced the allosteric activation of GDH1 oxidative
deamination. This physical association between these two
proteins facilitates the channeling of ␣-KG to PMP-BCATm
to generate the PLP-BCATm, which then restarts the cycle
(Fig. 7).
To see if GDH1 affected the other components of BCKDC,
overall BCKDC activity was measured in the presence or
absence of GDH1. GDH1 did not have a significant effect on
BCKDC (Table 5) activity. However, addition of GDH1 and
glutamate enhanced the calculated kcat value by 40–45% with
BCATm present when compared with BCKDC plus BCKAs
alone (Table 5). As with E1, Km values for BCAAs in the overall
reaction were lower in the presence of GDH1 and glutamate as
compared with native BCATm enzyme (27, 47). Neither the
CXXC mutants nor oxidized BCATm had any effect on the
overall kinetic parameters of BCKDC.
The interaction between GDH1 and BCATm can occur in
the absence of BCKDC. In the presence of a BCAA, glutamate,
DISCUSSION
BCATm, and GDH1, BCAA nitrogen is efficiently channeled
ϩ
In this study we have identified additional proteins in the to glutamate, and BCKA, ammonia, and NAD are the reaction
“BCAA metabolon.” Several metabolic enzymes such as GDH1, products. ␣-KG produced by GDH1-catalyzed oxidative
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Protein Complex in Branched-chain Amino Acid Metabolism
deamination of glutamate produces ␣-KG, which is converted gest the highest concentration of BCATm is actually in the
back to glutamate as it receives the BCAA nitrogen group and exocrine pancreas (acinar cells), not in the islets (52), and
no net change in ␣-ketoglutarate or glutamate concentrations KIC is also an insulin secretagogue. One might speculate that
occur. The interaction between BCATm and GDH1 also en- in vivo both leucine and KIC produced in the acinus play a
hances leucine stimulation of GDH1 activity. In the presence of role in the physiological response to leucine.
BCATm, leucine is an effective activator of GDH1 at physiolog-
We have shown that metabolon formation antagonizes
ically relevant concentrations of this BCAA (Յ1 mM, see Fig. 4). phosphorylation of E1 and inhibition of BCKDC activity
ϩ
In the presence of BCATm, all BCAAs could activate GDH1 while increasing the NADH/NAD ratio, and phosphoryla-
presumably by promoting an allosteric change in GDH1 that tion of E1␣ serves to shut down oxidation and promote
occurs on binding of PMP-BCATm formed during the first BCKA release. Thus, the presence of BCATm in a tissue
transamination half-reaction. Increased kinetic efficiency may play a role in regulating the phosphorylation state of
achieved by substrate channeling of the product ␣-KG to the BCKDC. In rat skeletal muscle where BDK expression is
active site of BCATm may also contribute. BCATm had no higher than in other rodent tissues, the enzyme is highly
effect on reductive amination. Thus the presence of BCATm phosphorylated at all times, which promotes BCKA release.
and BCAAs promotes glutamate nitrogen shuttling and ammo- There is active uptake of glutamate by skeletal muscle (53,
nia formation. Fixation of ammonia into glutamic acid yields 54). GDH1 is present in low concentrations in skeletal mus-
glutamine, and glutamine is used in the synthesis of purines and cle compared with liver (55), and alanine is the major prod-
pyrimidines through the formation of carbamyl phosphate. In uct of BCAA nitrogen transfer (56). Conditions that promote
the presence of active BCKDC, the BCKA that is produced in PMP-BCATm and GDH1 binding and activation of GDH1
the PLP-BCATm half-reaction is oxidized by BCKDC, which is could result in a transient increase in ammonia production.
facilitated by PLP-BCATm binding to the E1 decarboxylase. In the congenital hyperinsulinism syndrome that results
Because NADH used in the GDH reaction is regenerated in from mutation of the GDH1 GTP-binding site rendering the
the BCKDC reaction, the net reaction products of the com- enzyme insensitive to GTP inhibition, transient hyperam-
bined GDH1, BCATm, and BCKDC reactions are branched- monia has been observed in association with a protein meal,
chain acyl-CoAs and ammonia. Further oxidation of leucine and skeletal muscle was hypothesized as the main source of
produces acetyl-CoA and acetoacetate. Because glutamate is the excess ammonia (57). The clinical consequences of these
regenerated by reamination of the GDH1 product ␣-KG in the mutations suggest that GDH1 plays a significant role in the
PMP-BCATm half-reaction, the relative amount and activities overall control of amino acid catabolism and ammonia
of GDH1 and BCATm (availability of PMP-BCATm) will affect metabolism integrating responses to changes in intracellular
the net amount of ␣-ketoglutarate that is produced and avail- energy potential and amino acid levels (57).
