We have developed overexpression systems for the
human branched-chain aminotransferase isoenzymes. The enzymes function
as dimers and have substrate specificity comparable with the rat enzymes. The human cytosolic enzyme appears to turn over 2-5 times faster than the mitochondrial enzyme, and there may be anion and cation
effects on the kinetics of both enzymes. The two proteins demonstrate
similar absorption profiles, and the far UV circular dichroism spectra
show that no global structural changes occur when the proteins are
converted from the pyridoxal to pyridoxamine form. On the other hand,
the near UV circular dichroism spectra suggest differences in the local
environment surrounding tyrosines within these proteins. Both enzymes
require a reducing environment for maximal activity, but the
mitochondrial enzyme can be inhibited by nickel ions in the presence of
reducing agents, while the cytosolic enzyme is unaffected. Chemical
denaturation profiles of the proteins show that there are differences
in structural stability. Titration of -SH groups with
5,5'-dithiobis(2-nitrobenzoic acid) suggests that no disulfide bonds
are present in the mitochondrial enzyme and that at least two disulfide
bonds are present in the cytosolic enzyme. Two -SH groups are titrated
in the native form of the mitochondrial enzyme, leading to complete
inhibition of activity, while only one -SH group is titrated in the
cytosolic enzyme with no effect on activity. Although these proteins
share 58% identity in primary amino acid sequence, the local
environment surrounding the active site appears unique for each
isoenzyme.
 |
INTRODUCTION |
Branched-chain aminotransferases
(BCAT)1 catalyze reversible
transamination of the short chain aliphatic branched-chain amino acids,
leucine, isoleucine, and valine, to their respective
-keto acids.
Although transamination of branched-chain amino acids in animals and
microorganisms was observed in the 1950s (for a review, see Ref. 1), it
was not until 1966 that Ichihara and Koyama (2) and Taylor and Jenkins
(3) each reported independently that these reactions were catalyzed by
a single enzyme.
Bacteria generally contain a single BCAT (4-6), but in mammals it
has been established that there are two BCAT isoenzymes, a
mitochondrial (BCATm) and a cytosolic (BCATc) form (7). In humans and
rodents, BCATm is found in most tissues (1, 8). In contrast, BCATc is
found almost exclusively in the brain (1, 9). Each rat isoenzyme has
been purified (9, 10), and the cDNA sequences of the rat (11, 12)
as well as the human (12, 13) BCATc and BCATm are now available.
Recently, it has been reported that the mammalian BCATc gene may be
regulated by the c-myc oncogene product (14) and that the
BCAT may be a target for the anticonvulsant drug and nonmetabolizable
leucine analog, gabapentin (15). Furthermore, in yeast, deletion of
both BCAT genes results in severe growth retardation even after
supplementation with branched-chain amino acids, suggesting that these
enzymes perform an essential function in the cell (16). Nevertheless, the functional significance and structure of the individual eukaryotic BCAT isoenzymes is not yet known.
Another feature of the BCAT is their evolutionary relationship to the
bacterial enzyme D-amino acid aminotransferase (17). Based
on primary sequence comparisons, the BCAT and D-amino acid aminotransferase, which have opposite stereospecificity
(L- versus D-amino acids), and
another bacterial enzyme, 4-amino-4-deoxychorismate lyase, were placed
in a separate folding class (fold type IV) (11, 12, 18). With a few
exceptions, other known aminotransferases fall within the fold type I
or L-aspartate aminotransferase superfamily. A unique
feature of the fold type IV PLP-dependent enzymes is that
the proton is added to or abstracted from the C4' atom of the
coenzyme-imine or external aldimine intermediate on the re face instead of the si face of the PLP cofactor (19). The
crystal structure of the PMP form of D-amino acid
aminotransferase has been solved by Sugio et al. (20), and
the structure of the Escherichia coli BCAT in the pyridoxal
form has just been reported by Okada et al. (21). The
folding pattern of both enzymes is not only different from that of
other known PLP-dependent enzymes but also from that of
other known proteins (20). The E. coli BCAT enzyme consists
of six identical 34-kDa subunits that are an assembly of three dimer
units around a three-fold axis (21, 22). On the other hand, the
mammalian BCAT do not appear to be hexamers and have subunit molecular
masses ranging from 41 to 46 kDa (11, 12). Thus, cloning and
overexpression of both human BCAT isoenzymes has provided the tools
necessary to develop a molecular model of the mammalian BCAT that may
impact on our current understanding of this unusual class of
PLP-dependent enzymes.
 |
MATERIALS AND METHODS |
Plasmids, Bacterial Strains, Enzymes, and
Chemicals--
Plasmids pT7Blue(R) and pET-28a were purchased from
Novagen (Madison, WI). The pTrcHis vector was obtained from Invitrogen (Carlsbad, CA). Bacterial strains used were E. coli BL21DE3
and DH5-
. Restriction endonucleases and other DNA modification
enzymes were purchased from Promega, Inc. Nucleotide sequencing was
performed using the Sequenase version 2.0 7-deaza-dGTP sequencing kit
(U.S. Biochemical Corp.).
