The specificity and kinetic mechanism of branched-chain amino acid aminotransferase from Escherichia coli studied with a new improved coupled assay procedure and the enzyme's potential for biocatalysis

Article abstract of DOI:10.1111/febs.12609

Branched-chain amino acid aminotransferase (BCAT) plays a key role in the biosynthesis of hydrophobic amino acids (such as leucine, isoleucine and valine), and its substrate spectrum has not been fully explored or exploited owing to the inescapable restrictions of previous assays, which were mainly based on following the formation/consumption of the specific branched-chain substrates rather than the common amino group donor/acceptor. In our study, detailed measurements were made using a novel coupled assay, employing (R)-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans as an auxiliary enzyme, to provide accurate and reliable kinetic constants. We show that Escherichia coli BCAT can be used for asymmetric synthesis of a range of non-natural amino acids such as l-norleucine, l-norvaline and l-neopentylglycine and compare the kinetic results with the results of molecular modelling. A full two-substrate steady-state kinetic study for several substrates yields results consistent with a bi-bi ping-pong mechanism, and detailed analysis of the kinetic constants indicates that, for good 2-oxoacid substrates, release of 2-oxoglutarate is much slower than release of the product amino acid during the transamination reaction. The latter is in fact rate-limiting under conditions of substrate saturation.

Full text of DOI:10.1111/febs.12609

The specificity and kinetic mechanism of branched-chain
amino acid aminotransferase from Escherichia coli studied
with a new improved coupled assay procedure and the
enzyme’s potential for biocatalysis
1
2
1
Xuejing Yu , Xingguo Wang and Paul C. Engel
1
2
School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Ireland
Faculty of Life Science, Hubei University, Wuhan, China
Keywords
Branched-chain amino acid aminotransferase (BCAT) plays a key role in
the biosynthesis of hydrophobic amino acids (such as leucine, isoleucine
and valine), and its substrate spectrum has not been fully explored or
exploited owing to the inescapable restrictions of previous assays, which
were mainly based on following the formation/consumption of the specific
branched-chain substrates rather than the common amino group donor/
acceptor. In our study, detailed measurements were made using a novel
coupled assay, employing (R)-hydroxyglutarate dehydrogenase from Acid-
aminococcus fermentans as an auxiliary enzyme, to provide accurate and
reliable kinetic constants. We show that Escherichia coli BCAT can be used
for asymmetric synthesis of a range of non-natural amino acids such as
L-norleucine, L-norvaline and L-neopentylglycine and compare the kinetic
results with the results of molecular modelling. A full two-substrate steady-
state kinetic study for several substrates yields results consistent with a bi-
bi ping-pong mechanism, and detailed analysis of the kinetic constants
indicates that, for good 2-oxoacid substrates, release of 2-oxoglutarate is
much slower than release of the product amino acid during the transamina-
tion reaction. The latter is in fact rate-limiting under conditions of sub-
strate saturation.
branched-chain amino acid amino-
transferase; kinetic mechanism; non-natural
amino acids; rate-limiting step; substrate
specificity
Correspondence
P. C. Engel, School of Biomolecular and
Biomedical Science, Conway Institute,
University College Dublin, Belfield, Dublin 4,
Ireland
Fax: +353 1 283 7211
Tel: +353 1 716 6764
E-mail: paul.engel@ucd.ie
(
Received 9 August 2013, revised 3
November 2013, accepted 7 November
013)
2
doi:10.1111/febs.12609
Database
Branched-chain amino acid aminotransferase EC 2.6.1.42; (R)-2-hydroxyglutarate dehy-
drogenase EC 1.1.99.2
Introduction
The metabolism of amino acids typically involves a two-
stage interconversion between inorganic nitrogen, as
ammonia, and amino group nitrogen. Glutamate, and
sometimes alanine or glutamine, serve as the entry or
exit points for ammonia, utilizing a small number of
oxidoreductases of narrow specificity such as glutamate
dehydrogenase, alanine dehydrogenase and glutamate
synthase. The funnelling through these few enzymes
relies on interfacing with a set of aminotransferases of
wider specificity, which distribute amino groups to, or
gather amino groups from, a range of different carbon
skeletons. Apart from their evident metabolic impor-
tance, aminotransferases have biotechnological poten-
tial in view of their ability to handle a considerable
Abbreviations
BCAT, branched-chain amino acid aminotransferase; GDH, L-glutamate dehydrogenase; HGDH, (R) -2-hydroxyglutarate dehydrogenase; PLP,
pyridoxal 5′-phosphate.
