Báo cáo khoa học: Human kynurenine aminotransferase II – reactivity with substrates and inhibitors potx

Human kynurenine aminotransferase II – reactivity with
substrates and inhibitors
Elisabetta Passera
1
, Barbara Campanini
1
, Franca Rossi
2
, Valentina Casazza
2
, Menico Rizzi
2
, Roberto
Pellicciari
3
and Andrea Mozzarelli
1,4
1 Department of Biochemistry and Molecular Biology, University of Parma, Italy
2 DiSCAFF – Department of Chemical, Food, Pharmaceutical and Pharmacological Sciences, University of Piemonte Orientale A. Avogadro,
Novara, Italy
3 Department of Drug Chemistry and Technology, University of Perugia, Italy
4 National Institute of Biostructures and Biosystems, Rome, Italy
Introduction
Kynurenine aminotransferase (KAT, EC 2.6.1.7) is a
pyridoxal 5¢-phosphate (PLP)-dependent enzyme cata-
lyzing the irreversible transamination of l-kynurenine
(KYN) to produce kynurenic acid (KYNA). KYN is
the central metabolite in the kynurenine pathway
(Scheme 1), the main catabolic process of tryptophan
in most living organisms [1]. Kynurenine pathway
enzymes and metabolites (kynurenines) affect biologi-

cal functions of the immune and nervous systems
[2–6]. In particular, KYNA acts as a broad-spectrum
endogenous antagonist of all three ionotropic excit-
atory amino acid receptors in the central nervous
system (CNS), the ligand-gated ion channel receptors
N-methyl-d-aspartate (IC50 @ 8 lm) [7], and the
a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
and kainate receptors [8]. It has been reported that
Keywords
kynurenine aminotransferase II (KATII);
kynurenine pathway; PLP-dependent
enzymes; schizophrenia; tryptophan
metabolism
Correspondence
A. Mozzarelli, Department of Biochemistry
and Molecular Biology, University of Parma,
Viale GP Usberti 23 ⁄ A, 43100 Parma, Italy
Fax: +39 0521 905151
Tel: +39 0521 905138
E-mail:
(Received 22 November 2010, revised 8
March 2011, accepted 22 March 2011)
doi:10.1111/j.1742-4658.2011.08106.x
Kynurenine aminotransferase (KAT) is a pyridoxal 5¢-phosphate-dependent
enzyme that catalyzes the conversion of kynurenine, an intermediate of the
tryptophan degradation pathway, into kynurenic acid, an endogenous
antagonist of ionotropic excitatory amino acid receptors in the central ner-
vous system. KATII is the prevalent isoform in mammalian brain and a
drug target for the treatment of schizophrenia. We have carried out a spec-
troscopic and functional characterization of both the human wild-type

KATII and a variant carrying the active site mutation Tyr142 fi Phe. The
transamination and the b-lytic activity of KATII towards the substrates
kynurenine and a-aminoadipate, the substrate analog b-chloroalanine and
the inhibitors (R)-2-amino-4-(4-(ethylsulfonyl))-4-oxobutanoic acid and cys-
teine sulfinate were investigated with both conventional assays and a novel
continuous spectrophotometric assay. Furthermore, for high-throughput
KATII inhibitor screenings, an endpoint assay suitable for 96-well plates
was also developed and tested. The availability of these assays and spectro-
scopic analyses demonstrated that (R)-2-amino-4-(4-(ethylsulfonyl))-4-oxob-
utanoic acid and cysteine sulfinate, reported to be KATII inhibitors, are
poor substrates that undergo slow transamination.
Abbreviations
AAD, a-aminoadipate; AlaAT, alanine aminotransferase; AspAT, aspartate aminotransferase; BCA, b-chloroalanine; CNS, central nervous
system; CSA, cysteine sulfinate; ESBA, (R)-2-amino-4-(4-(ethylsulfonyl))-4-oxobutanoic acid; GOX, glucose oxidase; KAT, kynurenine
aminotransferase; KG, a-ketoglutarate; KYN,
L-kynurenine; KYNA, kynurenic acid; MPP
+
, 1-methyl-4-phenylpyridinium; 3-NPA, 3-nitropropionic
acid; OPS, O-phosphoserine; PLP, pyridoxal 5¢-phosphate; PMP, Pyridoxamine; SPC, S-phenylcysteine.
1882 FEBS Journal 278 (2011) 1882–1900 ª 2011 The Authors Journal compilation ª 2011 FEBS
KYNA also acts as a noncompetitive inhibitor of the
a7-nicotinic acetylcholine receptor [9–12] and is an
endogenous ligand of an orphan G-protein-coupled
receptor (GPR35) that is predominantly expressed in
immune cells [13]. The activation of glutamate recep-
tors is responsible for basal excitatory synaptic trans-
mission and for mechanisms that underlie learning and
memory, such as long-term potentiation and long-term
depression [14,15]. Any event that causes overactiva-
tion of glutamate receptors leads to a rise in intracellu-

lar Ca
2+
levels that promotes neuronal cell damage by
both activating destructive enzymes and increasing
the formation of reactive oxygen species [16,17].
Consequently, mechanisms capable of preventing glu-
tamate receptors from being overstimulated seem to be
essential for maintaining the normal physiological
condition in the CNS. KYNA is considered to be an
antiexcitotoxic agent, limiting neurotoxicity arising
from N-methyl-d-aspartate receptor overstimulation
[4]. Pharmacologically induced increases in KYNA
provide neuronal protection against ischemic damage
and anticonvulsive action [18–20]. However, an
increase in the endogenous levels of KYNA is associ-
ated with reduced glutamate release (glutamatergic
hypofunction) and, consequently, decreased extracellu-
lar dopamine levels [21], leading to impaired cognitive
capacity [22] and schizophrenia [23–26]. Furthermore,
KYNA levels are abnormally high in the brain and
cerebrospinal fluids of Alzheimer’s disease patients [22]
and in the frontal and temporal cortices of Down’s
syndrome patients [27].
On the basis of the intimate relationships between
abnormally high brain KYNA concentrations and neu-
rodegenerative diseases and psychotic disorders, the
Scheme 1. KYN pathway in mammalian
cells.
E. Passera et al. Reactivity of kynurenine aminotransferase
FEBS Journal 278 (2011) 1882–1900 ª 2011 The Authors Journal compilation ª 2011 FEBS 1883

enzymes involved in KYNA synthesis have been consid-
ered as potential targets for the development of com-
pounds with inhibitory activity [2–4,6,18,28–32]. It is
well established that KYN transamination to produce
KYNA in the CNS of mammals is carried out by at
least four distinct enzymes, constituting the KAT family
[33–39]: (a) KATI ⁄ glutamine transaminase K ⁄ cysteine
conjugate b-lyase 1; (b) KATII ⁄ a-aminoadipate (AAD)
aminotransferase; (c) KATIII ⁄ cysteine conjugate
b-lyase 2; (d) KATIV ⁄ glutamic-oxaloacetic transami-
nase 2 ⁄ mitochondrial aspartate aminotransferase
(AspAT).
KYNA does not cross the blood–brain barrier, and
is thus produced in the CNS [40]. Although all four
isoforms are present in the mammalian brain, to differ-
ent extents, only KATI and KATII have been thor-
oughly characterized with respect to their role in
cerebral KYNA synthesis [35,41]. These two isoforms
differ by substrate specificity, with KATI showing
lower KYN specificity than KATII [35]. The intrinsic
catalytic promiscuity of KATI is enhanced by a b-lyase
activity [42,43]. Therefore, KATII has been considered
to be the principal isoform responsible for the synthe-
sis of KYNA in the rodent and human brain
[34,35,41]. Crystallographic studies of KATs from dif-
ferent organisms, including humans, indicate that this
enzyme belongs to the a-family of PLP-dependent
enzymes [44] and to the fold type I group [45–49].
However, mammalian KATII and homologs from
yeast and thermophilic bacteria do not belong to any