Construction and Expression of phBCATm, phBCATc, and
phBCATc2--
For human BCATm, the sense primer
5'-AGCCATATGGCCTCCTCCAGTTTCAAG-3' contained an NdeI
restriction site. The last 18 nucleotides of the sense primer
corresponded to the first 18 bases encoding the mature BCATm protein
(12). The antisense primer 5'-GAGTGTCGCAACCACAT-3' corresponded to
nucleotides 1316-1332 of the full-length human cDNA (12). For
increased accuracy, a polymerase mixture of Taq and
Pwo polymerases (Boehringer Mannheim) was used. The
amplified product was cloned into the pT7 vector. The insert was
sequenced on both strands, and the fidelity was verified by comparison
with the original human BCATm cDNA clone. The purified cDNA was
ligated into the pET-28a vector cut with NdeI. Following
transformation, plasmid DNA isolated from single colonies was screened
for correct orientation of the cDNA insert, and then the construct
was used to transform BL21DE3 cells. Cells were grown to an
A600 between 0.6 and 0.9, and expression was
induced with 1 mM
isopropyl-
-D-thiogalactopyranoside. After 8 h,
expression was approximately 8 mg of BCATm/liter of culture with a
typical recovery of 6 mg of purified protein/liter of E. coli.
A similar strategy was used to construct the phBCATc expression vector.
Using the cDNA sequence in Ref. 13 (accession number U21551), a
sense primer was designed that contained an NheI site
immediately preceding the codon for the initiator methionine followed
by the nucleotides encoding the first 5 amino acids of the protein,
5'-TATGGCTAGCATGGATTGCAGTAACGGA-3'. The antisense primer
5'-TCAGGATAGCACAATTGTC-3' corresponded to nucleotides 1137-1155. Human
brain cDNA was used in the polymerase chain reaction, and the
amplified product was cloned into the pT7 vector and sequenced. The
purified cDNA was ligated into the pTrcHis vector cut with NheI and HindIII. The phBCATc2 plasmid was
engineered by ligating the full-length BCATc sequence into the pET-28a
vector cut with SalI and NheI. BL21DE3 cells were
grown and induced as described for phBCATm, and after 5-7 h,
expression was 2 and 8 mg of BCATc/liter with phBCATc and phBCATc2,
respectively. Typically, 50% of the expressed protein was recovered
after purification.
Protein Purification--
Nickel-NTA resin (Qiagen, Chatsworth,
CA) was used according to the manufacturer's instructions to purify
the histidine-tagged recombinant proteins. For BCATm, cells harvested
from 2 liters of culture were resuspended in extraction buffer
containing 0.1 M sodium phosphate, pH 8.0, 0.01 M Tris-HCl, and 5 mM
-mercaptoethanol. The
mixture was sonicated for 10 1-min intervals at 70% duty cycle using a
Branson model 250 sonifier. The extract was centrifuged for 8 min at
7800 × g at 4 °C. The supernatant was saved, and the pellet was resuspended in extraction buffer containing 4 M urea and sonicated as before. After centrifugation, the
second supernatant was combined with the first supernatant to which
urea had been added to 4 M. The total extract was incubated
with 7 ml of 50% nickel-NTA resin equilibrated in the extraction
buffer containing urea for 1 h at 4 °C with gentle stirring.
The mixture was loaded into a column, and the resin was washed with the
extraction buffer containing urea. The column was then washed with
buffer A containing 0.01 M Tris, pH 7.5 (HCl), 20%
glycerol, 150 mM NaCl, 5 mM
-mercaptoethanol
followed by buffer B, which contained 0.1 M sodium
phosphate (pH 6.0), 0.01 M Tris-HCl, 750 mM
NaCl, 10% glycerol. The column was washed further with buffer B
containing 50 mM imidazole. The protein was eluted with
buffer B containing 350 mM imidazole. The histidine tag was
removed by digestion with thrombin (100 NIH units) in 50 mM
Tris (pH 7.5), 150 mM NaCl for 1 h at 25 °C. The
final purification step was hydrophobic interaction chromatography as
described in Ref. 10 with the following modifications. The
BCATm-containing fraction from the nickel-NTA column was incubated with
5 mM
-ketoisocaproate in 100 mM potassium
phosphate at pH 7.5 on ice for 5 min before loading onto the column,
and the elution gradient began at 35% saturated ammonium sulfate. The
concentration of BCATm was determined from the absorbance at 280 nm
using the extinction coefficient of 67,600 M
1
cm
1 per monomer.
For human BCATc in either the pTrcHis or pET-28a vector, extraction and
nickel-NTA purification steps were identical to BCATm, except that urea
was not added to any buffers. Following nickel-NTA chromatography,
purification was carried out using hydroxyapatite chromatography. The
BCATc containing fraction from the nickel-NTA column was loaded onto
the hydroxyapatite column in 10 mM potassium phosphate, pH
7.0. Human BCATc was eluted with 200 mM potassium phosphate, pH 7.0. The final purification step was ion exchange chromatography using a Mono-Q column (Pharmacia Biotech Inc.) as
described in Ref. 10 except that the buffer was 10 mM
potassium phosphate, pH 8.0. The histidine tag remained on the
recombinant protein when expressed from the pTrcHis vector, but when
the pET-28a construct was used, the added histidines were cleaved with
thrombin as described for BCATm. The concentration of BCATc with and
without the histidine tag was determined from the absorbance at 280 nm using the extinction coefficients of 80,000 M
1 cm
1 and 86,300 M
1 cm
1 per monomer,
respectively.
Storage Conditions--
Human BCATm was extremely sensitive to
oxidation and required the presence of 100 mM DTT to remain
fully active. If samples were purged with argon every other day, the
enzyme retained significant activity for periods of up to 2 weeks at
4 °C. When stored in 50% glycerol at
20 °C with argon purging,
BCATm was stable for as long as 4 months. BCATc was not as sensitive to
oxidation and could be stored in the presence of 10 mM DTT
without argon purging. BCATc was stable for about 2 weeks at 4 °C
and for 3-4 months at
20 °C in 50% glycerol.