FEBS Journal 281 (2014) 391–400 ª 2013 FEBS
391

Branched-chain amino acid transamination kinetics
X. Yu et al.
range of substrates. In practice, they have been increas-
ingly applied to large-scale synthesis of non-natural
amino acids, which can be used as the building blocks
for peptidomimetic and other single-enantiomer drugs
in the pharmaceutical industry [1–4]. The most widely
investigated are the a-transaminases, e.g. aspartate ami-
notransferase, tyrosine aminotransferase, valine-pyru-
vate aminotransferase and branched-chain amino acid
aminotransferase of Escherichia coli K-12 [5]. These
enzymes catalyse reversible pyridoxal 5′-phosphate
assays to assess substrate specificity and also possible
changes in specificity through mutagenesis. The avail-
ability of a convenient and reliable continuous assay has
opened the door to ready kinetic analysis and to thor-
ough quantitative studies of mechanism and specificity.
In our study, the branched-chain amino acid amino-
transferase BCAT (EC 2.6.1.42) was used as a starting
point to explore the substrate range in order to assess
this enzyme’s full potential capacity for producing
non-natural amino acids. Classified as transaminase B
in 1979 [7], E. coli BCAT was initially characterized
by coupled assays employing glutamate dehydrogenase
(GDH) as auxiliary enzyme. However, significant
product inhibition of GDH was observed at low gluta-
mate concentration and this is inescapable since gluta-
mate serves as both substrate for BCAT and product
for GDH [6]. Further characterization was carried out
in 1988 by Inoue et al. [8], who determined detailed
kinetic parameters in the direction of glutamate forma-
tion by the method of Velick and Vavra [9], but only
for biological amino acids. They reported that E. coli
BCAT shows activity towards various aliphatic amino
acids, of which L-isoleucine was the best substrate,
showing the highest catalytic efficiency value (kcat/Km).
There is a need to obtain similarly accurate kinetic
parameters of BCAT in the opposite direction of reac-
tion and for a wider range of branched-chain 2-oxoac-
ids, including those corresponding to non-natural
amino acids of industrial interest. Such information
would be of importance for evaluating process technol-
ogy options [10]. Earlier studies [11–15] had alerted us
to the potential of BCAT as a biocatalyst for produc-
tion of high-value non-natural amino acids, such as
L-tert-leucine and L-3-hydroxyadamantylglycine. There-
fore, the overall substrate specificity and kinetic prop-
erties of E. coli BCAT have been revisited using the
novel coupled assay, which delivers reliable initial-rate
measurements. In our hands, BCAT showed broad
applicability to the synthesis of non-natural aliphatic
branched-chain amino acids, such as L-norleucine,
L-norvaline, L-tert-leucine and L-neopentylglycine,
which can be used as building blocks for pharmaceuti-
cal drug synthesis [16,17]. The detailed analysis has
also yielded new insight into the kinetic features of the
reaction which should guide thinking in future protein
engineering of this enzyme.
(
PLP) dependent transfer of an amino group to a
-oxoacid acceptor from a donor, most frequently
L-glutamate, yielding a new amino acid and the
-oxoacid product corresponding to the donor (2-oxo-
2
2
glutarate in the case of L-glutamate). Aminotransferases
have several advantages over other biocatalysts, includ-
ing high reaction rates and enantioselectivity and the
low cost of the amino donor, glutamate. Also, since the
PLP is a tightly bound prosthetic group there is no
requirement for bulk addition or recycling of a cofactor,
as with amino acid dehydrogenases. However, this last,
practically attractive feature of aminotransferase bioca-
talysis carries a penalty, as the overall reaction offers no
convenient spectrophotometric change to serve as the
basis of a continuous assay. Inevitably this complicates
studies of kinetics and in particular of substrate specific-
ity. We have recently addressed this problem by
introducing a novel coupled assay [6] using a bacterial
(
R)-2-hydroxyglutarate dehydrogenase (HGDH) as an
auxiliary enzyme (Fig. 1). Oxoglutarate generated in the
transamination reaction is continuously reduced by
NADH, and thus the decrease in A340 can be used to
monitor the reaction. Crucially, since the coupling reac-
tion involves the glutamate/oxoglutarate couple rather
than the other variable substrate pair, the assay not only
can be applied to many aminotransferases but also for
any given one can be used reliably for comparative
NH3+
O
COO–
–OOC
COO–
R
R
NH3+
BCAT
O
NADH + H+
–OOC
COO–
COO–
HGDH
OH
Results
NAD+
–OOC
COO–
Characterization of BCAT
Fig. 1. Coupled assay system to measure BCAT activity. The assay
procedure is based on NADH depletion in the HGDH reaction. R is
for example isopropyl, isobutyl or 2-methylbutyl [6].