of the seven subgroups of fold type I aminotransferas-
es [47], but rather form a distinct subfamily [47,50,51].
Furthermore, human KATII shows intriguing struc-
tural determinants [52], such as the conformation
adopted by the N-terminal region and the presence of
Tyr142 above the cofactor molecule. These features
are typical of PLP-dependent b-lyases [46,53], and hint
at additional PLP-dependent reactions catalyzed by
KATII [54].
Although KATII is considered to be interesting drug
target in the treatment of schizophrenia and other neu-
rological disorders [54,55], only a few inhibitors have
so far been developed [55–61]. They are depicted in
Scheme 2. From the point of view of drug develop-
ment, the existence in the human brain of at least four
KYNA-synthesizing enzymes, combined with the need
for fine-tuning of KYNA levels to avoid the poten-
tially harmful effects caused by a deficiency of this
metabolite in the CNS, requires the design of isozyme-
specific inhibitors [54]. The isozyme specificity of
a KATII inhibitor, 1-methyl-4-phenylpyridinium
(MPP
+
) [61], has been reported, and might be the
starting point for the development of potent and
specific inhibitors of the synthesis of KYNA in the
brain. Recently, the three-dimensional structure of the
complex between KATII and a fluoroquinolone deriva-
tive, BFF-122, has been solved at 2.1-A
˚

resolution,
allowing, in combination with spectroscopic and inhi-
bition studies, ascertainment of the mechanism of
action of this inhibitor [62]. BFF-122 forms a hydraz-
one adduct with PLP, and is thus an irreversible inhib-
itor, like the majority of the pharmacologically
relevant inhibitors of PLP-dependent enzymes [63].
In this study, we have characterized (a) the absorp-
tion and fluorescence properties and (b) the transamina-
tion and the b-elimination in the presence of substrates
and substrate analogs of recombinant human KATII
and a variant carrying the Tyr142 fi Phe mutation,
which is expected, on the basis of structural evaluations,
to exhibit a decreased propensity for b-elimination [52],
a side reaction common to transaminases. During this
investigation, two efficient and rapid assays were devel-
oped to screen KATII inhibitors: (a) a continuous assay
based on the absorbance of the natural substrate KYN;
Scheme 2. KATII natural substrates (KYN and AAD) and inhibitors:
ESBA [55,57], CSA [59], MPP
+
, 3-NPA [61], OPS [60], and BFF-122
[56,62].
Reactivity of kynurenine aminotransferase E. Passera et al.
1884 FEBS Journal 278 (2011) 1882–1900 ª 2011 The Authors Journal compilation ª 2011 FEBS
and (b) an endpoint assay, suitable for 96-well plates,
based on the coupling of KAT activity to reporter reac-
tions catalyzed by glutamate oxidase and peroxidase.
The latter assay is well suited for high-throughput
screening of KATII inhibitors.

Results
Spectroscopic characterization of KATII and
Tyr142 fi Phe KATII
Absorption spectroscopy
The absorption spectrum of human KATII (Fig. 1A)
at pH 7.5 exhibited, in addition to the band centered
at 278 nm, a band at 360 nm that is typical of a de-
protonated internal aldimine. The A
280 nm
⁄ A
360 nm
ratio was 5. The extinction coefficient calculated by the
method of Peterson [64] was found to be
9510 m
)1
Æcm
)1
. The absorption spectrum exhibited a
shoulder at about 420 nm that might be attributable to
the protonated internal aldimine (see below). KATII
instability at pH values lower than 6 precluded the
determination of the pH dependence of the proton-
ation of the internal aldimine. In the presence of
the natural, nonchromophoric substrate AAD [41]
(Scheme 2), the band at 360 nm disappeared and a
species absorbing maximally at 325 nm accumulated,
probably the pyridoxamine form of the cofactor
(Fig. 1A; Scheme 3, species 5). The shoulder at 420 nm
remained unmodified, suggesting the presence of an
inactive PLP enzyme species.

Tyr142 fi Phe KATII exhibited an absorption spec-
trum that was almost superimposable on that of the
wild-type enzyme, with an invariant A
280 nm
⁄ A
360 nm
ratio (data not shown). Because the extinction coeffi-
cient of the cofactor at 360 nm was found to be
9400 m
)1
Æcm
)1
, this invariant ratio can be explained by
a concomitant decrease in the extinction coefficient at
280 nm resulting from the Tyr fi Phe substitution.
The addition of AAD to the mutant caused spectro-
scopic changes similar to those observed for the wild-
type enzyme, with a less intense peak at 325 nm (the
wild-type ⁄ Tyr142 fi Phe ratio at 325 nm was 1.12;
data not shown).
Fluorescence spectroscopy
KATII contains three tryptophans. The emission spec-
trum upon excitation at 298 nm showed a band cen-
tered at 345 nm, indicative of tryptophans being
predominantly exposed to solvent. No energy transfer
occurred between tryptophan and PLP, as indicated by
the absence of peaks centered at either 420 or 500 nm
Fig. 1. Spectroscopic characterization of KATII. (A) Absorption
spectra of a solution containing 10 l
M KATII and 50 mM Hepes

(pH 7.5) at 25 °C, in the absence (solid line) and in the presence
(dotted line) of 10 m
M AAD. (B) Emission spectrum of a solution
containing 26 l
M KATII and 50 mM Hepes (pH 7.5) at 25 °C,
excited at 298 nm. (C) Emission spectra of a solution containing
26 l
M KATII and 50 mM Hepes (pH 7.5) at 25 °C, excited at
330 nm (continuous line), 360 nm (dotted line), and 420 nm (dash-
dotted line).
E. Passera et al. Reactivity of kynurenine aminotransferase
FEBS Journal 278 (2011) 1882–1900 ª 2011 The Authors Journal compilation ª 2011 FEBS 1885
(Fig. 1B), in contrast to observations on fold type II
enzymes, such as tryptophan synthase [65] and O-acet-
ylserine sulfhydrylase [66]. Direct excitation of PLP at
360 nm gave a structured emission with a maximum at
417 nm and a shoulder at 520 nm (Fig. 1C). The emis-
sion at 417 nm is typical of the enolimine tautomer of
the internal aldimine, whereas the emission at 520 nm
is typical of the ketoenamine tautomer [67,68]. The flu-
orescence emission spectrum of Tyr142 fi Phe KATII
was indistinguishable from that of wild-type KATII.
A new continuous spectrophotometric assay for
KATII activity
To overcome the limitations of the discontinuous KAT
assay [41,56–58,69,70], an assay for the continuous
monitoring of KYN transamination was developed.
The absorption spectrum of a solution containing
900 lm KYN, 10 mm a-ketoglutarate (KG) and 50 lm
PLP (pH 7.5, 37 °C) exhibited a maximum at 361 nm,

typical of KYN at neutral pH. The spectrum obtained
upon addition of KATII to the reaction mixture and
equilibration exhibited a band at 332 nm and a shoul-
der at 344 nm (Fig. 2A), typical of KYNA [71]. The
difference spectrum (Fig. 2A, inset) showed a positive
peak at about 340 nm and a negative peak at 360 nm.
Thus, at wavelengths lower than 352 nm, the accumu-
lation of KYNA could be monitored with good sensi-
tivity. Nonetheless, the extinction coefficient of KYN
at 340 nm was too high (3290 m
)1
Æcm
)1
) to allow for
initial velocity determinations at KYN concentrations
higher than 8 mm, the published K
m
for KYN being
about 5 mm [41]. Therefore, assays were carried out
with monitoring of the reaction at 310 nm, a wave-
length that represents a compromise between high sen-
sitivity and an extinction coefficient for KYN that is
low enough to monitor time courses with KYN con-
centrations up to about three-fold the expected K
m
.
The extinction coefficients at 310 nm for KYN and
KYNA were calculated to be 1049 m
)1
Æcm