Amino Acid Analysis--
The extinction coefficient for each
isoenzyme was calculated from the amino acid composition of each
protein. The N terminus of each recombinant protein was verified by
amino acid sequence analysis. Both composition analysis and N-terminal
sequencing were performed by the Protein Analysis Core Laboratory of
the Comprehensive Cancer Center of Wake Forest University.
Spectrophotometric Measurements--
Absorption spectra were
taken with a Beckman DU 640 spectrophotometer. CD measurements were
carried out with a JASCO J-720 spectropolarimeter equipped with a
variable temperature accessory. The enzymes were converted to the PLP
and PMP forms by incubation with the appropriate substrate followed by
dialysis. The instrument was calibrated with
ammonium-D-camphorsulfonate. CD spectra in the near UV
region were measured in a 1-cm quartz cylindrical cuvette at a protein
concentration of 1 mg/ml. In the far UV region, CD spectra were
acquired using a protein concentration of 0.5 mg/ml in 5 mM
potassium phosphate buffer and a 0.05-cm path length. The final spectra
were the average of four accumulations. The CD spectra of the PLP and
PMP forms of BCATm and BCATc were analyzed using the convex constraint
algorithm described in Refs. 23 and 24. In all cases, the targeted
protein CD spectrum was added to the appropriate data set, which
contained 30 proteins, and the enlarged spectral set (30 + 1) was
analyzed by CDANAL software (Jasco). Based on the quality of the fit,
the number of pure components was set either at three or four, and the
contribution of each element was obtained from the conformational
weight matrix. Sigma values varied from 0.8 to 1.5.
Sulfhydryl Group Titrations--
5-15 nmol of protein were
dissolved in 1 ml of buffer containing 50 mM Tris-HCl (pH
7), 1 mM EDTA, and 6 M guanidine hydrochloride. The reaction was initiated by the addition of 200 µl of 10 mM DTNB at room temperature, and the absorbance at 412 nm
was monitored for 15 min. The amount of free thiol was calculated from
the liberated 2-nitro-5-thiobenzoate anion using a molar extinction
coefficient of 14,150 (25). The same procedure was repeated in the
absence of denaturant to determine the number of cysteines accessible to solvent. Cysteine titrations were also performed in the presence of
either 10 mM
-ketoisocaproate or isoleucine.
Branched-chain Aminotransferase Assay and Steady State
Kinetics--
Branched-chain aminotransferase activity was measured at
37 °C in 25 mM potassium phosphate buffer, pH 7.8, using
1 mM
-keto[1-14C]isovalerate and 12 mM isoleucine as described (9, 10). A unit of enzyme
activity was defined as 1 µmol of valine formed per min at 37 °C.
In the kinetic studies, bovine serum albumin was omitted, and the assay
contained 2 mM DTT and 12.5 mM EDTA.
Reaction rates with the branched-chain amino acid/
-ketoglutarate
pairs were determined from the formation of glutamate, which was
assayed fluorimetrically by a slight modification of the method described by Williamson and Corkey (26). Briefly, excess
-ketoglutarate was removed from the neutralized sample by the
addition of either 10 mM (BCATm) or 15 mM
(BCATc) H2O2. Samples were incubated at room
temperature for 5-15 min before an aliquot (250 µl) was added to 1 ml of assay buffer. NADH fluorescence was determined with an excitation
wavelength of 340 nm using a FOCI Mark I spectrofluorimeter (Farrand
Optical Co., New York). The kinetic parameters of the amino acid
reactions were determined by holding the concentration of
-ketoglutarate constant at 5 mM for BCATm and 10 mM for BCATc. For the
-keto acid/glutamate pairs,
reaction rates were determined from the formation of
1-14C-labeled branched-chain amino acid as described
previously (7, 10). The glutamate concentration was kept constant at
100 mM as the high salt concentration affected the kinetic
parameters. The data were fit to the following velocity equation.
|
(Eq. 1)
|
The Km of
-ketoglutarate or glutamate was
first estimated by varying the concentration of each branched-chain
amino or
-keto acid at approximately 5 times Km.
For each substrate pair, data were collected from six or seven
concentrations of amino acid or
-keto acid. Kinetic parameters are
the means and standard errors of 3-5 separate determinations from two
different BCATm and BCATc preparations. In separate experiments, the
apparent Km for
-ketoglutarate and glutamate was
determined by varying the concentrations of both the amino acid
and
-keto acid substrates simultaneously. The data were fit to the
initial velocity rate equation pertaining to a ping-pong kinetic
mechanism (27) via nonlinear regression in the approach outlined by
Cleland (28).
|
(Eq. 2)
|
In this equation, the parameters Ka and
Kb represent the Michaelis constants for
substrates A (amino acid) and B (
-ketoacid), respectively.
Analytical Ultracentrifugation--
The equilibrium
sedimentation experiments were performed in the Analytical
Ultracentrifugation Facility at Wake Forest University. Using a Beckman
Optima XL-A analytical ultracentrifuge, BCATm and BCATc in 25 mM Tris (pH 7.5), 150 mM KCl were centrifuged at 7500, 9000, 11,000, 14,000, and 40,000 rpm. Data were gathered after
12 and 14 h at each speed. The data were examined to verify that
equilibrium was established. Partial specific volumes were calculated
based on the amino acid composition of each protein as described by
Laue et al. (29). Data analysis was performed using Origin
software provided by Beckman.