The recombinant BCAT, purified via a three-step pro-
cedure [6], gave a single band on an SDS/PAGE gel
3
92
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X. Yu et al.
Branched-chain amino acid transamination kinetics
(
mass (3.5 9 10 Da for the subunit). In all cases,
> 90% purity) with the correct apparent molecular
Molecular modelling of substrate binding in
BCAT
4
5
.82 9 10 Da, calculated by Lee-Peng et al. [7], was
1
In order to investigate the basis of substrate specificity,
the three-dimensional structure of the E. coli BCAT
complex with the substrate analogue 2-methylleucine
used as total protein molecular mass. Under the stan-
dard coupled assay conditions, initial velocities were
proportional to enzyme concentration in the range 0–
ꢀ
2.4 A resolution) (PDB Accession No. 1I1L) [20,21]
(
0
.08 lM (calculated on an oligomer basis). In all
was used as a template to model the binding of vari-
ous natural and non-natural branched-chain amino
acids: L-leucine, L-isoleucine, L-norleucine, L-norvaline,
L-tert-leucine (shown in Fig. 2). All amino acids were
modelled to sit in the active site of E. coli BCAT in
the most energetically favourable conformation. The
inner side of the pocket (shown in green and molecular
surface form) was demonstrated to bind the hydro-
phobic side chain and the a-carboxylate group of the
substrates. Various side chains of substrates could be
accepted because of the wide binding pocket. In the
earlier kinetics study, 2-oxoacids corresponding to
L-leucine and L-isoleucine (Fig. 2b,c), which gave maxi-
mal kcat values combined with the lowest Km values,
were considered as the best substrates of BCAT among
the above compounds. This indicates that a methyl
cases, 1 unit of enzyme activity is defined as the
amount of enzyme required to catalyse the oxidation
of 1 lmol NADH per minute in the 100 mM Tris/
HCl (pH 8.0) buffer containing 10 mM glutamate and
1
of purified BCAT in this assay system was 7.7
0 mM 2-oxoisocaproate at 37 °C. The specific activity
Uꢀmgꢁ1
.
Substrate specificity
As described in previous literature [18,19], transamin-
ases are subject to inhibition by substrate and product.
In order to avoid substrate inhibition, a low steady-
state concentration of L-glutamate (10 mM) was kept
throughout the assays to profile substrate specificity.
The apparent Km and kcat values for nine 2-oxoacids
as amino acceptor at this fixed concentration of L-glu-
tamate as amino donor are shown in Table 1. Under
the conditions applied, 3-methyl-2-oxopentanoate and
b
c
group in either the C position or the C position does
not impede the binding. L-norleucine, which has a car-
bon backbone longer by one carbon unit, was a some-
what poorer substrate, displaying a loose connection
with Y129 or F36 as shown in Fig. 2d. As for a smal-
ler molecule, such as L-tert-leucine, its shorter carbon
main chain results in weak interaction with the hydro-
phobic binding pocket (Fig. 2f).
2
-oxoisocaproate, the oxoacid salts corresponding to
L-isoleucine and L-leucine respectively, appeared to be
the best branched-chain substrates, with similar appar-
ent Km values (0.07 mM and 0.08 mM respectively) and
similar apparent kcat values. There was also remark-
able constancy in the kcat values for all oxoacid
substrates with at least a 5-carbon chain, with the
exception of 4,4-dimethyl-2-oxopentanoate (product
L-neopentylglycine), which is presumably somewhat
sterically hindered and gave a value ~ 50% lower. By
contrast, the specificity constant kcat/Km for trimethyl-
pyruvate (product L-tert-leucine) is nearly 81-fold
smaller than that towards natural substrate 3-methyl-
Validation of a ping-pong mechanism and
detailed kinetic analysis
For a more thorough analysis, yielding insight into the
kinetic mechanism, it is necessary to carry out a sys-
tematic variation of the concentrations of the two sub-
strates. Assuming that the dependence of the initial
reaction rate on substrate concentration follows simple
Michaelis–Menten behaviour for both substrates, the
initial-rate equation can be represented in its simple
reciprocal form [22,23]:
ꢁ
1
ꢁ1
ꢁ1 ꢁ1
2
-oxopentanoate (4.07 mM ꢀs versus 329 mM ꢀs ).
