Báo cáo khoa học: pH dependence, substrate specificity and inhibition of human kynurenine aminotransferase I ppt

To determine the possible transam-ination activity of hKAT-I to other amino acids, a different amino acid at varying concentrations 0.1–32 mM was used to replace kynurenine and 16 mMa-ke

pH dependence, substrate specificity and inhibition of human

kynurenine aminotransferase I

Qian Han, Junsuo Li and Jianyong Li

Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

Human kynurenine aminotransferase I/glutamine

trans-aminase K (hKAT-I) is an important multifunctional

enzyme This study systematically studies the substrates of

hKAT-I and reassesses the effects of pH, Tris, amino acids

and a-keto acids on the activity of the enzyme The

experi-ments were comprised of functional expression of the

hKAT-I in an insect cell/baculovirus expression system,

purification of its recombinant protein, and functional

characterization of the purified enzyme This study

demon-strates that hKAT-I can catalyze kynurenine to kynurenic

acid under physiological pH conditions, indicates

indo-3-pyruvate and cysteine as efficient inhibitors for hKAT-I, and

also provides biochemical information about the substrate specificity and cosubstrate inhibition of the enzyme hKAT-I

is inhibited by Tris under physiological pH conditions, which explains why it has been concluded that the enzyme could not efficiently catalyze kynurenine transamination Our findings provide a biochemical basis towards understanding the overall physiological role of hKAT-I in vivo and insight into controlling the levels of endogenous kynurenic acid through modulation of the enzyme in the human brain Keywords: cysteine; indo-3-pyruvate; kynurenic acid; kynurenine aminotransferase; pH effect

In mammals, kynurenine aminotransferase I/glutamine

transaminase K (EC 2.6.1.64; KAT-I) is a multifunctional

enzyme In vitro, the enzyme catalyzes the transamination of

several amino acids (e.g glutamine, methionine, aromatic

amino acids including kynurenine) and also possesses

cysteine S-conjugate b-lyase activity (EC 4.4.1.13) [1]

Kynurenic acid (KYNA), the stable product derived from

the kynurenine transamination pathway [2–4], is involved in

several physiological aspects of the central nervous system

(CNS) by acting as an antagonist at both the

glutamate-binding site and the allosteric glycine site of the

N-methyl-D-aspartate receptor and possibly by blocking the 7-nicotinic

acetylcholine receptor [5–8] Low KYNA levels in the central

nervous system are correlated to cerebral diseases such as

schizophrenia and Huntington’s disease [9–13] Only two

pyridoxal 5¢-phosphate (PLP)-dependent aminotransferases

that are able to catalyze the transamination of kynurenine to

KYNA, arbitrarily termed KAT-I and II, have been

described in rat and human brains [14–16]

In addition, KYNA is involved in maintaining physio-logical arterial blood pressure In rats, the region of the rostral and caudal medulla in the CNS plays an important role in regulating cardiovascular function [17–20] Sponta-neously hypertensive rats that have higher arterial blood pressure were found to have significantly lower KAT activity and KYNA content in their rostral and caudal medulla than the control rats [20] Injection of KYNA into the rostral ventrolateral medulla of these rats significantly decreased their arterial pressure [21], which suggests that KYNA is involved in maintaining physiological arterial blood pressure Recently, the mutant KAT-I from all the strains of spontaneously hypertensive rats displayed altered kinetics; lower initial velocity and Kmfor both kynurenine and pyruvate [22] This mutation may explain the enhanced sensitivity to glutamate and nicotine seen in spontaneously hypertensive rats, suggesting it may be related to an underlying mechanism of hypertension and increased sen-sitivity to stroke [22] However, Cooper suggested that another mechanism, i.e the involvement of altered gluta-mine transamination and sulfur and aromatic amino acid metabolism should also be considered [1]

Although a number of studies described the characters of the enzyme ([1] and references therein), only an insect homologue, Aedes aegypti kynurenine aminotransferase [23] and a bacterium homologue [24] were systematically characterized using purified recombinant proteins To compare the characteristics of KAT-Is in different living organisms, determine substrate specificity, and evaluate the possible effect of other amino acids and keto acids on hKAT-I, we expressed the enzyme in a baculovirus/insect cell protein expression system Our large scale hKAT-I expression and subsequent purification enabled us to obtain

a large amount (mg range) of pure hKAT-I for extensive biochemical characterization Our results revealed some

Correspondence to J Li, Department of Pathobiology, University of

Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana,

IL 61802, USA Fax: +1 217 2447421, Tel.: +1 217 2443913,

E-mail: jli2@uiuc.edu

Abbreviations: AeKAT, Aedes aegypti kynurenine aminotransferase;

CNS, central nervous system; hKAT-I, human kynurenine

amino-transferase I; HTS, high-titre viral stocks; KYNA, kynurenic acid;

aKMB, a-keto-methylthiobutyric acid; PLP, pyridoxal 5¢-phosphate;

Sf9, Spodoptera frugiperda insect cells.