able for oxidation. A high GDH1/PMP-BCATm favors glu-
Proteins show large structural heterogeneity at the molec-
tamate oxidation. If, for example, reaction rates are equal, ular level that permits them to recognize and adapt to the
then full operation of the cycle produces an increase in structure of the binding partners. The “induced fit” and
leucine catabolic metabolites. Regardless, BCAA nitrogen is “conformational selection” mechanisms are well developed
transferred into the large glutamate pool. However, actual hypotheses for conformational change in proteins bound in
net transfer of BCAA nitrogen occurs when BCKDC is active complexes as compared with unbound proteins (32, 58). The
(BCAAs are oxidized) and is promoted by BCATm-E1␣ conformational changes of GDH1 upon complex formation
complex formation and substrate channeling of BCKA from with PMP-BCATm support the conformational selection
BCATm to E1.
mechanism of complex formation between these two pro-
In humans and rodents, the pancreas has one of the highest teins. Detailed structural analysis of GDH1 using activators
concentrations of BCATm in the body (48). Leucine is a known and inhibitors has been described by Smith et al. (40) (see in
insulin secretagogue (19, 49). It has been hypothesized that Fig. 7C) (50). A special allosteric regulation site antenna
leucine stimulation of GDH1 activity and leucine metabolism domain regulates the open/closed conformation of the cata-
to acetyl-CoA and acetoacetate are both involved in the leucine lytic cleft. The six glutamate-binding domains form the cat-
stimulation of insulin secretion (51). Our results suggest that alytic core of this enzyme. Upon substrate and cofactor bind-
activation of GDH1 oxidative deamination of glutamate in ing, the NADH-binding domain clamps down on the
the mitochondria is likely modulated by leucine and BCATm glutamate-binding domain. Apo-GDH1 is usually in an open
concentrations as well as factors influencing complex forma- conformation. However, when substrate and cofactor bind,
ϩ
tion such as NAD /NADH, ADP, and GTP concentrations. it forms a ternary complex with an energetically favorable
Glutamate oxidation would be favored by limiting BCATm closed catalytic cleft. Activators, like ADP, have no direct
availability or stimulating glutamate nitrogen transfer via other effect on closing the catalytic cleft, rather they facilitate the
aminotransferases (alanine aminotransferase and aspartate opening of the catalytic cleft. Upon ADP binding, one of the
aminotransferase) to produce ␣-KG. Interestingly, in freshly arginine residues rotates toward the antenna region that
isolated rat islets, we found limited amounts of BCATm pro- facilitates the interaction with the ␤-phosphate of ADP and
tein on Western blots.3 Immunohistochemical results sug- ultimately enhances the opening of the active site (40).
Inhibitors like GTP bind to a different binding site and form
an energetically unfavorable abortive complex where the
3 A. J. Sweatt and S. M. Hutson, unpublished data.
catalytic cleft is in a super-closed state. Our results suggest
274 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 1•JANUARY 1, 2010

Protein Complex in Branched-chain Amino Acid Metabolism
17. Gao, Z., Young, R. A., Li, G., Najafi, H., Buettger, C., Sukumvanich, S. S.,
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bind GDH1. On the other hand, PMP-BCATm does not have
a direct effect on ADP binding to GDH1. These results sug-
gest that either PMP-BCATm binds to the GTP-binding site
of GDH1 or binding of PMP-BCATm changes the conforma-
18. Hutton, J. C., Sener, A., and Malaisse, W. J. (1980) J. Biol. Chem. 255,
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19. Sener, A., and Malaisse, W. J. (1980) Nature 288, 187–189
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PMP-BCATm and GDH1 during catalysis facilitates the
22. MacDonald, M. J., Smith, A. D., 3rd, Hasan, N. M., Sabat, G., and Fahien,
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open conformation of unphosphorylated E1␣ promotes
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GDH1? BCATm is a homodimeric protein containing two
active sites. The cofactor is at the bottom of the active site. The
PLP cofactor is bound to Lys-202 via a Schiff base. The PMP
form is held only by ionic interactions. The large conforma-
tional change of Lys-202 and rotation of PMP cofactor in the
active site make space for proper positioning of ␣-KG, which
only binds to the PMP form of the enzyme. The free amino
group of both PMP and the Lys-202 side chain remain in the
protonated state, which creates a net positive charge at the
active site of PMP-BCATm. The electrostatic nature of the res-
idues at the opening of the active site pocket and next to the
GTP-binding site, specifically Glu-439 and Asp-442, are nega-
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of PMP-BCATm and GDH1 may facilitate binding and transfer
of ␣-KG from PMP-BCATm to GDH1.
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276 JOURNAL OF BIOLOGICAL CHEMISTRY
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Metabolism and Bioenergetics:
Branched-chain Amino Acid Metabolon:
INTERACTION OF GLUTAMATE
DEHYDROGENASE WITH THE
MITOCHONDRIAL BRANCHED-CHAIN
AMINOTRANSFERASE (BCATm)
Mohammad Mainul Islam, Manisha Nautiyal,
R. Max Wynn, James A. Mobley, David T.
Chuang and Susan M. Hutson
J. Biol. Chem. 2010, 285:265-276.
doi: 10.1074/jbc.M109.048777 originally published online October 26, 2009
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