Surprisingly, the Km value of BCAT for 2-oxovalerate
product L-norvaline) was 7.5-fold higher than that for
-oxoisocaproate (0.6 mM versus 0.08 mM), while kcat
(
2
values were similar. In the case of pyruvate, it is note-
worthy that Inoue et al. [8] reported that this oxoacid
cannot serve as an amino acceptor for BCAT. In fact,
although its Km is very high (~ 57 mM), the kcat value
is only 5-fold lower than that for the best substrates.
In view of the demonstrated broad range of substrate
specificity for the larger substrates, BCAT shows
excellent potential for asymmetric synthesis of several
non-natural amino acids, for instance L-norleucine, L-
norvaline and L-neopentylglycine.
e
v
/A /B
/AB
¼
/ þ
þ
þ
(1)
o
½Aꢂ ½Bꢂ ½Aꢂ½Bꢂ
If A and B are designated as glutamate and 2-oxo-
acid respectively, then all four constants in Eqn (1)
can be obtained by carrying out initial-velocity mea-
surements at varying concentrations of B for a series
of fixed concentrations of A. The Lineweaver–Burk
plots of e/v against 1/[B] should be a series of lines,
and slopes and ordinate intercepts are measured for
FEBS Journal 281 (2014) 391–400 ª 2013 FEBS
393

Branched-chain amino acid transamination kinetics
X. Yu et al.
Table 1. Kinetic parameters of BCAT towards a range of natural and non-natural 2-oxoacids.
Corresponding amino
kcat/K
m
ꢁ1
(mMꢁ1ꢀsꢁ1
Substrate
acid
Structure
K
m
(mM)
k
cat (s
)
)
O
O
3
-Methyl-2-oxopentanoate
L-isoleucine
0.07 ꢃ 0.008 23.0 ꢃ 1.2
329
OH
OH
O
O
2
-Oxoisocaproate
L-leucine
L-valine
0.08 ꢃ 0.013 24.7 ꢃ 0.87 309
0.20 ꢃ 0.012 10.5 ꢃ 0.11 52.5
0.22 ꢃ 0.026 23.1 ꢃ 0.34 105
0.60 ꢃ 0.027 25.9 ꢃ 0.72 43.2
0.08 ꢃ 0.005 12.4 ꢃ 0.12 155
0.15 ꢃ 0.006 0.61 ꢃ 0.05 4.07
O
2
2
2
4
-Oxoisovalerate
-Oxohexanoate
-Oxovalerate
OH
O
O
L-orleucine
L-norvaline
OH
O
O
OH
O
O
O
,4-Dimethyl-2-oxopentanoate L-neopentylglycine
OH
Trimethylpyruvate
L-tert-Leucine
O
OH
O
O
Pyruvate
L-alanine
56.8 ꢃ 1.8
4.88 ꢃ 0.17 0.09
OH
O
O
2
-Oxobutyrate
L-a-Aminobutyric acid
3.37 ꢃ 0.28
13.9 ꢃ 0.61 4.12
OH
O
Assay mixtures (1 mL) contained 100 mM Tris/HCl (pH 8.0), 0.13 mM NADH, 10 mM glutamate, purified BCAT and coupling enzyme (HGDH)
plus 2-oxoacid at concentrations varying from 0.01 mM to 10 mM.
each one. The changing intercepts φ + φ /[A], recip-
is φ . The slopes can be similarly replotted to give
0
A
0
rocals of the apparent Vmax for each glutamate con-
centration, are replotted against 1/[A] in a secondary
plot, where the slope is φA and the ordinate intercept
φAB as the slope and φ as the intercept. All constants
can equally be obtained in different pairs by the alter-
native graphical procedure of plotting e/v initially
B
3
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X. Yu et al.
Branched-chain amino acid transamination kinetics
A
B
C
D
Fig. 2. Models of BCAT bound with
various amino acids. The active site region
in BCAT (as a molecular surface in green),
the protein residues (in red) that make
contact with the hydrophobic side chain of
the substrates and residues (in yellow)
that bind the a-carboxylate group of the
substrates are depicted. The cofactor PLP
E
F
(in dark blue) is shown in the ball and stick
form. The various substrates (in light blue)
are (A) 2-methylleucine; (B) L-leucine; (C) L-
isoleucine; (D) L-norleucine; (E) L-norvaline;
(F) L-tert-leucine (software YASARA).
against 1/[A] and then replotting slopes and intercepts
against 1/[B].