Miscellaneous Procedures--
SDS-PAGE was carried out according
to Laemmli (30) in 10% gels as described in Wallin et al.
(10).
 |
RESULTS AND DISCUSSION |
Expression and Purification--
The human BCATm and BCATc have
been cloned (12, 13). Mature BCATm consists of 365 amino acids with a
calculated molecular mass of 41,300 daltons, while BCATc consists of
385 amino acids with a calculated molecular mass of 42,800 daltons. A
comparison of the primary sequences of these proteins reveals that they
are 58% identical to each other (see Fig.
1) and 74% (BCATc) and 82% (BCATm)
identical to their respective rat enzymes. Identity is found throughout
the overlapping primary sequences including the C terminus. The most
significant differences in primary sequences are found in the
N-terminal portion (BCATc, residues 1-50) of the proteins.

View larger version (66K):
[in this window]
[in a new window]
|
Fig. 1.
Sequence alignment of recombinant human BCATc
and BCATm after cleavage of the histidine tag by thrombin. Deduced
amino acid sequences were aligned using the GAP analysis software
(Genetics Computing Group Sequence Analysis Software Package, version
8.0) (34). Additional residues unique to the recombinant proteins are
shown in lowercase italic type. The extra N-terminal
residues for histidine-tagged BCATc are mggshhhhhhgmas. The
active site lysine is boxed, and cysteine residues are
shaded.
|
|
In initial attempts to overexpress BCATm in a variety of expression
plasmids, low expression, inactive, or insoluble protein was observed.
The BCATm cDNA was then cloned into the pET-28a vector, which
introduced a six-histidine residue tag onto the N terminus of the
protein. High levels of expression of active protein were obtained, but
the majority of the protein was still found in inclusion bodies. In
addition, the soluble protein would not bind to the nickel-NTA resin.
This suggested that the N terminus of the BCATm fusion protein was
somewhat buried, making the histidine tag inaccessible to the resin.
Upon complete denaturation in 8 M urea, BCATm bound to the
resin; however, active enzyme could not be recovered either by gradual
refolding of the protein while it remained on the resin or by dialysis
versus urea-free buffer. In 4 M urea, the
tertiary or quaternary structure of the protein was altered enough to
permit binding of the N-terminal histidine tag to the resin. The far UV
CD spectrum of BCATm (data not shown) showed no detectable change upon
increasing urea concentrations from 0 through 4 M, thus
indicating that the secondary structure of BCATm remained intact. In 4 M urea, the specific activity of the enzyme was reduced
about 50% but could be restored after gradual removal of the
denaturant. The use of 4 M urea also increased the yield of
recombinant protein. Specific activity of purified BCATm after thrombin
cleavage was 88 ± 6 units/mg of protein (n = 3).
The N-terminal sequence of the recombinant protein carries the
additional amino acids gshm (these additional residues are listed throughout in lowercase italic type) as verified by amino acid
sequencing (see Fig. 1). SDS-PAGE of the purified recombinant BCATm
protein is shown in Fig. 2A.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 2.
SDS-PAGE of purified human BCATm and
BCATc. 3-5 µg of protein were loaded onto the gels, and
molecular mass standards are indicated. Panel A shows BCATm
after cleavage of the histidine tag by thrombin (41.7 kDa). Panel
B shows BCATc expressed using the pTrcHis vector (44.3 kDa), and
panel C shows BCATc expressed using the pET-28a vector
following removal of the histidine tag (43.4 kDa).
|
|
For BCATc, initial attempts with expression plasmids driven by the
highly processive T7 RNA polymerase were problematic, because the
protein was found primarily in inclusion bodies. With the lower level
of expression found with the pTrcHis construct, the solubility of the
recombinant protein was increased. Recently, we have expressed BCATc
using the pET-28a vector/T7 expression system and removed the histidine
tag. Although some protein was still found in inclusion bodies, the
high yield in the pET vector/T7 system permitted purification of
sufficient protein for kinetic and structural analysis. SDS-PAGE of the
purified recombinant BCATc proteins is shown in Fig. 2, B
and C. Unlike BCATm, recombinant BCATc readily bound to the
nickel-NTA resin under native conditions, indicating that the N
terminus of BCATc was exposed to the solvent, and urea addition (4 M) did not improve recovery. Thus, the behavior of the
recombinant proteins on the nickel-NTA resin suggests that in the
N-terminal portion of the proteins, the tertiary structure of BCATm and
BCATc is different. Specific activity of BCATc, with or without the
histidine tag, was 124 ± 9 units/mg of protein (n = 5). Recombinant BCATc expressed using the pTrcHis construct (phBCATc)
contained an additional 14 amino acid residues
(mggshhhhhhgmas) at the N terminus of the recombinant
protein that were contributed by the vector sequence. The protein
expressed using the pET-28a vector construct (phBCATc2) contained six
additional amino acids after thrombin cleavage. Both nucleotide
sequencing of the phBCATc2 construct and N-terminal amino acid
sequencing of the recombinant protein showed that the N-terminal
extension was gshmac (see Fig. 1). The N-terminal sequence
of the recombinant protein with the histidine tag (phBCATc) was
also confirmed by direct amino acid sequence analysis. Preliminary
experiments showed that the physical and kinetic properties of the two
recombinant enzymes were similar, and both enzymes were used in
subsequent experiments.