)1
and
4674 m
)1
Æcm
)1
, respectively, with a De at 310 nm of
3625 m
)1
Æcm
)1
. Under these conditions, reactions were
carried out as a function of KYN concentration
between 2 and 23 m m, in the presence of 10 mm KG
(Fig. 2B). This KG concentration was assumed to be
saturating on the basis of the previously determined
K
m
for KG of 1.2 mm [41]. Control measurements
showed that V
0
values were independent of KG con-
centration down to 0.2 mm (Fig. 2B, inset). At the
lowest KG concentration (0.02 mm) and at high KYN
concentrations, the rate of the reverse reaction from
PMP to PLP became rate-limiting (Fig. 2B, inset).
Data points, reported in the typical Michaelis–Menten
plot (Fig. 2B), were fitted to K¢ values of 10 ± 1 mm,
V¢
max

values of 0.022 ± 0.001 mmÆmin
)1
, and a k
cat
of
25 min
)1
. In order to directly compare the rate deter-
mined with the continuous assay with the rate reported
for the discontinuous assay, which was carried out in
Scheme 3. General reaction mechanism of aminotransferases, including tautomeric and protonation equilibria. The absorption maxima for
the catalytic intermediates are reported. The b-elimination side reaction is boxed. Adapted from [44,74,118].
Reactivity of kynurenine aminotransferase E. Passera et al.
1886 FEBS Journal 278 (2011) 1882–1900 ª 2011 The Authors Journal compilation ª 2011 FEBS
the presence of 200 mm potassium phosphate, 5 mm
KG, and 0.04 mm PLP (pH 7.5, 45 °C) (116), we
assayed the enzyme under the same experimental con-
ditions. The continuous assay gave a K
m
(mm) of 2.09
and a k
cat
(min
)1
) of 110. The discontinuous assay
gave a K
m
(mm) of 0.96 and a k
cat
(min

)1
) of 186. The
k
cat
difference is mostly attributable to the method
used for evaluation of the protein concentration. In
fact, for the published data (116), the protein concen-
tration was determined with the Bradford method,
whereas we measured the bound PLP concentration
with the alkali method. We determined that the PLP
method led to a 1.38-fold higher value for active site
concentration than the Bradford method. Thus, the
actual k
cat
(min
)1
) for the discontinuous assay was
134, only 1.21-fold higher than the value determined
with the continuous assay.
b-Lyase activity of KATII and Tyr142 fi Phe KATII
It is well established that transaminases, owing to the
chemistry of the catalyzed reaction, are prone to
b-elimination as a side reaction when the substrate con-
tains a good b-leaving group [43,72–74]. In fact, the
quinonoid intermediate formed upon a-proton removal
can follow two pathways (Scheme 3): (a) protonation
on the imine nitrogen to form the ketimine (transamina-
tion pathway); and (c) elimination of the b-substituent
to form the a-aminoacrylate Schiff base (b-elimination
pathway), which spontaneously and irreversibly hydro-

lyzes to pyruvate and ammonia. In turn, these products
may inhibit or inactivate the enzyme.
First, we analyzed the reaction of KATII in the
presence of 5 mm b-chloroalanine (BCA), a substrate
that contains chloride as a good b-leaving group [75].
Transamination of BCA in the presence of 10 mm KG
was found to be negligible, as measured by the glucose
oxidase (GOX)-coupled assay, which monitors the for-
mation of glutamate (see Experimental procedures,
and below). In contrast, a series of spectra recorded as
a function of time exhibited the accumulation of a spe-
cies with maximum absorbance at 330 nm (Fig. 3A),
which progressively shifted to about 315–320 nm.
Upon reaction completion, the concentration of pyru-
vate was estimated on the basis of the absorbance at
315 nm, and was found to be 3.7 mm. The concentra-
tion of ammonia, determined by Nessler’s assay, was
3.8 mm. This indicates that a significant amount of
BCA had undergone a b-elimination reaction, with
formation of a-aminoacrylate, which decomposes to
pyruvate and ammonia. The same assay carried out
on Tyr142 fi Phe KATII indicated a reduced effi-
ciency of the mutant in the b-elimination of chloride
in the presence of BCA. In fact, only $ 1.8 mm
ammonia was produced from 5 mm BCA, under the
same conditions. The initial rate of pyruvate formation
catalyzed by KATII and Tyr142 fi Phe KATII in the
Fig. 2. Reactivity of KATII towards KYN. (A) Absorption spectra of
KYN and KYNA formed upon reaction in the presence of KATII and
KG. The reaction mixture contained 10 m

M KG, 40 lM PLP, 900 lM
KYN and 50 mM Hepes (pH 7.5) at 37 °C (solid line). The reaction,
carried out in 0.1-cm pathlength cuvettes, was started by the addi-
tion of 9.4 l
M KATII. A spectrum was collected at equilibrium, which
was reached $ 150 min after enzyme addition (dashed line). Inset:
difference spectrum of the reaction mixture before enzyme addition
and upon equilibration. (B) Dependence of the rate of reaction of
KATII on KYN in the presence of KG. The reaction mixture contained
870 n
M KATII in 50 mM Hepes, 10 mM KG, and 40 lM PLP (pH 7.5),
and variable concentrations of KYN. The reaction was carried out at
25 °C in 0.1-cm pathlength cuvettes. The solid line through data
points represents fitting to the Michaelis–Menten equation with
V ¢
max
= 0.022 ± 0.001 mMÆmin
)1
and K ¢
m
=10±1mM. Inset: the
reaction was carried at 10 m
M KG (closed circles), 2 mM KG (open
triangles), 0.2 m
M KG (open squares), and 0.02 mM KG (open dia-
monds).
E. Passera et al. Reactivity of kynurenine aminotransferase
FEBS Journal 278 (2011) 1882–1900 ª 2011 The Authors Journal compilation ª 2011 FEBS 1887
presence of BCA (Fig. 3B) allowed determination of
specific activities of 5 nmolÆlg

)1
Æmin
)1
and 0.22 nmo-
lÆlg
)1
Æmin
)1
, respectively. The formation of pyruvate
was characterized by a fast linear phase (Fig. 3B), fol-
lowed by a slow phase. The deviation from linearity in
the reaction occurred at a concentration of pyruvate
that was less than 1% of the total substrate concentra-
tion. This deviation is not generated by the lack of
adherence to steady-state conditions, is strongly
suggestive of an inactivation process taking place as a
consequence of the b-elimination reaction. Two possi-
ble mechanisms can be invoked to explain enzyme
inhibition: covalent modification of the enzyme, and
product inhibition. In the latter case, removal of the
products from the reaction mixture should lead to the
recovery of enzymatic activity, whereas covalent modi-
fication causes permanent inactivation of the enzyme.
It is known that, during b-lytic reactions, some amin-
otransferases become covalently inactivated by a syn-
catalytic mechanism involving the cofactor and a basic
residue in the active site [72,76] (see also Scheme 4).
To determine whether this is the case for KATII, the
residual activity of the enzyme was measured upon
reaction with BCA. KAT II (174 lm), incubated with