A series of experiments was accordingly performed
to measure initial rates of reaction, systematically
varying the concentration of 2-oxoisocaproate (0.01–
0
.5 mM) at each of a series of fixed concentrations of
L-glutamate (2–8 mM) in a final volume of 1 mL.
Apart from the substrate concentrations, all other con-
ditions were kept constant. An unambiguous parallel-
line pattern in the Lineweaver–Burk double reciprocal
plots of rate versus 2-oxoisocaproate concentrations at
different L-glutamate concentrations (Fig. 3) indicates
a ping-pong mechanism, as found for most amin-
otransferases [9,22,23]. In Fig. 3, where each line is for
a different fixed glutamate concentration, φB emerges
as the constant slope of the parallel lines. The con-
stancy of the slope means that in Eqn (1) the φAB
term is missing (φAB is zero). The changing intercepts
Fig. 3. Double reciprocal plots of reaction rates for variable
concentrations of 2-oxoisocaproate at various concentrations of
L-glutamate. 2-Oxoisocaproate was varied from 0.01 mM to 0.5 mM
as amino acceptor for each of four different concentrations of
L-glutamate: 2 mM, 4 mM, 6 mM and 8 mM. The concentration of
NADH for the coupling reaction was fixed at 0.13 mM. Reaction
velocities were measured from the decrease in absorbance of
NADH at 340 nm.
are given by φ + φ2-oxoisocaproate/[2-oxoisocaproate],
0
so that, in the secondary replot against 1/[2-oxoisocap-
roate] (Fig. 4A), the slope is φ2-oxoisocaproate and the
ordinate intercept is φ . The three constants are given
0
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395

Branched-chain amino acid transamination kinetics
X. Yu et al.
A
both substrates simultaneously, is in full agreement
with earlier results both for BCAT and for other amino-
transferases [8,9]. The kinetic mechanism is illustrated in
Scheme 1.
The corresponding steady-state rate equation is
given in full by
e
v
1
1
k2 þ k3 k6 þ k7
¼
þ
þ
þ
(2)
k7 k3 k1k3½Aꢂ k5k7½Bꢂ
Accordingly the φ constants are
1
1
k3 þ k7
k3k7
k2 þ k3
k1k3
k6 þ k7
k5k7
B
/0
¼
þ
¼
/ ¼
/ ¼
A
B
k7 k3
In order to further explore the kinetic mechanism,
a similar analysis was carried out for two other good
substrates, 2-oxohexanoate (Fig. S1, Table S1) and
4
,4-dimethyl-2-oxopentanoate (Fig. S2, Table S2).
Both gave a similar pattern to that seen above for
-oxoisocaproate. The resulting kinetic constants are
2
shown in Table 3. The first test that may be applied
to these results applies to any ping-pong mechanism:
one substrate, glutamate, is kept the same for all the
reactions and the constant φA is given by (k + k )/
Fig 4. Reaction of 2-oxoisocaproate (B) with L-glutamate (A). (a)
2
3
Replots of primary intercepts (φ
b) Replots of primary intercepts (φ
oxoisocaproate].
0
+ φ
B
/[B]) against 1/[glutamate].
k k , referring to the rate constants in Scheme 1.
1 3
(
0
+ φ /[A]) against 1/[2-
A
Since these only involve the free enzyme E and the
complex E-glutamate, it follows that φA should be
the same regardless of the choice of the other sub-
strate. This indeed appears to be borne out, with val-
ues of ~ 0.41, 0.38 and 0.44 mMꢀs for the three
substrates used. This constancy contrasts with a more
than 20-fold variation in φB for the same three sub-
strates and provides a further demanding test of the
ping-pong mechanism.
in Table 2. They can equally be obtained by the alter-
native graphical procedure of plotting e/v initially
against 1/[2-oxoisocaproate] and then replotting inter-
cepts against 1/[glutamate] (Fig. 4b). The resulting esti-
mates of the constants are also given in Table 2 and
the close agreement between the two estimates of each
constant gives a good indication of the reliability of
the graphical analysis. From this analysis, the extrapo-
The next observation, however, is not a necessary
general prediction for a ping-pong mechanism. Inspec-
ꢁ
1
tion of the values of φ shows that these also show
lated kcat, 1/φ , is 53.5 s . The extrapolated Michaelis
0
0
near-constancy (~ 0.019, 0.018 and 0.017 s). Referring
constants for glutamate and 2-oxoisocaproate, φ /φ
A
0
again to Scheme 1, φ is given by 1/k + 1/k . If we
and φ /φ respectively, are 21.8 mM and 0.17 mM.