Enzymes: kynurenine aminotransferase I/glutamine transaminase K

(EC 2.6.1.64); cysteine 5-conjugate b-lyase (EC 4.4.1.13).

(Received 23 August 2004, revised 13 October 2004,

accepted 21 October 2004)

interesting biochemical characteristics of hKAT-I, which

have not been systematically addressed before For example,

the pH profile of hKAT-I exhibits high activity under

neutral conditions, which contrasts its reported pH profile

showing that the enzyme exhibited high activity only at

basic pH values [15,25] Tris buffer significantly inhibited

enzyme activity at neutral conditions, but showed no

inhibition under basic conditions, which might explain

why previous studies have reported that hKAT-I had

limited activity at physiological pH conditions and

displayed optimum activity at fairly basic conditions

Moreover, for the first time, we found that cysteine and

indo-3-pyruvate are effective inhibitors of the enzyme

in vitro Our data provide a better overall picture of the

enzyme and should be helpful in a comprehensive

under-standing of the role of hKAT-I, especially in KYNA

biosynthesis in the human brain

Experimental procedures

Enzyme expression and purification

Construction of recombinant transfer vectors The coding

sequence of hKAT-I was amplified from first strand human

liver cDNA (Clontech, Palo Alto, CA, USA) using a

specific forward (5¢-CTCGAGATGGCCAAACAGCTG

CAG) and reverse primer (5-AAGCTTAGAGTTCCAC

CTTCCACTT) containing a XhoI and a HindIII restriction

site (underlined sequence), respectively The PCR products

were cloned into a TOPO TA cloning vector and then

subcloned into a baculovirus transfer vector pBlueBac4.5

(Invitrogen, Carlsbad, CA, USA) Recombinant transfer

vectors were sequenced and confirmed to ensure that the

inserted DNA sequences were in frame

Production of recombinant baculoviruses Recombinant

pBlueBac4.5 transfer vectors were cotransfected with

linearized Bac-N-BlueTM Autographa californica multiple

nuclear polyhedrosis virus DNA in the presence of

InsectinPlusTMinsect cell-specific liposomes to Spodoptera

frugiperda(Sf9) insect cells (Invitrogen) The recombinant

baculoviruses were purified through the plaque assay

procedure Blue putative recombinant plaques were

trans-ferred to 12-well microtitre plates and amplified in Sf9 cells

Viral DNA was isolated for PCR analysis to determine the

purity of the recombinant viruses High-titre viral stocks

(HTS) for individual recombinant viruses were generated by

amplification in Sf9 cell suspension culture

Recombinant protein expression Sf9 insect cells were used

for protein expression The cells were cultured at 27C in an

Ultimate InsectTMserum-free medium (Invitrogen)

supple-mented with 10 unitsÆmL)1heparin (Sigma, St Louis, MO,

USA) in culture spinner flasks and constant stirring at

80 r.p.m When the cell density reached 2· 106cellsÆmL)1,

they were inoculated with the HTS of recombinant

baculoviruses at a multiplicity of infection of six viral

particles per cell

Purification of recombinant hKAT-I Sf9 cells in 2 L of

cell culture were harvested on the fourth day after

hKAT-I recombinant virus inoculation by centrifugation

(800 g for 15 min at 4C) and the cell pellets were dissolved in a lysis buffer containing 25 mM phosphate, 0.1 mM pyridoxal 5¢-phosphate (PLP), 2 mM dithiothre-itol, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and 150 mM NaCl with a final pH of 7.4 After incubation on ice for 30 min, cell lysates were centrifuged

at 18 000 g for 20 min at 4C and the supernatant was collected and assayed for KAT-I activity Soluble proteins in the supernatant were precipitated with 65% saturation of ammonium sulfate Protein precipitate was then redissolved in 10% saturated ammonium sulfate and applied to a phenyl sepharose column A linear gradient of ammonium sulfate from 10% to 0% in

10 mM sodium phosphate buffer (pH 7.0) was used for protein elution The hKAT-I active fractions were pooled, dialyzed and then separated by DEAE Sepharose chromatography with a linear NaCl gradient (0–500 mM)

in the same phosphate buffer The active fractions were collected, concentrated and then separated by a Super-dexTM 200 gel filtration column The hKAT-I active fractions were collected and concentrated The purity of the enzyme was assessed by SDS/PAGE analysis hKAT-I content was determined by a Bio-Rad (Hercules,