50 mm BCA for 20 min at 25 °C, was assayed upon
200-fold dilution, using 10 mm KYN and 10 mm KG.
The activity was found to be only 3%, indicating that
a significant amount of the enzyme was inactivated as
a consequence of the occurrence of the b-elimination
reaction.
BCA is considered to be the best substrate to test
for b-elimination reactions. However, KATII b-elimi-
nation activity was also evaluated with S-phenylcyste-
ine (SPC). SPC was chosen because cysteine
S-conjugates are good substrates for the b-lytic activity
of the related enzyme KATI [43,77]. Cysteine S-conju-
gate b-lyase side reactions can have both negative and
positive physiological consequences. Adverse effects
may occur as a result of cysteine S-conjugate b-lyases
catalyzing reactions that generate toxic sulfur-contain-
ing fragments, whereas possible beneficial conse-
quences of cysteine S-conjugate b-lyases activity
include pharmacological applications in cancer therapy
via the bioactivation of prodrugs into antiproliferative
and proapoptotic agents [42,43,78–80]. It was found
that the reaction of KATII in the presence of 3 mm
SPC produced 210 lm ammonia, whereas the b-lytic
activity of Tyr142 fi Phe KATII was undetectable.
We also evaluated whether the natural substrate KYN
underwent b-elimination by KATII. The specific
activity measured with 20 mm KYN was 9 · 10
)6
lmolÆmin
)1

Ælg
)1
, which is four orders of magnitude
lower than that measured with BCA.
Reactivity of KATII with cysteine sulfinate
(CSA) and (R)-2-amino-4-(4-(ethylsulfonyl))-4-oxo-
butanoic acid (ESBA)
In vivo experiments have indicated that both CSA and
ESBA are inhibitors of KATII [55,57,59]. However,
their structures (Scheme 3) suggest that they might be
substrates for transamination or ⁄ and b-elimination.
Fig. 3. Reactivity of KATII towards BCA. (A) Reaction of KATII with
BCA. The reaction mixture contained 15 l
M KATII and 50 mM
Hepes (pH 7.5) at 25 °C, in the absence (solid line) and presence
(dotted lines) of 5 m
M BCA, after 1, 5, 10 and 28 min of mixing
(dotted lines). (B) Time courses of pyruvate formation by KATII and
Tyr142 fi Phe KATII. The reaction mixture contained either 64 n
M
KATII (solid black line) or 64 nM Tyr142 fi Phe KATII (dotted black
line) and 5 m
M BCA and 100 mM K
2
PO
4
(pH 7.5) at 25 °C. The solid
dashed lines represent fitting to linear equations with slopes of
17 l
MÆmin

)1
and 0.74 lMÆmin
)1
for KATII and Tyr142 fi Phe KATII,
respectively.
Reactivity of kynurenine aminotransferase E. Passera et al.
1888 FEBS Journal 278 (2011) 1882–1900 ª 2011 The Authors Journal compilation ª 2011 FEBS
Indeed, CSA is a known substrate of AspAT that is
able to catalyze both its transamination [81,82] and
b-elimination, with production of sulfinate [74].
The spectra of KATII (Fig. 4A) and Tyr142 fi Phe
KATII (data not shown) in the presence of CSA
exhibited a decrease in the intensity of the band at
360 nm, with the concomitant accumulation of a spe-
cies absorbing at 330 nm, probably pyridoxamine
(PMP). In the presence of KG, CSA transaminated to
b-sulfinylpyruvate [81], as demonstrated by the GOX-
coupled assay (data not shown). To further investigate
the CSA mechanism of action, KATII activity assays
were carried out at 4 mm and 20 mm CSA (Fig. 4B).
It was found that CSA inhibited the KATII transami-
nation reaction. Data points were fitted to Eqn (4)
with an apparent V
max
of 0.025 ± 0.001 mmÆmin
)1
,an
apparent K
m
of 12.3 ± 1.5 mm, and a K

ii
of
17.2 ± 3.5 mm. The corresponding K
i
, calculated from
Eqn (5), was 13 lm. The IC
50
value reported from
in vivo experiments on rats [59] is $ 2 lm.
ESBA is an aromatic compound (Scheme 3) that is
structurally analogous to KYN. ESBA absorbed at
287 nm with an extinction coefficient of 2050 m
)1
cm
)1
(Fig. 5A). ESBA might be either a pure inhibitor, as
previously proposed [55], or, more likely, a substrate
analog. We evaluated both the transamination and the
b-lytic activities of KATII on ESBA, in the absence and
presence of oxoacids, with monitoring of the reaction
products, including ammonia. The reaction of ESBA
with KATII, in the absence of 2-oxoacids, led to marked
changes in the absorption spectrum, with an intensity
increase at 283 nm and at $ 330 nm (Fig. 5A). In the
presence of 10 mm KG, a species absorbing maximally
at 338 nm accumulated (Fig. 5A). The amount of ESBA
transaminated by KATII at equilibrium was assessed by
the GOX-coupled assay, and found to be about 90%.
Thus, the main product of the reaction was 4-[4-(ethyl-
sulfonyl)phenyl]-2,4-dioxobutanoic acid, which is char-

acterized by an extinction coefficient at 338 nm of
15 400 m
)1
Æcm
)1
. Kinetic parameters for the reaction of
ESBA with KATII were determined by monitoring the
change in absorbance at 338 nm, caused by 4-[4-(ethyl-
sulfonyl)phenyl]-2,4-dioxobutanoic acid accumulation,
as a function of time, at different ESBA concentrations.
Data were fitted to the Michaelis–Menten equation with
K¢
m
= 4.5 ± 0.9 mm and V¢
max
= 7.8 ± 0.6 lmÆmin
)1
(Fig. 5B). The k
cat
value for the reaction of KATII with
ESBA was 9 min
)1
, only about 2.5-fold lower than the
value of 25 min
)1
for the reaction with KYN.
She rate of b-elimination was determined by moni-
toring the formation of ammonia as a function of time
for a solution containing KATII, 8 mm ESBA, and
12 mm KG. The reaction was linear within 180 min,

with a slope of 2.5 lmÆmin
)1
ammonia (e.g. the specific
activity was 25 pmolÆlg
)1
Æmin
)1
). This rate is expected
to be a lower limit, because, for substrates with poor
leaving groups, the transamination reaction, in the
presence of 2-oxo acids, is favored with respect to the
b-elimination reaction. As a comparison, the reaction
of KATII with 5 mm BCA gave a specific activity of
5 nmolÆlg
)1
Æmin
)1
, indicating that ESBA is a poor
substrate for b-elimination.
Scheme 4. Proposed mechanism for the reaction of KATII with BCA and the syncatalytic inactivation at the stage of the a-aminoacrylate
intermediate. X is a nucleophilic amino acid in the active site of the enzyme. Adapted from [72].
E. Passera et al. Reactivity of kynurenine aminotransferase
FEBS Journal 278 (2011) 1882–1900 ª 2011 The Authors Journal compilation ª 2011 FEBS 1889
We also determined whether ESBA or its reaction
products inactivated KAT II, as was observed with
BCA. A solution of KAT II (174 lm) was incubated
with 8 mm ESBA for 60 min at 25 °C. The reaction
was diluted 200-fold in an assay solution containing
10 mm KYN and 10 mm KG. KATII reacted with
ESBA was found to be two-fold less active than the

