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: 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 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 [...]... 102, 10 3–1 11 Guidetti P, Okuno E & Schwarcz R (1997) Characterization of rat brain kynurenine aminotransferases I and II J Neurosci Res 50, 45 7–4 65 Han Q, Li J & Li J (2004) pH dependence, substrate specificity and inhibition of human kynurenine aminotransferase I Eur J Biochem 271, 480 4–4 814 Han Q, Robinson H, Cai T, Tagle DA & Li J (2009) Biochemical and structural properties of mouse kynurenine aminotransferase. .. aminotransferase III Mol Cell Biol 29, 78 4–7 93 Okuno E, Nakamura M & Schwarcz R (1991) Two kynurenine aminotransferases in human brain Brain Res 542, 30 7–3 12 Schmidt W, Guidetti P, Okuno E & Schwarcz R (1993) Characterization of human brain kynurenine aminotransferases using [3H ]kynurenine as a substrate Neuroscience 55, 17 7–1 84 Yu P, Li Z, Zhang L, Tagle DA & Cai T (2006) Characterization of kynurenine aminotransferase. . .Reactivity of kynurenine aminotransferase E Passera et al 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 [8 3–8 5], is attributed to a Schiff base of the active site lysine (in KATII, Lys263) with a deprotonated imine... known KAT isoenzymes, AAD is efficiently used only by KATII [41,103] Reactivity of kynurenine aminotransferase Reactivity towards natural and non-natural substrates KATII was previously indicated to be an ADD aminotransferase 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 transamination... reagent were 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- FEBS Journal 278 (2011) 188 2–1 900 ª 2011 The Authors Journal compilation ª 2011 FEBS 1895 Reactivity of kynurenine aminotransferase E Passera et al added to the mixture, and the absorbance of the solution... study on bovine and rat KAT II reported absorption spectra with two main peaks at 32 0–3 30 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 tautomers of the internal aldimine of PLP [35] PLP is a probe of the active site environment, and the tautomeric... surrounding the cofactor, and thus to changes in the conformation and ligation state of the active site [9 0– 92] Several studies have investigated the function and dynamics of aminotransferases with fluorescence techniques [67,68,9 3–1 00] Unlike those of other PLPdependent enzymes [91], the emission spectra of KATII and Tyr142 fi Phe KATII do not show any energy transfer between tryptophans and the cofactor (Fig... 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 isoformspecific 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]... aminotransferase III, a novel member of a phylogenetically conserved KAT family Gene 365, 11 1–1 18 FEBS Journal 278 (2011) 188 2–1 900 ª 2011 The Authors Journal compilation ª 2011 FEBS 1897 Reactivity of kynurenine aminotransferase E Passera et al 40 Fukui S, Schwarcz R, Rapoport SI, Takada Y & Smith QR (1991) Blood–brain barrier transport of kynurenines: implications for brain synthesis and metabolism... 200 7–2 017 41 Han Q, Cai T, Tagle DA, Robinson H & Li J (2008) Substrate specificity and structure of human aminoadipate aminotransferase ⁄ kynurenine aminotransferase II Biosci Rep 28, 20 5–2 15 42 Cooper AJ & Pinto JT (2006) Cysteine S-conjugate beta-lyases Amino Acids 30, 1–1 5 43 Cooper AJ, Pinto JT, Krasnikov BF, Niatsetskaya ZV, Han Q, Li J, Vauzour D & Spencer JP (2008) Substrate specificity of human . Human kynurenine aminotransferase II – reactivity with substrates and inhibitors Elisabetta Passera 1 , Barbara. 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