CA, USA) protein assay kit using bovine serum albumin

as a standard The concentration of hKAT-I stock solution was adjusted to 20 mgÆmL)1 in 20 mM phos-phate buffer (pH 7.5), aliquoted into 200 lL microcen-trifuge tubes with 10 lL in each and frozen at )80 C Biochemical characterization

hKAT-I activity assay All chemicals were purchased from Sigma Chemical Company unless otherwise specified hKAT-I activity assay was based on methods described in previous reports [26,27] Briefly, a typical reaction mixture

of 50 lL containing 5 mM kynurenine, 2 mM a-ketobuty-rate, 40 lMPLP and 2 lg hKAT-I was prepared using a

200 mM phosphate buffer, pH 7.5 The reaction mixture was incubated for 10 min at 45C, and the reaction was stopped by adding an equal volume of 0.8Mformic acid Supernatant was obtained by centrifugation of the reaction mixture at 15 000 g at 4C for 10 min and analyzed by HPLC-UV at 330 nm for KYNA The amount of KYNA formed in the reaction mixture was calculated based on a standard curve generated using authentic KYNA and the specific activity of the enzyme was expressed as lmolÆmin)1Æmg)1

Effect of buffer and pH on hKAT-I To determine the effect of pH on hKAT-I activity, a buffer mixture consisting

of 100 mMphosphate and 100 mMboric acid was prepared and the pH of the buffer was adjusted to 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, respectively The buffer mixture was selec-ted to maintain a relatively consistent buffer composition and ionic strength, yet have sufficient buffering capacity for relatively broad pH range A typical reaction mixture containing 5 mM kynurenine, 2 mM a-ketobutyrate, and

2 lg hKAT-I was prepared using the buffer mixture at different pHs The reaction mixture was incubated and analyzed as described in the hKAT-I activity assay Initial results showed that the specific activity of hKAT-I in the reaction mixture prepared in Tris buffer was much lower

than in the above buffer mixture To compare hKAT-I

activity in different buffers, 200 mMphosphate alone with

pH 6.0–8.0, and 200 mMTris alone with pH 7.5–8.5 were

prepared and used for hKAT-I activity assays under the

same conditions

Spectral analysis PLP has an absorption peak in the

visible region under neutral or weak basic conditions Its

visible peak shifts towards longer wavelengths when

associated with transaminase and diminishes upon

forma-tion of pyridoxamine after reacting with amino group

donors (the half reaction of the overall

transaminase-mediated reactions) The spectrum of hKAT-I in phosphate

buffer, pH 7.5, was analyzed using a Hitachi U2001

double-beam spectrophotometer and compared to the spectrum of

free PLP in the same buffer The same spectral analysis was

also used to evaluate the potential interaction of PLP and

Tris through comparison of the spectral characteristics of

PLP in Tris buffer at different pHs with those of PLP in

phosphate buffer at the same pH conditions

Substrate specificity To determine the possible

transam-ination activity of hKAT-I to other amino acids, a different

amino acid at varying concentrations (0.1–32 mM) was used

to replace kynurenine and 16 mMa-ketobutyrate used as an

amino group acceptor in the typical reaction mixture (50 lL

total volume prepared in 200 mMphosphate buffer, pH 7.5)

specified in the hKAT-I activity assay The mixture was

incubated for 10 min at 45C The product was quantified

based on the detection of o-phthaldialdehyde thiol

(OPT)-amino acid product conjugate by HPLC with fluorescent

detection (excitation: 325 nm; emission: 465 nm) after their

corresponding reaction mixtures were derived by OPT

reagent [28] To determine the substrate specificity for

a-keto acids, 16 individual a-keto acids were tested for their

ability to function as the amino group acceptor for hKAT-I

In the assays, a different a-keto acid at varying

concentra-tions (0.25–16 mM) was used to replace a-ketobutyrate in

the presence of 15 mMkynurenine in the typical reaction

mixture and the rate of KYNA production was determined

as described in the hKAT-I activity assay

Presence of other amino acids or other keto acids on

hKAT-I catalyzed KYNA formation.Analysis of substrate

specificity revealed that a number of amino acids and a-keto

acids can serve as the amino group donor and acceptor,

respectively, for hKAT-I To determine the effect of a

competing amino acid or keto acids on hKAT-I catalyzed

KYNA production from kynurenine, a different amino acid

(with a final concentration of either 2 mMor 32 mM) or a

different a-keto acid (with a final concentration of 2 mM)

was incorporated into the typical reaction mixture

contain-ing 5 mM (for testing amino acids) or 15 mM (for testing

a-keto acids) kynurenine, 2 mMa-ketobutyrate, and 2 lg

hKAT-I in a total volume of 50 lL and the enzyme

activity was assayed in the same manner as described for

the hKAT-I activity assay

All assays were performed in at least triplicate The results

for the effects of keto acids and amino acids were analyzed

by using the Student’s t-test The kinetic parameters of the

recombinant enzyme towards different amino acids or

a-keto acids were calculated by fitting the experimental

data to the Michaelis–Menten equation using theENZYME KINETICS MODULE(SPSS Science, Chicago, IL, USA)