unreacted enzyme, suggesting that b-lytic activity of
ESBA leads to partial syncatalytic inactivation of the
enzyme. The mechanism of inhibition of ESBA on
Fig. 4. Reactivity of KATII towards CSA. (A) Reaction with KATII
monitored by absorption spectroscopy. The reaction mixture con-
tained 7 l
M KATII in 50 mM Hepes (pH 7.5) (solid line) at 25 °C, in
the presence of 1.8 m
M CSA. Spectra were taken 5 min (dotted
line), 10 min (short dashed line), 15 min (dash-dotted line) and
60 min (long dashed line) after the addition of CSA. (B) Determina-
tion of the mechanism of inhibition. The inhibitory effect of CSA on
KATII was determined by monitoring the rate of reaction in a mix-
ture containing 870 n
M KATII in 50 mM Hepes (pH 7.5) in the pres-
ence of 10 m
M KG, 40 lM PLP, and concentrations of KYN from
2.5 to 10 m
M. The reaction was carried out at 25 °C in 0.1-cm path-
length cuvettes, either in the absence (closed circles) or the pres-
ence of 4 m
M (open squares) and 20 mM CSA (open triangles). The
solid lines through data points represent global fitting to Eqn (4)
with V
max app
= 0.025 ± 0.001 mMÆmin
)1
, K
m app
= 12.3 ± 1.5 mM,

and K
ii
= 17.2 ± 3.5 mM.
Fig. 5. Reactivity of KATII towards ESBA. (A) Absorption spectra
recorded for a solution containing 8 l
M KATII in 50 mM Hepes
(pH 7.5) (solid line) at 25 °C in the presence of 100 l
M ESBA
(dashed dotted line) after 11 min from reaction start and at equilib-
rium ($ 3 h) upon addition of 10 m
M KG (dotted line; the spectrum
has been divided by 2). A spectrum of a solution containing 100 l
M
ESBA in 50 mM Hepes (pH 7.5) is shown for comparison (dashed
line). (B) Dependence of the rate of reaction of KATII on ESBA con-
centration in the presence of KG. The reaction mixture contained
870 n
M KATII in 50 mM Hepes (pH 7.5) in the presence of 10 mM
KG and variable concentrations of ESBA. The reaction was carried
out at 25 °C in 0.1-cm pathlength cuvettes. The solid line through
data points represents fitting to the Michaelis–Menten equation
with V
max
= 7.8 ± 0.6 lMÆmin
)1
and K
m
= 4.5 ± 0.9 mM.
Reactivity of kynurenine aminotransferase E. Passera et al.
1890 FEBS Journal 278 (2011) 1882–1900 ª 2011 The Authors Journal compilation ª 2011 FEBS

KATII could not be determined, owing to the interfer-
ence of the ESBA spectrum with the spectroscopic sig-
nals used to monitor KATII activity. However,
inhibition parameters were further evaluated by an
endpoint assay (see below).
A 96-well plate assay for high-throughput
screening of KATII inhibitors
Because KATII is a potential target for schizophrenia
and other neurological disorders, a high-throughput
screening assay was developed to identify KATII inhib-
itors, and implemented on a 96-well plate format. The
assay is based on the determination of the endpoint
absorbance intensity at 500 nm, generated from the
coupled enzymatic reactions of glutamate oxidase and
peroxidase in the presence of o-dianisidine, acting on
glutamate produced in the transamination of AAD or
other substrates in the presence of KG. This assay is
well suited to monitor the transamination of potential
substrates and the inhibition caused by the screened
compounds. The results of a typical assay are shown in
Fig. 6. Incubation of a mixture containing 2.2 lm
KATII and 10 mm KYN for 30 min led to the forma-
tion of 810 ± 91.9 lm glutamate; that is, 8.1 ± 0.9%
of KYN was transaminated within the incubation time.
When the reaction was carried out in solution and the
transamination was determined directly by the absorp-
tion intensity of KYNA (see above), the same degree
of KYN transamination was measured. The transami-
nation reaction in the presence of 10 mm AAD
(Fig. 6) generated a higher amount of glutamate

(1.1 ± 0.0997 mm), owing to the higher catalytic effi-
ciency of KATII towards AAD than to KYN [41]. A
mixture of 1 mm ESBA, 200 mm CSA and 2 mm
O-phosphoserine (OPS) gave measurable levels of
transamination, which were approximately 8 ± 0.78%,
1.4 ± 0.07% and 41 ± 4.2%, respectively, of the level
of transamination with AAD (Fig. 6B). Transamina-
tion in the presence of either 5 mm BCA or 50 mm
3-nitropropionic acid (3-NPA) was found to be negligi-
ble (Fig. 6B). Furthermore, the assay allows identifica-
tion of compounds that inhibit KATII activity. It was
found that the presence of either 1 mm or 100 lm
ESBA led to 71 ± 4.9% and 63 ± 1.4% KATII activ-
ity inhibition, respectively (Fig. 6B), in good agreement
with data previously obtained (64% inhibition at 1 mm
ESBA) [57]. CSA, BCA, 3-NPA and OPS inhibition of
KATII was also measured (Fig. 6B), and found to be
in good agreement with data reported in the literature,
showing an IC
50
value of approximately 2 lm for CSA
[59], and inhibition of 24% and 38% with 5 mm
3-NPA [61] and 1 mm OPS [60], respectively.
Fig. 6. Ninety-six-well plate assay for substrates and inhibitors of
KATII. (A) Representative 96-well plate assay. Each reaction well
contained 10 m
M KG, 40 lM PLP and 50 mM Hepes (pH 7.5) at
25 °C. Reactions were allowed to proceed for 30 min, and stopped
with phosphoric acid to a final concentration of 14 m
M. A solution

containing 0.75 m
M o-dianisidine, 0.015 U of GOX and 2.25 U of
peroxidase was then added to the reaction mixture. The reaction
was allowed to develop for 90 min at 37 °C, and stopped with
3.66
M sulfuric acid. Each reaction well was duplicated (odd and
even lines). Wells in lines 1 and 2 were used to construct a calibra-
tion curve, with the following glutamate concentrations: 0 (a),
10 l
M (b), 50 lM (c), 100 lM (d), 200 lM (e), 400 lM (f), and 800 lM
(g). The effect of the tested molecules on the KAT reaction is
shown in lines 3 and 4. Each well contained 400 l
M glutamate and
10 m
M KYN (a), 10 mM AAD (b), 1 mM ESBA (c), 200 lM CSA (d),
5m
M BCA (e), 50 mM 3-NPA (f), and 2 mM OPS (g). Wells in lines
5 and 6 are blanks containing only tested molecules at the higher
concentration. The transamination activity of 10 m
M KYN (a),
10 m
M AAD (b), 1 mM ESBA (c), 200 lM CSA (d), 5 mM BCA (e),
50 m
M 3-NPA (f) and 2 mM OPS (g) in the presence of 2.2 lM
KATII is shown in lines 7 and 8. In lines 9–12, each molecule was
tested for inhibition of the transamination reaction in the presence
of 10 m
M AAD and 2.2 lM KATII, with the following concentrations
of inhibitors: 1 m
M ESBA (a9–10), 100 lM ESBA (b9–10), 200 lM

CSA (c9–10), 20 lM CSA (d9–10), 5 mM BCA (e9–10), 500 lM BCA
(f9–10), 50 m
M 3-NPA (a11–12), 5 mM 3-NPA (b11–12), 2 mM OPS
(c11–12), and 200 l
M OPS (D11–12). (B) Transamination activity of
KATII in the presence of either AAD, KYN, ESBA, CSA, BCA,
3-NPA, and OPS (black bars), or AAD, ESBA, CSA, BCA, 3-NPA,
and OPS (red bars), at the concentrations shown in the figure. The
activities are expressed as a percentage of the degree of transami-
nation measured in the presence of 10 m
M AAD.
E. Passera et al. Reactivity of kynurenine aminotransferase
FEBS Journal 278 (2011) 1882–1900 ª 2011 The Authors Journal compilation ª 2011 FEBS 1891
Discussion
Spectroscopic properties of KATII and the
Tyr142 fi Phe mutant
The absorption spectra of KATII and Tyr142 fi Phe
KATII show a band at 360 nm that, on the basis of
previous studies on aminotransferases [83–85], is
attributed to a Schiff base of the active site lysine (in
KATII, Lys263) with a deprotonated imine nitrogen
(Fig. 1A; Scheme 3, species 1a). A previous study on
bovine and rat KAT II reported absorption spectra
with two main peaks at 320–330 nm and 400 nm [86].
Furthermore, KATI shows an absorption spectrum
with two bands centered at 335 nm and 422 nm,
indicative of a mixture of the PLP and PMP forms
of the enzyme or of enolimine and ketoenamine tau-
tomers of the internal aldimine of PLP [35]. PLP is a
probe of the active site environment, and the tauto-