Sedimentation Equilibrium--
Upon size exclusion chromatography,
purified rat BCATm behaved anomalously and appeared to be a monomer,
while rat BCATc was clearly a homodimer (9, 10). To determine
unequivocally the subunit composition of the human isoenzymes,
equilibrium sedimentation experiments were performed with the
recombinant proteins. After the data were subjected to curve fitting
analysis, BCATm and BCATc showed average molecular weights of
81,500 ± 2510 and 83,900 ± 4800, respectively. These values
are approximately twice the calculated monomer molecular weights of the
recombinant proteins after thrombin cleavage, 41,700 (BCATm) and 43,400 (BCATc), identifying both enzymes as dimers in solution.
Spectral Analysis of Recombinant Human BCAT Isoenzymes--
The
absorption spectra of the recombinant enzymes at pH 7.5 are shown in
Fig. 3. In addition to the peak at 280 nm, two peaks characteristic of bound cofactor are observed at 416 and
326 nm, indicating the pyridoxal and pyridoxamine forms, respectively. In the visible range, the spectra for each isoenzyme are essentially superimposable. The higher absorption of BCATc at 280 nm probably reflects the higher tyrosine and tryptophan content of this protein. Extinction coefficients at 280 nm were determined for each protein. Values were 67,600 M
1 cm
1 per
BCATm monomer and 86,300 M
1 cm
1
and 80,000 M
1 cm
1 per BCATc
monomer, without and with the histidine tag, respectively.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3.
UV-visible spectra of human BCATc and BCATm
in the PLP and PMP forms. The proteins were converted to the PLP
and PMP forms as described under "Materials and Methods." Proteins
(0.01 mM) were in 10 mM phosphate buffer, pH
7.5. Solid line, BCATc PLP form; dashed line,
BCATc PMP form; dotted line, BCATm PLP form;
dash-dot-dash line, BCATm PMP form.
|
|
Fig. 4 shows the far and near UV CD
spectra of the proteins. The far UV CD spectra of both BCATc and BCATm
in their PLP and PMP forms have similar features with minima at 219 and
210 nm and a maximum at 193 nm (see Fig. 4A).
-Helical
structure is normally characterized by the presence of two minima at
222 and 208-210 nm and a maximum at 191-193 nm (31); therefore,
-helices are present in both of these proteins.
-Sheets are also
present because a minimum occurs at 219 nm, which is close to the
minimum (216-218 nm) normally observed for
-forms (31). The
similarity of the far UV CD spectra of the two proteins indicates that
their secondary structure content is similar. In addition, secondary structure estimates calculated using the far UV CD spectra of the PLP
form of the enzymes suggested an
-helix content of 38% for BCATm
and 35% for BCATc. Predicted
-sheet was on the order of 20-24%.
As shown in Fig. 4, no major change occurred in the global structure of
the proteins upon conversion of the PLP form to the PMP form, and
predicted
-helix content was similar for the PMP forms (33% for
BCATm and 36% for BCATc). The crystal structure of the PLP form of the
smaller E. coli BCAT (34 kDa) revealed a secondary structure
content of 39%
-helix and 41%
-sheet (21). The similarity of
the predicted
-helix content in the human BCAT proteins and observed
-helix content in the bacterial enzyme structure is also consistent
with the hypothesis that all of these forms of the enzymes have the
same basic folded structure.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 4.
Circular dichroism spectra of BCATc and BCATm
in the PLP and PMP forms. Spectra from the far UV range are shown
in panel A, and spectra from the near UV range are shown in
panel B. Conditions were as described under "Materials and
Methods." Lines used are the same as in Fig. 3.
|
|
The near UV CD spectra of the PLP form of both BCATm and BCATc were
dominated by a band at 421 nm (see Fig. 4B). This peak resulted from the presence of the pyridoxal group in an asymmetric environment. Despite the similarity of this peak, there was a major
difference in the spectrum of BCATc compared with BCATm around 280 nm.
While BCATc showed a peak at 285 nm with a positive ellipticity, BCATm
showed a peak at 270 nm with a negative value. Upon conversion of the
PLP form to the PMP form, the peak at 421 nm disappeared for both
proteins. For BCATm, this was concomitant with the appearance of a
weaker band at 326 nm as well as a major increase in the intensity of
the band at 277 nm. When the PLP form of BCATc was converted to the PMP
form, the positive ellipticity of the peak at 285 nm was changed to a
negative value. Since the far UV CD spectra of the PLP form of BCATm
and BCATc were very similar to their respective PMP forms (see Fig.
4A), the conversion of PLP to PMP did not seem to affect the
global structure of the proteins. Consequently, the changes observed
around 280 nm could be assigned to an altered asymmetric environment of
the aromatic residues in the active site. Furthermore, based on the
difference in intensity and sign of the ellipticity of peaks around 280 nm, the structure of the active site in BCATc appears different from BCATm.
DTNB Titration--
Like their rat counterparts (9), human BCATm,
unlike human BCATc, must be stored in a reducing environment, and all
four enzymes require the addition of DTT to the assay for maximal
activity, whereas the bacterial protein from Salmonella
typhimurium, which contains three cysteine residues, is not
sensitive to sulfhydryl reagents (32). The requirement for the presence
of a reducing agent indicates a unique property of the mammalian
aminotransferases, i.e. a relationship between the state of
reduction of the cysteines in the proteins and activity of the enzymes.
Therefore, to evaluate whether modification of sulfhydryl groups within
these proteins altered their catalytic capacity, DTNB titrations were
performed on both human enzymes.