0
3
7
B
0
assume that the value of φ is indeed effectively con-
This kinetic pattern, supporting a ping-pong mecha-
nism, in which there is no ternary complex containing
0
stant for these substrates and that the small differences
Table 2. Kinetic parameters determined by initial-rate measurements of the reaction between 2-oxoisocaproate and glutamate.
ꢁ1
2-oxoisocaproate
m
glutamate
m
φ
0
(s)
φ2-oxoisocaproate (mMꢀs)
φ
glutamate (mMꢀs)
k
cat (s
)
K
(mM)
K
(mM)
a
0.0186 ꢃ 0.0013
0.0188 ꢃ 0.0006
0.0187
0.00313 ꢃ 0.0002
0.0032 ꢃ 0.0001
0.0032
0.408 ꢃ 0.0009
0.407 ꢃ 0.0011
0.407
53.7 ꢃ 2.13
53.2 ꢃ 1.10
53.5
0.168 ꢃ 0.001
0.170 ꢃ 0.001
0.169
21.9 ꢃ 1.7
b
21.6 ꢃ 0.81
Mean
21.8
Kinetic data were determined by the use of primary plots against reciprocals of both glutamate concentration (a) and 2-oxoisocaproate con-
centration (b). Each of these sets of primary plots yields estimates of the three φ constants in the initial-rate equation. The alternative meth-
ods of plotting yield similar values of the constants and the bottom row in the table shows the mean of the two estimated values.
3
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X. Yu et al.
Branched-chain amino acid transamination kinetics
Table 3. Kinetic constants determined by initial-rate measurements of the reaction between glutamate and two other 2-oxoacid substrates.
glutamate
substrate
K
m
φ
glutamate
K
m
φ
substrate
ꢁ1
Substrate
φ
0
(s)
k
cat (s
)
(mMꢀs)
(mM)
(mMꢀs)
(mM)
2
4
2
-oxoisocaproate
0.0187
0.0180
0.0167
53.5
55.8
59.9
0.407
0.375
0.441
21.8
20.9
26.5
0.0032
0.085
0.169
4.74
2.69
,4-dimethyl-2-oxopentanoate
-oxohexanoate
0.0449
P
k7
Q
2-oxohexanoate as amino group acceptor, substrate
inhibition by glutamate became apparent at 500 mM
and 200 mM respectively. Moreover, inhibition by the
k5[B]
k6
k1[A]
k2
k3
E
E
EA
E'
E'B
2
-oxoacid amino acceptor became apparent at much
lower concentrations. For instance, in the presence of
0 mM glutamate, increasing the concentration of 4,4-
Scheme 1. Reaction scheme for
a
two-substrate ping-pong
mechanism.
1
dimethyl-2-oxopentanoate also to 10 mM decreased the
rate by 65% compared with the observed maximum
(Fig. S3), causing severe departure from linearity in
Lineweaver–Burk plots. For three other 2-oxoacids
similar inhibition became apparent at lower concentra-
tions, making it impossible to obtain linear plots and
good kinetic parameters according to Eqn (1).
merely reflect experimental error, the implication is
that 1/k (unlikely to be constant) must be much smal-
7
ler than 1/k , in other words that release of 2-oxoglut-
3
arate is much slower than release of the product
amino acid and is in fact rate-limiting under condi-
tions of substrate saturation. Since both φA and φ0
appear to be constant for these substrates, their ratio
φ /φ , which is the true K for glutamate at saturat-
In this context, the fact that serious inhibition by
glutamate does not supervene until concentrations of
at least 200 mM are reached is of considerable practical
significance. In application to the synthesis of non-nat-
ural amino acids, one of the drawbacks of the amino-
transferase reaction is that the equilibrium constant is
evenly poised. It is therefore important to be able to
drive the reaction forward with high concentrations of
glutamate.
A
0
m
ing concentration of the other substrate, is necessarily
also constant.
Finally we can now return to Table 1 where we
noted earlier the striking constancy of the kcat value
for all the best substrates. In this case kcat is only an
‘
apparent’ constant since it only applies at the fixed
non-saturating concentration of L-glutamate (10 mM)
employed in the experiment. However, since by defini-
tion the oxoacid concentration is saturating, and there-
Discussion
fore eliminates the φB term in Eqn (1) and since we
,
have seen that the other two φ constants in the equa-
tion are independent of the nature of the oxoacid, we
can now also predict that kcat will be constant with
changing oxoacid substrate, not only for 10 mM gluta-
mate but for any arbitrary fixed glutamate concentra-
tion. This is a consequence of the deduced inequality
k ≫ k . We may assume that for the poorer substrates
The broad substrate specificity of the branched-chain
aminotransferase of E. coli is well established [1,2,5].