meric distribution therefore reflects the active site
polarity. The deprotonated form of the internal aldi-
mine of PLP is typical of aminotransferases, e,g,
AspAT [87]. It is well established that the deprotonat-
ed form of PLP in transaminases is stabilized by a
hydrogen bond with the hydroxyl group of a tyrosine
that lowers the pK
a
of the imine nitrogen by two
orders of magnitude [87–89]. On the basis of the
KATII structure [52], Tyr233 is the conserved tyro-
sine that plays this stabilizing role. In aminotransfe-
rases, the deprotonated species is in equilibrium with
a protonated form that can be present as either the
enolimine tautomer, absorbing at $ 330 nm, or the
ketoenamine tautomer, absorbing at 420 nm
(Scheme 3, species 1b). The pK
a
of this equilibrium is
6.3 for AspAT [84]. The pK
a
of the protonation equi-
librium of the internal aldimine of KATII was not
determined, owing to enzyme instability, but was esti-
mated to be lower than 6.
PLP is an intrinsic fluorescent probe that is often
exploited in vitamin B
6
-dependent enzyme spectros-
copy, because of its sensitivity to the environment sur-

rounding the cofactor, and thus to changes in the
conformation and ligation state of the active site [90–
92]. Several studies have investigated the function and
dynamics of aminotransferases with fluorescence tech-
niques [67,68,93–100]. Unlike those of other PLP-
dependent enzymes [91], the emission spectra of KATII
and Tyr142 fi Phe KATII do not show any energy
transfer between tryptophans and the cofactor
(Fig. 1B). Analysis of the KATII three-dimensional
structure [52] reveals that three tryptophans are within
30 A
˚
of the cofactor, so the absence of energy transfer
is an indication of an unfavorable orientation between
tryptophan rings and PLP. The deprotonated internal
aldimine, absorbing at 360 nm, is fluorescent, with a
structured emission showing a maximum at 420 nm
and a shoulder at about 500 nm (Fig. 2C). This is in
agreement with findings on model PLP Schiff bases
and AspAT [67]. Only the emission at 420 nm is
directly attributed to the fluorescence emission of the
deprotonated internal aldimine; the band at 500 nm
might result from direct excitation of the band absorb-
ing at 420 nm. This mechanism is confirmed by the
emission spectrum upon excitation at 420 nm, which is
centered at 500 nm. Excitation at 330 nm gives a struc-
tured emission with a shoulder at about 380 nm, which
might originate from the excitation of residual PMP
[67], and a band centered at about 420 nm, which origi-
nates from the excitation of the deprotonated internal

aldimine band.
Development of activity assays for KATII
KAT activity has usually been assayed by a cumber-
some discontinuous method coupled with HPLC deter-
mination or liquid scintillation spectrometry of KYNA
[41,56–58,69]. The development of a continuous assay
for KATII with KYN as substrate has been hampered
by its suboptimal spectral properties. In fact, KYN
strongly absorbs at 361 nm (e = 4350 m
)1
Æcm
)1
) [71],
which does not allow for assays at concentrations
higher than 2 mm, the estimated K
m
of the enzyme
being about 5 mm [41]. Our strategy for the develop-
ment of a continuous assay was based on the observa-
tion that only at 310 nm is the absorbance of KYN low
enough to allow assays with up to 23 mm KYN in 0.1-
cm pathlength cuvettes. At the same time, at 310 nm
the absorbance difference between KYN and KYNA is
significant, making transamination time courses easily
detectable. Kinetic traces collected at 310 nm were lin-
ear over the time needed to calculate initial velocities
(about 10 min), and showed no lag or burst phases. The
extinction coefficient used to calculate V
0
was

3625 m
)1
cm
)1
; that is, for each molecule of KYN con-
sumed in the reaction (1049 m
)1
cm
)1
), one molecule of
KYNA (4674 m
)1
cm
)1
) is produced. The amount of
KYN consumed during the linear portion of the kinet-
ics is less than 5%, thus fulfilling the requirements for
steady state. Furthermore, this assay allows for the
determination of V
0
with a range of substrate concen-
trations, from 0.3 to more than 2.5 K
m
, that is accept-
able for accurate determination of V
max
and K
m
[101].
We also checked that the concentration of KG was sat-

urating under our experimental conditions. We esti-
mated K
m
KG
to be lower than 20 lm. At saturating KG
concentrations, V¢
max
is equal to V
max
, which has a
Reactivity of kynurenine aminotransferase E. Passera et al.
1892 FEBS Journal 278 (2011) 1882–1900 ª 2011 The Authors Journal compilation ª 2011 FEBS
value of 0.022 mmÆmin
)1
, and K¢
m
is equal to aK
m
KYN
,
which has a value of 10 mm, where a = k
4
⁄ k
2
.
In many cases [102], the rate-limiting step for trans-
aminases is the a-proton abstraction (Scheme 3); thus,
k
4
> k

2
, and K¢ represents an upper limit to K
m
KYN
.
Consequently, K
m
KYN
is in the millimolar range, in
agreement with a previous study on KATII [41], where
a value of $ 5mm was reported. The k
cat
value, which
we found to be about 110 min
)1
, is in good agreement
with the value previously reported, $ 134 min
)1
[41].
The difference is probably associated with the less pre-
cise determination of enzyme active sites with the
Bradford method than with cofactor release by alkali
denaturation, a method that is accepted to be the most
accurate for PLP-dependent enzymes. We should
also point out that a continuous assay is usually more
precise than a discontinuous assay, especially when
the concentration of the product has to be determined
via HPLC analysis. In summary, the continuous
assay allows the fast determination of enzyme activ-
ity, and, more importantly, of inhibition mechanisms

(see below). As a further advantage, the amount of
enzyme needed for each assay is low, about 9 lg,
which is compatible with the low yields of KATII
expression.
The high-throughput search for specific and potent
enzyme inhibitors takes great advantage of fast, sim-
ple, cheap and reliable activity assays. The 96-well
plate assay that we have developed for KATII allows
fast evaluation of libraries of compounds via an initial
visual inspection. The assay can also be exploited for
the quantitative comparison of substrate preference
and inhibitor selectivity with different isoforms of the
KAT superfamily. The assay is very sensitive and is
linear in an 80-fold concentration range of glutamate
(between 10 and 800 lm). Furthermore, very low
amounts of enzyme are used for the assay. The assay
was optimized for $ 20 lg of KATII per assay and an
incubation of 30 min. With increases in the incubation
time, the amount of enzyme can be proportionally
decreased. Additionally, the assay works with both
AAD and KYN, which makes it specific for KATII.
In fact, one of the main drawbacks of the discontinu-
ous KATII assay is the lack of isoform specificity
[103]. Unlike KYN, which can be used as the amino
group donor by all four known KAT isoenzymes,
AAD is efficiently used only by KATII [41,103].
Reactivity towards natural and non-natural substrates
KATII was previously indicated to be an ADD ami-
notransferase. Indeed, the catalytic efficiency of the
enzyme towards AAD is slightly higher than that