Based on the primary sequence, BCATm contains six cysteine
residues/monomer (see Fig. 1). As shown in Table
I, two -SH groups/monomer were titrated
with native enzyme. Upon denaturation, 5.7 -SH groups were titrated,
indicating that no disulfide bonds were present. However, upon storage
of BCATm for a few days in the absence of DTT, the number of titratable
-SH groups decreased to 3.7, suggesting that a disulfide bond was
gradually being formed. This process was accompanied by a decrease in
the specific activity of the enzyme. Fig.
5 shows an SDS-PAGE of BCATm stored in
the absence of DTT. Lane A shows that a more compact
structure gradually forms upon storage without a reducing agent. As
shown in lane B, the addition of DTT prior to loading the
sample on the gel led to the disappearance of the lower band; thus,
this faster migrating band results from the formation of a disulfide
bond. This is consistent with the observation that the number of
titratable cysteines decreases over time in the absence of DTT.
View this table:
[in this window]
[in a new window]
|
Table I
DTNB titration of BCATm and BCATc
Proteins (2 µM) were incubated with a 100-fold molar
excess of DTNB in the absence (native) or presence (denatured) of 6 M guanidine hydrochloride. Titrations were performed at
25-27 °C in 50 mM Tris-HCl, pH 7.0. Results are the
average of 3-6 different titrations. Total number of cysteine residues
per enzyme is taken from the deduced amino acid sequence for each
protein.
|
|

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 5.
SDS-PAGE of human BCATc and BCATm under
reducing and nonreducing conditions. Proteins were stored for 3-5
days in the absence of reducing reagent. 3-5 µg of protein/lane were
loaded onto the gel, and 5 mM DTT was added to the
indicated samples. STD, molecular mass markers; lane
A, BCATm (41.7 kDa) without DTT; lane B, BCATm plus
DTT; lane C, BCATc (44.3 kDa) without DTT; lane
D, BCATc plus DTT.
|
|
The cytosolic isoenzyme contains 10 cysteines/monomer, yet only one -SH
group was titrated in the native form of the histidine-tagged BCATc
(see Table I). The phBCATc2 construct added an extra cysteine residue
immediately preceding the N-terminal methionone (see Fig. 1). An
additional -SH group was titrated in the native form after removal of the histidine tag, suggesting that this cysteine was accessible to DTNB. In the denatured form, 4.6 (histidine-tagged) and
6.6 (without histidine tag) -SH groups were seen, indicating that at
least two disulfide bonds are present in BCATc. This conclusion is
supported by the electrophoretic movement of the protein (see Fig. 5,
lanes C and D). In the absence of DTT, BCATc
migrated faster than when DTT was present. This increased mobility
under nonreducing conditions suggests that the enzyme maintains a more compact structure due to the presence of disulfide bonds (33).
The activity of rat BCATm is known to be inhibited by sulfhydryl
reagents (9), so the effect of DTNB on activity of the human enzymes
was investigated. Spectrophotometric titration of BCATm with a 100-fold
molar excess of DTNB occurred rapidly, and human BCATm activity was
inhibited 90-100% after titration of both accessible -SH groups. By
reducing the ratio of DTNB to enzyme to 5, the reaction could be slowed
such that essentially one -SH group was titrated after 6 min. To
determine the effect of this titration on BCATm activity, the protein
was incubated with DTNB for 6 min in assay buffer without DTT. Then
substrate was added, and activity was followed for 20 min. Without DTT,
the enzyme does not have maximal activity, so a sample under identical
assay conditions without DTNB was used as a control. As shown in Fig. 6, BCATm activity was decreased 40%
after incubation with DTNB. Therefore, it appears that labeling of one
-SH group by DTNB is not enough to fully inactivate BCATm. When
substrate was added along with DTNB in the spectrophotometric assay, no
-SH groups were titrated. Further, after incubation with DTNB in the
presence of substrate, BCATm activity was unaltered (see Fig. 6),
indicating that substrate protects against labeling and inhibition.
Therefore, the modified -SH group may lie in or near the active site of
BCATm, and studies are now in progress to verify its location. As shown in Fig. 6, titration of the single accessible -SH group in the histidine-tagged BCATc did not affect enzyme activity. Similar results
were obtained with BCATc without the histidine tag (data not shown).
This suggests that the modified residue is away from the active site.
Since it is known that the catalytic mechanism of transamination does
not require a cysteine residue, one could speculate that the loss of
activity seen with BCATm is due to steric hindrance from the
introduction of the bulky DTNB molecule in the active site. Another
possibility is that this effect results from a conformational change in
the enzyme. Possible candidate cysteines in BCATm are the two cysteine
that are not conserved in the BCATc sequence (Cys108
and Cys168, see Fig. 1). Ultimately, however, understanding
the effects of sulfhydryl reagents on catalytic function and protein
stability (see below) will require isolation of the derivatized
peptides and knowledge of the crystal structure of both proteins.

View larger version (65K):
[in this window]
[in a new window]
|
Fig. 6.