Results Spectral characteristics of the recombinant hKAT-I Purified hKAT-I showed a single protein band on SDS/ PAGE with a relative molecular mass of 46 kDa (Fig 1A, insert), which closely matches its calculated mass (47 875 Da) Spectral analysis of the purified enzyme in phosphate buffer (pH 7.5) revealed an absorption peak in the visible region with a kmaxat 422 nm, which corresponds

to protein associated PLP The protein associated PLP was easily distinguished from free PLP that had its visible absorption peak with a kmaxat 388 nm (Fig 1A) Based on protein concentration in comparison with the absorbance

of protein associated PLP, an approximate 1 : 1 ratio of the protein against PLP was established The 422 nm

A

B

Fig 1 Spectral characteristics of hKAT-I (A) The absorption peak of protein associated PLP in the visible region in phosphate buffer,

pH 7.5, has a k max at 422 nm, which can be distinguished from the corresponding absorption peak of free PLP that has a k max at 388 nm Insert is SDS/PAGE gel that illustrates soluble protein from hKAT-I recombinant baculovirus infected insect cells (lane 1), purified hKAT-I (lane 2) and protein standard (lane 3), respectively (B) Spectral changes of hKAT-I following the addition of 10 m M glutamine into hKAT-I solution The concomitant decrease of the 422 nm peak and increase of the 335 nm peak indicate the conversion of the enzyme associated PLP to pyridoxamine The reaction mixture was scanned at

1 min intervals following glutamine addition.

absorption peak was rapidly diminished upon incorporation

of glutamine into the reaction mixture with the concomitant

formation of a new absorption peak with a kmaxat 335 nm

(Fig 1B), indicating the formation of pyridoxamine from

the enzyme associated PLP Addition of kynurenine

dimin-ished the 422 nm peak, but the absorption peak of

kynurenine overlapped with the pyridoxamine peak and

partially overlapped with the 422 nm peak (not shown)

Incorporation of a-ketobutyrate or other a-keto acid

concomitantly decreased and increased the 335 nm and

422 nm peak, respectively, and the relative dimensions of

the pyridoxamine peak (335 nm) and enzyme-PLP peak

(422 nm) were dependent on the molar ratio between the

amino group donor and the acceptor in the mixture These

results established that the expressed recombinant protein is

folded properly with its PLP prosthetic group and retains its

biochemical activities

Effect of buffer and pH on hKAT-I activity

When the phosphate and borate buffer mixture, adjusted

to pH 6.0–9.5, was used to prepare hKAT-I/kynurenine/

a-ketobutyrate reaction mixtures, hKAT-I showed little activity with pH 6.0, became fairly active at pH 6.5–7.0, and displayed high activity at pH 7.5–9.0 (Fig 2A) The high activity of hKAT-I in catalyzing the kynurenine to KYNA

Fig 2 Effect of pH and Tris buffer on hKAT-I activity hKAT-I was

incubated in the presence of 5 m M kynurenine and 2 m M a-ketobutrate

as described in Experimental procedures (A) hKAT-I activity profiles

at different pHs in a buffer mixture containing 100 m M phosphate and

100 m M boric acid The pH ranged from 6.0 to 9.5 (B) Inhibition of

hKAT-I activity by Tris at neutral and weak basic condition: ) ) ),

hKAT-I activity in a reaction mixture prepared using 200 m M Tris

buffer; ÆÆÆÆ, hKAT-I activity in a reaction mixture prepared using

200 m phosphate buffer.

Fig 3 Interaction of free PLP with Tris amine under different pH conditions One hundred microliters of either 20 m M phosphate buffer

or Tris buffer at pH 7.0, 7.5, 8.0, or 8.5 was mixed with 400 lL of 0.25 m M PLP prepared in distilled water and the spectra of the mixtures were recorded using a Hitachi U2001 double-beam spectro-photometer 2.0 min after buffer addition (A) Spectra of PLP in the presence of phosphate, pH 8.0 (trace 1) and Tris, pH 8.0 (trace 2) (B) Spectra of PLP in Tris pH 7.0 (trace 1), 7.5 (trace 2), 8.0 (trace 3) or 8.5 (trace 4) (C) Spectra of PLP in phosphate pH 8.0 before (trace 1) and after incorporation of 4 m M glutamine (trace 2), which serves as a control for suggesting a similar interaction of Tris amine with PLP as that of amino acid substrate.

Table 1 Kinetic parameters of hKAT-I towards different amino acids and comparison with the rate of purified brain hKAT-I and the k cat /K m of other aminotransferases The activities were measured as described in Experimental procedures The a-ketobutyrate concentrations were 16 m M in the presence of different concentrations of amino acids The kinetic parameters of the enzyme to amino acids were calculated by fitting the experimental data to the Michaelis–Menten equation using the ENZYME KINETICS MODULE BGlnAT, bacterium homologue, glutamine:phenylpyruvate aminotransferase from Thermus thermophilus HB8 [24]; AeKAT, insect homologue, from Aedes aegypti [23]; RGlnAT, rat glutamine transaminase K/KAT-I [30]; BhKAT-I, hKAT-I, purified from human brain [29]; 3-HK, 3-hydroxykynurenine.

