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

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 Tyr142fi Phe.. The transamination an

substrates and inhibitors

Elisabetta Passera1, Barbara Campanini1, Franca Rossi2, Valentina Casazza2, Menico Rizzi2, Roberto Pellicciari3and Andrea Mozzarelli1,4

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: andrea.mozzarelli@unipr.it

(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 Tyr142fi 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.

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 Ca2+ 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.

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 extdiffer-ents, 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 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-eliminatransamina-tion in the presence of substrates and substrate analogs of recombinant human KATII and a variant carrying the Tyr142fi 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].

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

Tyr142fi 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 A280 nm⁄ A360 nm

ratio was 5 The extinction coefficient calculated by the

method of Peterson [64] was found to be

9510 m)1Æcm)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

Tyr142fi Phe KATII exhibited an absorption

spec-trum that was almost superimposable on that of the

wild-type enzyme, with an invariant A280 nm⁄ A360 nm

ratio (data not shown) Because the extinction

coeffi-cient of the cofactor at 360 nm was found to be

9400 m)1Æcm)1, this invariant ratio can be explained by

a concomitant decrease in the extinction coefficient at

280 nm resulting from the Tyrfi 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 m M 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 m M Hepes (pH 7.5) at 25 C, excited at 298 nm (C) Emission spectra of a solution containing

26 l M KATII and 50 m M Hepes (pH 7.5) at 25 C, excited at

330 nm (continuous line), 360 nm (dotted line), and 420 nm (dash-dotted line).

(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 Tyr142fi 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, 37C) 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)1Æcm)1) to allow for initial velocity determinations at KYN concentrations higher than 8 mm, the published Km 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 Km The extinction coefficients at 310 nm for KYN and KYNA were calculated to be 1049 m)1Æcm)1 and

4674 m)1Æcm)1, respectively, with a De at 310 nm of

3625 m)1Æcm)1 Under these conditions, reactions were carried out as a function of KYN concentration between 2 and 23 mm, in the presence of 10 mm KG (Fig 2B) This KG concentration was assumed to be saturating on the basis of the previously determined

Km for KG of 1.2 mm [41] Control measurements showed that V0 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 kcatof

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].

the presence of 200 mm potassium phosphate, 5 mm

KG, and 0.04 mm PLP (pH 7.5, 45C) (116), we

assayed the enzyme under the same experimental

con-ditions The continuous assay gave a Km(mm) of 2.09

and a kcat (min)1) of 110 The discontinuous assay gave a Km(mm) of 0.96 and a kcat(min)1) of 186 The

kcat 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 kcat (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 Tyr142fi 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) elimina(transamina-tion 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 Tyr142fi 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 Tyr142fi 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 l M PLP, 900 l M

KYN and 50 m M 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 m M Hepes, 10 m M KG, and 40 l M 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 m M Æmin)1and K ¢ m = 10 ± 1 m M Inset: the

reaction was carried at 10 m M KG (closed circles), 2 m M KG (open

triangles), 0.2 m M KG (open squares), and 0.02 m M KG (open

dia-monds).

presence of BCA (Fig 3B) allowed determination of

specific activities of 5 nmolÆlg)1Æmin)1 and 0.22

nmo-lÆlg)1Æmin)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 25C, 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 Tyr142fi 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)1Ælg)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 m M

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 n M Tyr142 fi Phe KATII (dotted black

line) and 5 m M BCA and 100 m M K2PO4(pH 7.5) at 25 C The solid

dashed lines represent fitting to linear equations with slopes of

17 l M Æmin)1and 0.74 l M Æmin)1for KATII and Tyr142 fi Phe KATII,

respectively.

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 Tyr142fi 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 Vmaxof 0.025 ± 0.001 mmÆmin)1, an

apparent Km of 12.3 ± 1.5 mm, and a Kii of

17.2 ± 3.5 mm The corresponding Ki, calculated from

Eqn (5), was 13 lm The IC50 value reported from

in vivoexperiments 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)1cm)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)1Æcm)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 kcatvalue for the reaction of KATII with ESBA was 9 min)1, only about 2.5-fold lower than the value of 25 min)1for 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)1ammonia (e.g the specific activity was 25 pmolÆlg)1Æmin)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)1Æmin)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].

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 25C 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 m M 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 inhibiDetermina-tion 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 m M Hepes (pH 7.5) in the

pres-ence of 10 m M KG, 40 l M 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 m M CSA (open triangles) The

solid lines through data points represent global fitting to Eqn (4)

with Vmax app= 0.025 ± 0.001 m M Æmin)1, Km app= 12.3 ± 1.5 m M ,

and K ii = 17.2 ± 3.5 m M

Fig 5 Reactivity of KATII towards ESBA (A) Absorption spectra recorded for a solution containing 8 l M KATII in 50 m M 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 m M 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 m M Hepes (pH 7.5) in the presence of 10 m M

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 Vmax= 7.8 ± 0.6 l M Æmin)1and Km= 4.5 ± 0.9 m M

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 IC50value 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 l M PLP and 50 m M 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 concentracalibra-tions: 0 (a),

10 l M (b), 50 l M (c), 100 l M (d), 200 l M (e), 400 l M (f), and 800 l M

(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 m M AAD (b), 1 m M ESBA (c), 200 l M CSA (d),

5 m M BCA (e), 50 m M 3-NPA (f), and 2 m M 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 m M ESBA (c), 200 l M CSA (d), 5 m M BCA (e),

50 m M 3-NPA (f) and 2 m M OPS (g) in the presence of 2.2 l M

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 l M KATII, with the following concentrations

of inhibitors: 1 m M ESBA (a9–10), 100 l M ESBA (b9–10), 200 l M

CSA (c9–10), 20 l M CSA (d9–10), 5 m M BCA (e9–10), 500 l M BCA (f9–10), 50 m M 3-NPA (a11–12), 5 m M 3-NPA (b11–12), 2 m M 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.