The effect of incubation with DTNB on the
activity of BCATm and histidine-tagged BCATc. Enzymes were
preincubated with a 5-fold molar excess of DTNB for either 6 min
(BCATm) or 12 min (BCATc). Substrate was added, and product formation
was followed for 20 min. Values are expressed as a percentage of the
control, which contained no DTNB, after 20 min. Product formation was
also measured after preincubation with substrate and DTNB added
simultaneously. Open bar, control sample; filled
bar, DTNB only; hatched bar, substrate and DTNB. In a
separate spectrophotometric titration assay, it was determined that
after 6-min (BCATm) and 12-min (BCATc) incubation with a 5-fold excess
of DTNB, one -SH group was titrated in either enzyme.
|
|
The Effect of Ni2+ Cations on the Activity of BCATc and
BCATm--
Affinity chromatography using nickel-NTA resin is based on
the interaction of the histidine tag in the protein with nickel cations
chelated to the resin. After elution from this column, human BCATm had
low specific activity, which did not increase even after the second
purification step of hydrophobic interaction chromatography. However,
when EDTA was added to the assay, the specific activity increased to
about 75 µmol/min/mg of protein or higher. Further, the addition of
Ni2+ cations to the assay led to 67% inactivation of the
purified enzyme, while subsequent addition of EDTA restored activity
completely. On the other hand, when DTT or
-mercaptoethanol was
omitted from the assay, added Ni2+ had no inhibitory effect
on BCATm activity. One possible explanation for these results is that
one or more disulfide bonds are reduced by either DTT or
-mercaptoethanol, allowing interaction of Ni2+ with
cysteine residues within the active site. This seems unlikely, because
in freshly purified BCATm, titration with DTNB suggests that no
disulfide bonds are present. Alternatively, one can hypothesize that
empty coordinates of Ni2+ cations bound in or near the
active site of BCATm interact with the thiol groups of DTT or
-mercaptoethanol, leading to either steric hindrance or a
conformational change.
Chemical Denaturation of BCATc and BCATm--
Since BCATc contains
two or three disulfide bonds, one expects to see a higher stability for
this protein compared with BCATm; therefore, chemical denaturation
experiments were performed to compare the two proteins. Fig.
7 shows the urea denaturation of BCATc
and BCATm as monitored by CD spectroscopy. Human BCATm remains partially folded up to a urea concentration of 8 M,
indicating high structural stability. On the other hand, the
denaturation curve of BCATc appeared as a relatively broad transition
with a midpoint of 4.1 M urea. Surprisingly, this suggests
that the structural stability of BCATc is lower than BCATm. It is
generally accepted that disulfide bonds can make a substantial
contribution to the stability of proteins (33); however, for BCATc, it
seems that this is not the case. Moreover, the broadness of the
transition indicates that unfolding of BCATc is less cooperative than
BCATm. Since chemical denaturation using urea or guanidine
hydrochloride is irreversible, the free energy of denaturation could
not be calculated. Nonetheless, the data suggest that there are
differences in the stability of the secondary structure of the two
proteins.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 7.
The effect of urea denaturation on the
circular dichroism spectra of BCATc and BCATm. The PLP form of
each enzyme at 0.1 mg/ml was incubated overnight at room temperature
with various concentrations of urea in 10 mM potassium
phosphate buffer, pH 8.0. Open circles represent BCATm, and
filled circles represent BCATc.
|
|
Kinetic Parameters--
The steady state kinetics of BCATm and
BCATc were examined, and the calculated Michaelis-Menten parameters are
summarized in Table II. Transamination of
branched-chain amino acids with
-ketoglutarate exhibited kinetics
similar to what has been reported for the rat enzymes. Values
calculated for the Km for leucine and isoleucine
were around 1.0 mM or less with a significantly higher
Km observed with valine. Differences between the two
isoenzymes were also found. First, the Km values for
leucine and valine were lower for BCATc than for BCATm. Observed kcat values suggested that the mitochondrial
enzyme turns over faster using leucine than isoleucine and valine, but
kcat/Km values were higher
for isoleucine with both enzymes, suggesting preference for this
branched-chain amino acid. For reamination of the branched-chain
-keto acids, generally Km values for the
-ketoacids were about 2-fold lower for BCATc than for BCATm. With
both isoenzymes, the Km for KIC was similar with
either fixed substrate (glutamate or isoleucine), whereas the
Km for KIV paired with glutamate was 2.5-fold higher than when the fixed substrate was isoleucine.
View this table:
[in this window]
[in a new window]
|
Table II
Kinetic constants for the reactions of human branched-chain
aminotransferase isoenzymes
Branched-chain aminotransferase activity was measured as described
under "Materials and Methods." The fixed substrates were 10 × Km. Glutamate was fixed at 100 mM.
Km and Vmax values (used to
calculate kcat) were obtained from hyperbolic plots
fit to the Michaelis-Menten equation. Means and S.E. values are from
3-5 separate determinations.
|
|
The possibility that the observed differences in kinetic parameters
with KIV/Glu and KIV/Ile were a result of differences in ionic strength
was investigated by adding KCl to the
-ketoacid/Ile assay buffer
(see Table II). The addition of 150 mM KCl raised the
apparent Km values for KIV and KIC 2-fold or more. Smaller effects were observed on kcat, resulting
in a decrease in kcat/Km for
both isoenzymes. The effect of added KCl on branched-chain amino acid
deamination was also examined in a single set of experiments. Increases
of 40-50% in the Km values for Leu and Ile were
found with BCATm. Both Km and
kcat were increased with BCATc (data not shown).