Amino acid

K m m M k cat min)1

k cat /K m

min)1Æm M )1 k cat /K m

min)1Æm M )1 k cat /K m

min)1Æm M )1 k cat /K m

min)1Æm M )1 rate

lmolÆmin)1Æmg)1 Glutamine 2.8 ± 0.5 440.5 ± 28.7 157.3 4 147.8 0.04 1.8

Phenylalanine 1.7 ± 0.3 91.0 ± 4.8 53.5 13 80.9 0.17 0.37

Leucine 7.6 ± 3 339.9 ± 43.1 44.7 1.3 · 10)4 22 < 0.05

Methionine 6.4 ± 0.9 215.4 ± 14.4 33.7 6.3 116.6 0.012 < 0.05

Tyrosine 3.2 ± 0.4 91.0 ± 4.8 28.4 200 154.7 0.0031 < 0.05

Histidine 5.4 ± 1 143.6 ± 14.4 26.6 1.2 · 10)3 112 < 0.05

Amino-butyrate 21.3 ± 4.7 38.3 ± 4.8 1.8 3.4

Asparagine 23.1 ± 5.7 14.4 ± 0.14 0.6 2.2 · 10)3 37.8

Glycinea

Lysine a

Isoleucineb

Valineb

a

The enzyme activity towards these amino acids at 32 m M are 0.06–0.4 lmolÆmin)1Æmg)1, K m > 32 m M bThe enzyme activity towards these amino acids at 32 m M was < 0.05 lmolÆmin)1Æmg)1, and the enzyme shows no detectable activity towards 3-hydroxykynurenine.

Fig 4 Transamination activity of hKAT-I towards different amino acids with a-ketobutyrate as an amino acceptor Purified recombinant hKAT-I was incubated with each of the 24 amino acids at 20 m M (A), except 3-hydroxykynurenine, which was not tested at 20 m M due to its low solubility in aqueous solution, and 2 m M (B) in the presence of 16 m M a-ketobutyrate or oxaloacetate (for activity towards aminobutyrate), respectively as described in Experimental procedures The activity was quantified by the amount of aminobutyrate or aspartate produced in the reaction mixture.

pathway under physiological conditions contrasted with

previous reports that mammalian KAT-I had extremely

limited activity in catalyzing the production of KYNA

from kynurenine under neutral conditions [14,15,25,29]

However, when the same reaction mixtures were prepared

in Tris buffer alone, as described in some previous reports,

hKAT-I showed extremely low activity at pH 7.5, and

essentially no activity at pH 8.0, but became active at

pH 8.5 (Fig 2B, dashed line), which is quite similar to

earlier studies [14,15,25,29] It is intriguing that the pH

profiles of enzyme activity were so different between Tris

and phosphate buffer (Fig 2B, dotted line)

The aldehyde group of PLP can react with a primary

amine to form a fairly stable Schiff base, so the inhibition of

Tris on hKAT-I might be due to competition of the amino

group on Tris molecules with enzyme associated PLP When 400 lL of a PLP solution, prepared in distilled water

at 0.25 mM, was mixed with 100 lL of 20 mMphosphate buffer or 20 mMTris buffer at pH 8.0, the visible absorption peak of PLP was shifted towards longer wavelengths after the addition of Tris buffer, as compared to that of PLP after the addition of phosphate buffer (Fig 3A) Other than a slight change in peak dimension, pH changes of phosphate buffer (pH 6.5–8.0) did not lead to a noticeable spectral shift

of the PLP absorption peak (not shown), but apparent spectral shift towards longer wavelengths was observed in the PLP solution after the addition of Tris at pH 7.5, 8.0, and 8.5, respectively, as compared to the addition of Tris at

pH 7.0 (Fig 3B) The same spectral shift was also observed when glutamine was added to the phosphate prepared PLP

Fig 5 Cosubstrate specificity of hKAT-I hKAT-I was incubated in the presence of kynurenine at 15 m M and a different amino group acceptor (a-keto acid) at concentrations ranging from 0.1 to 16 m M as described in Experimental procedures The activity was quantified by the amount of KYNA produced in the reaction mixture.