towards KYN [41]. The reaction of KATII with
AAD in the absence of ketoacids leads to the trans-
amination of PLP to form PMP (Fig. 1A), which
accumulates in the presence of KG. Thus, at equilib-
rium, the PMP form of KATII is the most stable
enzyme form; that is, the rate of its formation is
higher than the rate of its consumption. The high
absorbance of KYN in the spectral range where PLP
and PMP intermediates also absorb hampers perfor-
mance of the same analysis for the reaction between
KYN, KG, and KATII.
PLP-dependent enzymes are reported to be quite pro-
miscuous, with respect to both substrate and the types
of reaction catalyzed. It is known that many PLP-
dependent enzymes, including tryptophan synthase
[104], 3,4-dihydroxyphenylalanine decarboxylase [105],
AspAT [106], and serine racemase [107,108], can cata-
lyze side reactions, some of which have been suggested
to play physiological roles [107,108]. In particular, am-
inotransferases are known to be (relatively) efficient in
catalyzing b-elimination reactions, especially on sub-
strates with good leaving groups, such as BCA and,
more interestingly, on S-substituted cysteine derivatives
[42,43,72,73,85,109,110]. In the case of KATII, the
exploration of b-lyase activity was particularly intrigu-
ing, in that structural comparisons suggested the pres-
ence of features normally observed in b-lyases, such as
the conformation of the N-terminal region and a tyro-
sine at position 142, which, in most aminotransferases,
is occupied by a tryptophan or a phenylalanine [52].

Thus, we investigated the b-lytic activity of KATII and
Tyr142 fi Phe KATII with BCA and SPC. Whereas
the Tyr142 fi Phe mutation does not change the reac-
tivity of KATII towards AAD and KYN in the pres-
ence of KG, it heavily influences the b-lytic activity of
the enzyme. In fact, both the wild type and the mutant
are able to eliminate chloride from BCA, with the pro-
duction of ammonia and pyruvate, but the
Tyr142 fi Phe mutant is 20-fold less efficient. One pos-
sible explanation is that the tyrosine at position 142
plays a role in the balance between b-elimination and
transamination. In the conditions tested here, we could
E. Passera et al. Reactivity of kynurenine aminotransferase
FEBS Journal 278 (2011) 1882–1900 ª 2011 The Authors Journal compilation ª 2011 FEBS 1893
not reach saturation in a plot of initial velocity against
BCA concentration. This is consistent with the observa-
tion that K
m
values of transaminases for BCA are usu-
ally very high, preventing the determination of kinetic
parameters. This also hampers the calculation of cata-
lytic efficiency for chloride elimination by KATII and
Tyr142 fi Phe KATII. For this reason, we cannot rule
out the possibility that the lower rate of b-elimination
observed for Tyr142 fi Phe KATII is partly attribut-
able to a higher K
m
value. Although a detailed charac-
terization of the b-lytic activity of this mutant is outside
the scope of this work, we checked the b-lytic activity

of wild-type KATII and the mutant enzyme in the pres-
ence of SPC. Also in this case, we observed b-elimina-
tion with production of ammonia by the wild-type
enzyme but no activity of the Tyr142 fi Phe mutant, a
further indication of a reduction of b-lytic activity
brought about by the mutation. Interestingly, the
b-elimination, although with a very low ratio with
respect to transamination, also takes place on the natu-
ral substrate KYN. This unusual b-elimination reaction
should produce o-aminobenzaldehyde, as already
reported in a controversial paper on the activity of ky-
nureninase [111]. At present, it is not known whether
this reaction has any physiological significance or is reg-
ulated by any effector, as is the case, for example, with
the mammalian serine racemase [107,108]. However,
one should bear in mind that the b-elimination reaction
requires the formation of an a-aminoacrylate intermedi-
ate that, in the case of KATII, as demonstrated by
experiments with BCA, leads to concomitant syncata-
lytic inactivation of the enzyme. Furthermore, it is unli-
kely that a very inefficient reaction, when compared to
the main one, would have any physiological signifi-
cance, unless it is tuned by effectors and ligands. The
understanding of this aspect of the KATII mechanism
of action is beyond the aim of this work, but deserves
further attention.
As expected on the basis of the ESBA structure,
KATII catalyzes both transamination and b-elimina-
tion of this compound. A rough estimate of the cata-
lytic efficiency of the b-elimination reaction, based on

specific activity at a fixed substrate concentration, indi-
cates that ESBA, like KYN, is a poor substrate for
b-elimination, when compared with BCA. However,
both ESBA and BCA are capable of permanently inac-
tivating KATII through a mechanism that probably
involves the formation of a covalent adduct between
the active site lysine and the a-aminoacrylate interme-
diate, as already reported for alanine aminotransferase
(AlaAT) [72] and AspAT [73] (Scheme 4). We were
unable to recover a PLP–ESBA derivative, either after
ultrafiltration or after gel filtration on microspin col-
umns (data not shown). This hampered the determina-
tion of the type of modification by MS. The
dependence of the percentage of initial activity of
KATII on time of incubation in the presence of 5 mm
BCA (data not shown) gives an exponential decay with
k
obs
= 0.7 min
)1
(t
1 ⁄ 2
$ 1 min). In the case of AlaAT,
the pseudo-first-order rate constant for inactivation
was 0.36 min
)1
, with t
1 ⁄ 2
$ 2 min, in the presence of
5mm BCA [72]. The partition ratio (moles of product

per mole of inactivated enzyme) is about 500, a value
comparable with those found for AlaAT, 1050 [72]
and for kynureninase, 530 [112].
ESBA and CSA – inhibitors and/or substrates?
Because of the potential role of KATII as a drug tar-
get in the treatment of psychiatric disorders, such as
schizophrenia, many efforts have been made to design
selective inhibitors. However, only a few molecules
have been proved to inhibit KATII activity: CSA on
brain slices of rats [59], ESBA in reverse dialysis exper-
iments on rat hippocampus [55], MPP
+
and 3-NPA
on both cortical brain slices and partially purified
KAT [61], and BFF-122 [56] (Scheme 2). Although
MPP
+
was shown to be able to discriminate between
KATI and KATII, stimulating the design of isoform-
specific inhibitors, the use of this compound triggers
Parkinsonian symptoms [113]. Thus, at present, ESBA
and BFF-122 are the only available specific and potent
KATII inhibitors [57]. Moreover, ESBA was found to
be pharmacologically active on rats but almost inactive
on humans [55,57]. It has been supposed that this dif-
ference in inhibitory activity may arise from the pres-
ence in the catalytic site of human KATII of two
hydrophobic residues, Leu40 and Pro76, which are
replaced by polar serines in the rat enzyme [57].
Recently [114], a human KATII double mutant har-

boring the serines characterizing the rat ortholog active
site was generated in order to investigate the molecular
basis for ESBA species specificity. The site-directed
mutagenesis approach did not provide any experimen-
tal support to explain the striking difference in ESBA
inhibitory efficiency towards rat and human KATII,
and underlined the need for more in-depth biochemical
investigations aimed at deciphering the mechanism of
ESBA inhibition. The mechanisms of ESBA and CSA
inhibition have not been previously investigated. We
have demonstrated that both CSA and ESBA are sub-
strate analogs, and not purely competitive inhibitors.
In the case of ESBA, the mechanism of inhibition
could not be determined, owing to its strong interfer-
ence with almost all of the assays that we used. How-
ever, the spectroscopic signal generated by the
Reactivity of kynurenine aminotransferase E. Passera et al.
1894 FEBS Journal 278 (2011) 1882–1900 ª 2011 The Authors Journal compilation ª 2011 FEBS
accumulation of the product of the reaction of ESBA
with KAT was exploited to calculate the kinetic
parameters and, thus, an approximate affinity of
ESBA for the enzyme. Although the identity of the
product could not be assessed by MS, it seems likely,
from its spectroscopic properties, that it represents the
a-ketoacid generated by ESBA transamination. An
apparent K
m
of 4.5 mm is in good agreement with
64% inhibition at 1 mm ESBA [57].
CSA is a physiological substrate of AspAT