The present study, however, has provided quantitative
detail with regard to the efficiency of handling a range
of both natural and non-natural substrates in the
direction of reaction most likely to be of practical
application, i.e. the chiral synthesis using L-glutamate
as amino donor. This has been possible because of the
efficiency of the coupling system employing 2-hydroxy-
glutarate dehydrogenase to monitor production of
7
3
in Table 1 this inequality no longer applies and that
release of product amino acid is largely rate-limiting.
2
-oxoglutarate [6]. The previous detailed kinetic study
Substrate inhibition
of the enzyme [8] was for the opposite direction of
reaction.
Detailed kinetic analysis of the type described relies on
strict adherence to Michaelis–Menten behaviour over
an adequate range of substrate concentrations. This
was not achievable for all substrates. Even with the
substrates shown in Table 3, departure from Michael-
is–Menten kinetics was seen over wider ranges of
concentration; with 4,4-dimethyl-2-oxopentanoate and
Increasingly in the field of biocatalysis, enzymes ca-
talysing a reaction between two or more substrates are
analysed by superficial application of a single-substrate
approach, in which apparent Vmax and Km values are
determined only for one fixed concentration of the
other substrate(s). The present study emphasizes how
FEBS Journal 281 (2014) 391–400 ª 2013 FEBS
397

Branched-chain amino acid transamination kinetics
X. Yu et al.
much is to be gained from a more thorough study.
Extraction of the full initial-rate equation and the rele-
vant constants has allowed us to give due attention to
the striking constancy of the true extrapolated maxi-
mum rate for several good substrates and to interpret
this in the light of the kinetic mechanism. This in turn
makes available critically important information for
anyone setting out to improve the catalytic perfor-
mance of the enzyme. For a currently ‘poor’ substrate
it would be relevant to concentrate on the handling of
that oxoacid/amino acid pair by the enzyme; however,
for a good substrate improvement can only be
achieved by concentrating on the way in which BCAT
handles L-glutamate and the product 2-oxoglutarate.
In general, the availability of a good coupled assay
should make it much easier from now on to assess
exactly what has been achieved in any protein engi-
neering experiments with this enzyme.
NADH, 10 mM glutamate, 10 mM 2-oxoisocaproate (or
other corresponding 2-oxoacids) and coupling enzyme
HGDH. The reactions were initiated by the addition of
BCAT and the oxidation of NADH was monitored at
ꢁ
1
ꢁ1
3
40 nm (e = 6220 M ꢀcm ).
In the standard assays, stock solutions of the coupling
enzymes kept in a ꢁ20 °C freezer were thawed, diluted
ꢁ
1
with 100 mM Tris/HCl (pH 8.0) containing 2 mgꢀmL
bovine serum albumin and stored on ice. The activity mea-
surement of HGDH was carried out spectrophotometri-
cally at 37 °C by monitoring the decrease of A340 caused
by oxidation of NADH. The activity was determined in
1
00 mM Tris/HCl buffer (pH 8.0) containing 0.13 mM
NADH and 10 mM 2-oxoglutarate with a final volume of
mL. At least three replicate readings were performed for
1
each assay and averaged. The range of the three readings
was never more than 5% and for the majority of readings
was within 2%.
Unless otherwise stated, reactions were started by the
addition of purified BCAT, which was diluted from stock
solutions (containing 50% glycerol) with 100 mM Tris/HCl
Materials and methods
ꢁ
1
(
pH 8.0) containing 2 mgꢀmL bovine serum albumin. All
substrates and NADH solutions were freshly prepared.
Reaction solutions were adjusted to pH 8.0 ꢃ 0.05 with
NaOH or HCl, and incubated at 37 °C during the measure-
ments. An appropriate activity range of added coupling
enzyme [6] is 80–130 U. When an excess amount of HGDH
was thus used, after a short initial lag phase the reaction
traces were linear and the steady-state rates of the coupled
assays were found to be directly proportional to the con-
centrations of BCAT.