solution (Fig 3C) These results suggest that the amino

group of the Tris molecule probably interacts with enzyme

associated PLP under basic conditions, thereby decreasing

its transaminase activity As a similar spectral shift of PLP

towards a longer wavelength was observed in Tris buffer

at pH 8.5, other competing mechanisms between the Tris

amine and the amino acid substrates may be involved as

well, which requires further elucidation

Substrate study of hKAT-I

hKAT-I was tested for aminotransferase activity towards

different amino acids using a-ketobutyrate as a primary

amino group acceptor The selection of a-ketobutyrate as

the amino group acceptor was based on initial results that

this keto acid showed no substrate inhibition at saturating

concentrations (discussed below) hKAT-I showed

detect-able activity towards aromatic amino acids (including,

kynurenine, phenylalanine, tryptophan and tyrosine); sulfur

containing amino acids (including, methionine and cysteine)

and other aliphatic amino acids (including, glutamine,

leucine, histidine, and aminobutyrate) This is different from

a previous study reporting that hKAT-I exhibited relatively

high activity to only four amino acid substrates [29]

(Table 1) hKAT-I also exhibited low activity to other

tested amino acids at high concentrations (Fig 4) Kinetic

results provided a better view regarding the efficiency of

hKAT-I towards the individual amino acids (Table 1)

Based on the parameter of kcat/Km, it is apparent that

hKAT-I is efficient in catalyzing the transamination of a

number of amino acids, including glutamine, phenylalanine,

leucine, kynurenine, methionine, tyrosine, histidine,

cys-teine, and aminobutyrate (Table 1) Although KAT-I

enzymes from different species, including insect kynurenine

aminotransferase from Aedes aegypti (AeKAT) [23], rat

glutamine transaminase K/KAT-I [30] and hKAT-I,

behave in a similar manner, they display apparent

differ-ences in substrate preference For example, hKAT-I is most

efficient in catalyzing the transamination of glutamine,

AeKAT is most efficient toward cysteine and tyrosine,

bacterium enzyme is most efficient toward tyrosine, and rat

KAT-I is most efficient to phenylalanine (Table 1) These

differences suggest that the enzyme may have different

functional priorities in different species

Sixteen a-keto acids were tested for their potential as the

amino group acceptor for hKAT-I with 15 mMkynurenine

as the amino group donor Among them, 12 a-keto acids

displayed detectable activity after 10 min of incubation

(Fig 5) and five (a-ketoglutarate, a-ketoiosleucine,

indo-3-pyruvate, a-ketoadipate and a-ketovaline) showed

detect-able activity only when incubation time lasted for an hour

Among the 12 a-keto acids capable of functioning as amino

group acceptors for hKAT-I, a-ketobutyrate,

mecapto-pyruvate and oxaloacetate showed no substrate inhibition

at saturating concentrations, but the others, especially

p-hydroxy-phenylpyruvate, aKMB and a-ketovalerate,

showed substrate inhibition at relatively low concentrations

(Fig 5) Although pyruvate has been the most commonly

used amino group acceptor for KAT-I activity assays, it

was much less efficient as the amino group acceptor for

hKAT-I than a number of other a-keto acids listed in

Table 2 Due to substrate inhibition, the K and k /K

could not be determined for p-hydroxy-phenylpyruvate, aKMB, a-ketovalerate, and a-ketocaproic acid (Table 2 and Fig 5), but based on reaction rates, they should also be more efficient than pyruvate as the amino group acceptor for hKAT-I

Effects of other amino acids on hKAT-I catalyzed kynurenine transamination

Based on the Kmof hKAT-I towards different amino acids, tryptophan, glutamine, phenylalanine, methionine, histi-dine, tyrosine, cysteine and leucine have either similar affinity or better affinity to hKAT-I than kynurenine; accordingly, the presence of any of these amino acids in the kynurenine/hKAT-I/a-keto acid mixture should lead to the competitive inhibition of hKAT-I activity towards kynur-enine When 32 mMof tryptophan, glutamine, phenylalan-ine, cystephenylalan-ine, methionphenylalan-ine, histidphenylalan-ine, tyrosine or leucine was incorporated into the kynurenine/hKAT-I/a-keto acid reaction mixture with 5 mMkynurenine, the rate of KYNA formation was significantly decreased (Fig 6B) When

2 mMof a different amino acid was incorporated into the kynurenine/hKAT-I/a-keto acid reaction mixture, the rate

of KYNA production was decreased only by tryptophan, glutamine, phenylalanine, and cysteine at 70%, 60%, 60%, and 30%, respectively (Fig 6A) Apparently, the decrease

in the rate of KYNA production in the hKAT-I/kynure-nine/a-keto acid reaction mixture in the presence of a different amino acid was due to competitive inhibition, because the extent of inhibition approximately matched the

Kmvalue of the corresponding competing amino acid in the reaction mixture Cysteine derivatives were proposed to be modulators for KYNA production in the mammalian brain

Table 2 Kinetic parameters of hKAT-I towards a-keto acids The activities were measured as described in Experimental procedures The

K m and k cat for amino acceptors were derived by using varying con-centrations of individual amino acceptors in the presence of 15 m M of kynurenine The parameters were calculated by fitting the experimental data to the Michaelis–Menten equation using the ENZYME KINETICS MODULE (Fig 5).