[115,116], owing to its structural similarity with aspar-
tate. Inhibition of KATII by CSA was determined to
be uncompetitive, a quite unexpected finding, consider-
ing that CSA is also a substrate of KATII, converting
the PLP form of the enzyme to the PMP form.
Uncompetitive inhibition involves the exclusive (or
predominant) binding of the inhibitor to the enzyme–
substrate complex or to any intermediate downstream
of it. In the case of CSA, uncompetitive inhibition
arise from preferential binding of CSA to the PMP
form of KATII, suggesting that CSA might mimic KG
better than KYN or AAD.
In conclusion, although both CSA and ESBA show
good inhibitory properties on KATII, with, at least
for CSA, inhibition constants in the micromolar
range, these molecules are actually substrates. New
hints for the development of a KATII-specific inhibi-
tor come from the recent observation that large mole-
cules with bulky substituents specifically bind to the
II-isoform of the enzyme [61,62], probably as a conse-
quence of the higher mobility of the N-terminal
domain of KATII than those of other aminotransfe-
rases [62]. We believe that these observations, together
with the tools developed here for the high-throughput
screening of KATII-specific inhibitors, will aid in the
development of anti-schizophrenic and precognitive
drugs.
Experimental procedures
Chemicals
All chemicals were purchased from Sigma–Aldrich (St Louis,

MO, USA), and were used as received. ESBA was synthe-
sized as previously described [55,57]. Experiments, unless
otherwise specified, were carried out in 50 mm Hepes buffer
(pH 7.5) at 25 °C.
Protein expression and purification
Human KATII and the mutant Tyr142 fi Phe were
expressed and purified as previously described [52]. The
enzyme was fully saturated with PLP by addition of a 10-
fold molar excess of cofactor, followed by extensive dialysis
against a solution containing 20 mm Hepes and 50 mm
NaCl (pH 8.0), and stored in small aliquots at )80 °C.
Spectroscopic measurements
Absorption spectra were collected with a Cary 400 spectro-
photometer (Varian, Cary, NC, USA). Spectra were cor-
rected for buffer contributions. The extinction coefficients
of wild-type KATII and Tyr142 fi Phe KATII were calcu-
lated from the amount of PLP released upon alkali dena-
turation, by the method of Peterson [64].
Fluorescence emission spectra were collected with a Spex
Fluoromax-2 fluorimeter (Jobin-Yvon, North Edison, NJ,
USA) by exciting the emission of tryptophans at 298 nm or
the emission of the cofactor at 330, 360 and 420 nm. Excita-
tion and emission slits were set at 1.5 nm (k
ex
= 298 nm),
3.5 nm (k
ex
= 330 nm and k
ex
= 360 nm), and 4 nm (k

ex
=
420 nm). Spectra were corrected for buffer contributions.
Assays
KAT activity
A continuous assay for the measurement of transamination
of KYN by KATII with KG as the acceptor was developed
(see Results). KYN and KYNA concentrations were
calculated from the absorbance in the UV–visible range,
using published extinction coefficients [71] (for KYN, e
257 nm
= 6750 m
)1
Æcm
)1
, and e
361 nm
= 4350 m
)1
Æcm
)1
; for KYNA,
e
332 nm
= 9800 m
)1
Æcm
)1
, and e
344 nm

= 7920 m
)1
Æcm
)1
).
b-Lytic activity
b-Elimination reactions carried out by aminotransferases
involve formation of an a-aminoacrylate intermediate that
rapidly decomposes to pyruvate and ammonia. Pyruvate
accumulation was measured at 220 nm. Time courses were
monitored at 25 °C in 0.1-cm pathlength cuvettes. The
assay was carried out in 100 mm phosphate buffer
(pH 7.5), to minimize the background signal at 220 nm.
Nessler’s assay
The ammonia concentration formed in reaction mixtures
was determined with Nessler’s assay [117], using a ready-to-
use solution (Fluka, Buchs, Switzerland; code 72190). At
pH 7.5, 98% of ammonia is in the NH
4
+
form, and the
solubility of NH
3
in water at 25 °C is about 50% (w ⁄ w);
thus, no significant loss of ammonia by evaporation was
expected under these experimental conditions. A calibration
curve was constructed with ammonium sulfate concentra-
tions in the range 25 lm to 4 mm. Forty microliters of a
solution containing ammonia was diluted with 860 lLof
water. One hundred microliters of Nessler’s reagent were

E. Passera et al. Reactivity of kynurenine aminotransferase
FEBS Journal 278 (2011) 1882–1900 ª 2011 The Authors Journal compilation ª 2011 FEBS 1895
added to the mixture, and the absorbance of the solution
was immediately recorded at 436 nm.
KAT activity measured with a GOX-coupled assay
A reaction mixture containing 10 mm AAD or 10 mm
KYN, 10 mm KG, 40 lm PLP, 2.3 lm KATII and 50 mm
Hepes (pH 7.5) was incubated at 25 °C for 30 min. The
reaction was stopped by addition of 14 mm phosphoric
acid. Then, 0.015 units of GOX (GOX-Sigma G5921,
St. Louis, MO, USA), 2.25 units of peroxidase (perox-
Sigma P8375) and 0.75 mm o-dianisidine were added to the
solution. The mixture was incubated at 37 °C for 90 min.
Sulfuric acid (3.36 mm) was added, and the absorbance
was measured at 530 nm. This assay was adapted to a
96-well plate format, with absorbance intensities being
measured at 500 nm with a home-made plate reader.
Blanks contained the same reagents as for the assay, except
that KATII solution was replaced by the same volume of
buffer. A calibration curve was constructed with known
concentrations of glutamate, ranging from 10 to 800 lm,in
the presence of 10 mm KG, 40 lm PLP, and 14 mm phos-
phoric acid. Inhibition assays were set up, with either CSA,
BCA, ESBA, 3-NPA or OPS being added to the mixture.
CSA, CSA, 3-NPA, OPS and ESBA at the same concentra-
tions used in the inhibition assay were also tested for trans-
amination activity in the presence of 10 mm KG.
Data fitting
Data fitting was carried out with sigma plot software,
release 9.0. Initial velocities as a function of KYN concen-

tration at a constant KG concentration (10 mm) were fitted
to a hyperbolic equation:
V
0
¼
V
0
max
Á KYN½
K
0
þ KYN½
ð1Þ
where:
V
0
max
¼
V
max
Á KG½
K
KG
m
þ KG½
ð2Þ
and
K
0
¼

aK
KYN
m
Á KG½
K
KG
m
þ KG½
ð3Þ
where V¢
max
and K¢ are apparent V
max
and K
m
, a is k
4
⁄ k
2
(see reaction mechanism in Discussion), and K
m
KYN
and
K
m
KG
are the Michaelis constants for KYN and KG, respec-
tively [101].
The parameters for the reaction of KATII in the presence
of CSA were determined by globally fitting data to Eqn (4):

V
0
¼
V
max appÁ
½KYN
K
m app
þ½KYNÁ 1 þ
½CSA
K
ii

ð4Þ
where K
m app
and V
max app
are apparent K
m
and apparent
V
max
, and K
ii
is the dissociation constant for dissociation of
the inhibitor from the enzyme–substrate complex. Because
the KYN concentration used in the assay is close to K
m
,an

approximated value of K
i
can be obtained from the following:
K
i
¼
K
ii
2 Á 1 þ
KG½
K
KG
m

ð5Þ
Acknowledgements
This work was supported by a grant from MIUR (CO-
FIN to A. Mozzarelli).
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