While the molecular basis for the effects of K+ and/or
Cl
on the kinetic parameters of the BCAT isoenzymes is
not yet understood, there is precedence for both cation and anion
effects on PLP-dependent enzymes (35-39). The structural
basis for the cation stimulation of tryptophan synthase (35),
dialkylglycine decarboxylase (40), tryptophanase (41), and tyrosine
phenol-lyase (42) have been investigated using x-ray
crystallography, and cation binding sites have been defined. In both
tryptophanase (43, 44) and tyrosine phenol-lyase (45) from E. coli, cations like K+ were shown to induce and
stabilize active conformations of these enzymes. With aspartate
aminotransferase, at alkaline pH, it was shown that chloride anions
(38) as well as dicarboxylic acids (46) can act as competitive
inhibitors, possibly by ion pairing with positively charged residues in
the active site that serve to bind the
-carboxylate group of the
substrate.
In the absence or presence of KCl, kcat and
kcat/Km values for the
deamination of the branched-chain amino acids were higher for BCATc
than BCATm, suggesting that BCATc turns over 2-5 times faster than
BCATm. For reamination, generally kcat values
between the two enzymes are similar; however, the markedly lower
Km values for
-keto acids found with BCATc result in calculated kcat/Km values
that are approximately 2-fold higher for KIC and 3-4-fold higher for
KIV than found with BCATm. An equilibrium constant of 1.4 ± 0.1 was also determined with BCATm, and this value agrees quite well with
the value of 1.7 originally reported by Taylor and Jenkins (3).
Substrate Specificity--
The amino acid preference of BCATc and
BCATm was determined by examining the relative rate of transamination
of [1-14C]KIC with the branched-chain amino acids,
branched-chain amino acid analogs, and other amino acids. Rates
relative to leucine are presented in Table
III. As expected, branched-chain amino
acids were clearly the preferred substrates. Glutamate was a better substrate for BCATc than BCATm, 82% of control versus 38%,
respectively. This difference could be attributed to a lower
Km for glutamate for BCATc (13 mM)
versus BCATm (24 mM). Human BCATc appeared to
accept the five-carbon straight chain analog, norvaline, twice as
readily as BCATm. Depending on the isoenzyme,
L-alloisoleucine was transaminated at about 50-80% of the
rate of the natural substrate, L-threoisoleucine.
D-Isoleucine, methionine, the aromatic amino acids,
glutamine, alanine, and aspartate were not accepted by either
enzyme.
View this table:
[in this window]
[in a new window]
|
Table III
Substrate specificity of BCATc and BCATm for amino acids
Branched-chain aminotransferase activity was determined as described
under "Materials and Methods" using [1-14C]KIC and 10 mM amino acid. The [1-14C]KIC concentration was
set at 10 × Km, 0.6 and 1.7 mM, for BCATc and BCATm, respectively. Aminotransferase activity is expressed relative to 10 mM leucine and was 131 µmol of
leucine formed/mg of protein/min for both isoenzymes. Means and S.D.
values from three separate determinations are presented.
|
|
The range of
-keto acid substrates is shown in Table
IV. Rates are shown relative to KIC and
[1-14C]leucine. The pattern of
-keto acids accepted by
the human isoenzymes is virtually identical. As expected, the hydroxy
acids of leucine (
-hydroxyisocaproate) and valine
(
-hydroxyisovalerate) were not substrates (see Table IV). The
straight chain
-keto acids were good substrates, with the
five-carbon
-ketovalerate preferred over the six-carbon
-ketocaproate. With five carbons,
-ketovalerate has the same
chain length as isoleucine and leucine but without branching.
-Ketobutyrate was transaminated poorly compared with KIV, showing
that, when the carbon length was 4, branching increased affinity for
the enzyme. Both
-ketocaproate and
-keto-
-methiobutyrate have
a carbon length of 6, but
-keto-
-methiobutyrate has a sulfur atom
substituted at the fifth carbon position.
-Ketocaproate was favored
over the
-keto acid of methionine, suggesting that the introduction
of the bulky sulfur atom decreases the affinity of the enzyme. Pyruvate
and phenylpyruvate were not accepted by either human isoenzyme. This
pattern of amino and
-keto acid preferences is very similar to what
has been reported for the rat BCAT isoenzymes (9). Thus, although
differences in apparent substrate affinity and rates of transamination
between BCATm and BCATc suggest localized variations in active site
architecture, overall structure appears conserved within the mammalian
BCAT.
View this table:
[in this window]
[in a new window]
|
Table IV
Substrate specificity of BCATc and BCATm for selected -keto acids
Branched-chain aminotransferase activity was determined as described
under "Materials and Methods" using 10 mM
[1-14C]leucine and saturating -keto acid at 0.6 and 1.7 mM for BCATc and BCATm, respectively. Radiolabeled
CO2 was trapped as described in Ref. 7. Aminotransferase
activity is expressed relative to the rate with KIC and was 163 and 130 µmol of KIC formed/mg of protein/min for BCATc and BCATm,
respectively. Means and S.D. values from three separate determinations
are presented.
|
|
 |
CONCLUSIONS |
This paper presents the first overexpression and characterization
of the human branched-chain aminotransferase isoenzymes. Our data show
that the human BCAT share the basic characteristics of other known BCAT
with respect to substrate specificity (1). Physical comparison of the
recombinant proteins has also revealed subtle kinetic and physical
differences in the two proteins with regard to stability, tertiary
structure of the N-terminal regions of the proteins, and structure of
the active sites. Although the structure of the E. coli
protein has provided insight into the structure of the human BCAT,
neither the E. coli nor the D-alanine aminotransferase structure appears to have sufficiently high sequence homology for use as a model in molecular
replacement.2 Thus,
understanding the molecular basis for the differences between the BCATc
and BCATm proteins and their bacterial counterparts will require
knowledge of the crystal structure of the mammalian BCAT. These
experiments are currently in progress.