a-Keto acids

K m

m M

k cat

(min)1)

k cat /K m

(min)1Æm M )1 ) a-Ketoleucine 1 ± 0.3 296.8 ± 28.7 247.4 Glyoxylate 1.5 ± 0.5 263.3 ± 28.7 175.5 Phenylpyruvate 0.8 ± 0.4 110.1 ± 19.2 137.6 Mercaptopyruvate 2.5 ± 0.4 234.6 ± 14.4 93.8 a-Ketobutyrate 3 ± 0.4 234.6 ± 9.6 78.2 Oxaloacetate 4.2 ± 0.4 143.6 ± 9.6 34.2 Pyruvate 12.1 ± 4.9 28.7 ± 4.8 2.4 a-Ketocaproic acid

a-Ketovalerate aKMB

High activity, but unable to calculate kinetic parameters because of their substrate inhibition at low concentration p-Hydroxy-phenylpyruvate

a-Ketoglutarate a-Ketoisoleucine a-Ketoadipate

Activity was detectable only when incuba-tion time lasted at least an hour, ranking

as listed.

a-Ketovaline Indo-3-pyruvate

[31,32] and cysteine had an intriguing effect on AeKAT

[23], so its effect on hKAT-I mediated kynurenine

transamination was tested more thoroughly Cysteine

showed apparent inhibition of hKAT-I-catalyzed KYNA

production from kynurenine at concentrations of 2 mM

(Fig 6C), but did not stimulate hKAT-I acitivity at low concentration as seen in the AeKAT catalyzed reaction [23] For most of the other tested amino acids, no inhibition on KYNA production was observed (Fig 6A,B), which could

be explained by their rather low affinity to the enzyme

Effects of other keto acids on hKAT-I activity Ability to function as the amino group acceptor for a number of biologically relevant keto acids and their substrate inhibition above certain concentrations (Fig 5) indicated that the presence of two keto acids might have either a positive or negative impact on hKAT-I mediated KYNA production, as compared to the presence of a single amino group acceptor To test this hypothesis, a different keto acid was added to the kynurenine/hKAT-I/a-ketobu-tyrate mixture and the rate of KYNA production in the reaction mixture was compared with that of the control reaction mixture with a-ketobutyrate alone A number of a-keto acids significantly increased the rate of KYNA production, but indo-3-pyruvate showed significant inhibi-tion on the enzyme, and a-ketoglutarate, a-ketoiosleucine,

Fig 6 Effect of other amino acids on hKAT-I activity Assay

condi-tions were similar to those described in Experimental procedures,

except kynurenine and a-ketobutyrate concentrations were 5 m M and

2 m M , respectively The concentrations of amino acids tested were

2 m M (A) and 32 m M (B), respectively Cysteine was tested from

0.3 m M to 16 m M (C) The activity was quantified by the amount of

KYNA produced in the reaction mixture *P < 0.5, **P < 0.01

significant difference from the control; 3-HK, 3-hydroxykynurenine.

Fig 7 Effect of the multiple a-keto acids on hKAT-I-catalyzed KYNA production The activities were quantified by the amount of KYNA produced in the reaction mixtures The volume of the reaction mixture was 50 lL consisting of 2 lg of hKAT-I, 15 m M kynurenine and two different a-keto acids Other conditions were similar to those described

in Experimental procedures (A) Rate of KYNA production in the hKAT-I and kynurenine mixture in the presence of 2 m M of a-ketobutyrate and 2 m M of a different a-keto acid *P < 0.5,

**P < 0.01, significant difference from the control (B) Effect of different concentrations of indo-3-pyruvate on hKAT-I activity p-HPP, p-hydroxyphenylpyruvate.

a-ketoadipate, a-ketovaline, pyruvate, and phenylpyruvate

had no significant effect on the rate of KYNA production

by hKAT-I (Fig 7A) Because indo-3-pyruvate has not

been reported as an inhibitor to hKAT-I in other studies, its

inhibition of hKAT-I at a broad range of concentrations

was further tested Analysis of hKAT-I activity in the

presence of varying concentrations of indo-3-pyruvate

showed that the compound inhibited hKAT-I activity at a

very low concentration of 0.08 mM, and completely

abol-ished the enzyme activity at 5.0 mM(Fig 7B)