Materials
In most cases analytical grade reagents were used. 2-
Oxoisocaproate (sodium salt) was purchased from Degussa
(Munich, Germany). Other oxoacids and L-amino acids
were purchased from Sigma Chemical Co. (St Louis, MO,
USA). NADH was obtained from Apollo (Manchester,
UK). Fluka (Buchs, Switzerland) supplied PLP. The recom-
binant E. coli BL21(DE3) cells carrying the gene encoding
HGDH (EC 1.1.99.2) from Acidaminococcus fermentans
were a kind gift from W. Buckel (Philipps Universit €a t,
Marburg, Germany).
Initial-rate kinetic analysis of BCAT
First, apparent kinetic constants of BCAT at a fixed con-
centration of glutamate were determined for nine 2-oxoacid
substrates by measuring rates of transamination in 1 mL
reaction mixtures containing 100 mM Tris/HCl (pH 8.0),
0.13 mM NADH, 10 mM glutamate, purified BCAT and
coupling enzyme (HGDH) plus 2-oxoacid at concentrations
varying from 0.01 mM to 10 mM. The reactions were initi-
ated by addition of 10 lL 2-oxoacids. The whole set of
bi-bi kinetic studies was carried out three times with each
substrate in order to optimize experimental design. In each
case the results reported are for the third optimized experi-
ment. Apparent Km and Vmax values were calculated by
fitting the Michaelis–Menten equation to the experimental
data using the non-linear least-squares fit mode of PRISM 4
Cloning, expression and purification
A recombinant plasmid containing the ilvE gene (Genbank
Accession No. NC_000913.2) encoding E. coli BCAT was
constructed using the expression vector pET23a(+) and
transformed into E. coli BL21(DE3) cells. The overexpres-
sion and purification of BCAT has been described previ-
ously [6]. Likewise, HGDH was expressed and purified as
detailed in the same paper. This coupling enzyme is now
generally available through Enzolve Technologies, Dublin,
Ireland.
Coupled assays of BCAT
(GraphPad Software, San Diego, CA, USA).
In the coupled assay for BCAT [6] the activity was mea-
sured with a Cary 50 UV–visible spectrophotometer (Agi-
lent Technologies, Cork, Ireland). The assay was conducted
at 37 °C in 1 cm light-path cuvettes with 1 mL reaction
mixture containing 100 mM Tris/HCl (pH 8.0), 0.13 mM
Acknowledgements
We are grateful to Professor Wolfgang Buckel (Phi-
lipps Universit €a t, Marburg, Germany) for his kind gift
3
98
FEBS Journal 281 (2014) 391–400 ª 2013 FEBS

X. Yu et al.
Branched-chain amino acid transamination kinetics
of recombinant E. coli cells carrying the gene encoding
(
for kinetic model parameter estimation for biocatalytic
reactions. Biotech Progr 28, 1186–1196.
R)-2-hydroxyglutarate dehydrogenase from A. fermen-
tans. Financial support from Wuhan Science and
Technology Bureau (to X.-G.W.) and Enterprise Ire-
land, the Royal Irish Academy and the Science Foun-
dation Ireland (to P.C.E.) is gratefully acknowledged.
X.Y. was supported by a postgraduate scholarship
under the EMBARK scheme of the Irish Research
Council for Science, Engineering and Technology and
also by the Chinese Scholar Scheme operating between
University College Dublin and the Chinese Scholar
Council.
11 Seo YM & Yun H (2011) Enzymatic synthesis of
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1
1
1
2 Li T, Kootstra AB & Fotheringham IG (2002)
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Branched-chain amino acid transamination kinetics
X. Yu et al.
Supporting information
L-glutamate.
Fig. S3. Substrate inhibition curve of 4,4-dimethyl-2-
oxopentanoate in the BCAT catalysed transamination
reaction in the presence of 10 mM L-glutamate.
Table S1. Kinetic parameters determined by initial-rate
measurements for substrates 2-oxohexanoate and glu-
tamate.
Additional supporting information may be found in
the online version of this article at the publisher’s web
site:
Fig. S1. Double reciprocal plot of reaction rates for
variable fixed concentrations of 2-oxohexanoate at var-
ious fixed concentrations of L-glutamate.
Table S2. Kinetic parameters determined by initial-rate
measurements for substrates 4,4-dimethyl-2-oxopent-
anoate and glutamate.
Fig. S2. Double reciprocal plot of reaction rates
for variable fixed concentrations of 4,4-dimethyl-
2
-oxopentanoate at various fixed concentrations of
4
00
FEBS Journal 281 (2014) 391–400 ª 2013 FEBS