Discussion

Analysis of substrate specificity confirmed that hKAT-I is a

multifunctional aminotransferase Kinetic analysis of the

enzyme towards different amino acids showed that the

enzyme is efficient in catalyzing the transamination of

glutamine, phenylalanine, leucine, kynurenine, tryptophan,

methionine, tyrosine, histidine, cysteine and

amino-butyrate, which contrasts an earlier report showing that

purified hKAT-I exhibited high activity to only four

individual amino acids (kynurenine, glutamine,

phenyl-alanine, and tryptophan) [29] (Table 1) The large spectrum

of amino acid substrates of KAT-I supports the proposed

role of the enzyme in sparing the essential amino acids

methionine, histidine, phenylalanine and tyrosine [1] and

providing a mechanism to maintain a continual equilibrium

among the amino acids [33] Moreover, the high activity

of hKAT-I towards kynurenine under physiological pH

conditions provides the basis for suggesting its function in

brain KYNA synthesis

By studying the pH profile of the enzyme, we

demon-strated, for the first time, that hKAT-I has a broad optimal

pH range, and is capable of efficiently catalyzing the

kynurenine to KYNA pathway under physiological

condi-tions, which contrasts the published pH profile of hKAT-I

[15,25] The inability of hKAT-I to catalyze efficient

transamination reactions in previous studies was probably

caused by Tris inhibition of the enzyme In transaminases,

PLP is bound in a Schiff base linkage with the e-NH2group

of an active site lysine residue [34,35] The mechanisms

regarding PLP-mediated transamination reactions (i.e the

protonation of the Schiff base, removal of the Ca proton

from the amino acid substrate, electron relocalization and

rearrangement of the Schiff base double bond from the

pyridoxal aldehyde carbon to the Ca of the amino acid

substrate, etc.) have been discussed in numerous reports

[36,37] Spectral shift of free PLP towards longer

wave-lengths in the presence of Tris at weak basic conditions

suggests that the amine can interact with the enzyme

associated PLP, which may lead to the formation of a Schiff

base through an initial nucleophilic addition to the carbonyl

group of PLP, followed by rapid proton transfer, leading to

water elimination and the formation of an imine The

apparent spectral shift of PLP in Tris buffer at weak basic

pH, the absence of such a spectral shift of PLP in a

phosphate buffer of pH 7.0–9.0, in conjunction with the

same spectral shift of PLP in phosphate buffer upon

incorporation of glutamine and the high activity of hKAT-I

in phosphate buffer at both neutral and weak basic

conditions (pHs 7.0–8.0), provides a reasonable basis for

suggesting that the extremely low activity of KAT-I under

physiological pH is due to KAT-I inhibition by Tris amine Our data established that hKAT-I is quite capable of catalyzing the kynurenine to KYNA pathway at physiolo-gical conditions

Data concerning the effect of other amino acids on hKAT-I catalyzed KYNA production determined that KYNA production is not seriously affected by most amino acids, except for tryptophan, glutamine, phenylalanine and cysteine, which decreased KYNA production by 30–70%, which is consistent with a previous report, i.e tryptophan, glutamine and phenylalanine are inhibitors of the enzyme [29] Cysteine was reported to be a good substrate for glutamine transaminase K [38], but its effect on hKAT-I activity has not been tested In mammals, endogenous cysteine displays neuroexcitatory actions similar to those of glutamate [39,40] Cysteine derivatives, homocysteine, cys-teine sulfinate, homocyscys-teine sulfinate and cysteate, were able to reduce the production of KYNA in cortical slices in rats, due presumably to their interaction with KATs, and they were considered endogenous modulators of KYNA formation in the brain [31,32] However, because there are potentially two KATs in the brain, which one is inhibited by cysteine derivatives has not been fully understood, although the possible inhibition of KAT-II has been proposed [31] The inhibition of hKAT-I by cysteine in vitro suggests that the reduction of KYNA production in cortical slices in the presence of cysteine or cysteine derivatives could be due to their inhibition to KAT-I

Most naturally occurring a-keto acids can serve as the amino group acceptor for hKAT-I Pyruvate has been the most commonly used amino group acceptor for character-izing hKAT-I [14,15,25,29] Through kinetic analysis, it is clear that pyruvate is not an efficient amino group acceptor due to its high Km (Fig 5) The results dealing with cosubstrate profiles in a previous study [29] could have been different if cosubstrate inhibition had been taken into consideration Our data confirmed that glyoxylate, aKMB, p-hydroxy-phenylpyruvate, a-ketovalerate, a-ketocaproic acid, a-ketoleucine, mercaptopyruvate, a-ketobutyrate, pyenylpyruvate and oxaloacetate are more efficient amino group acceptors than pyruvate for hKAT-I (Fig 5 and Table 2), which is similar to the previously given information about glutamine transaminase K, i.e wide a-keto acid specificity, but high activity with a-KMB and glyoxylate, strong substrate inhibition with phenylpyruvate and poor affinity toward alanine and pyruvate [30,41]

In summary, by systematically studying the potential substrates, amino acids and a-keto acids for hKAT-I, a new substrate map for hKAT-I is obtained This study con-firmed that hKAT-I is a multifunctional enzyme New pH profiles of hKAT-I were described and the reasons why it is different from the reported pH profile were discussed Indo-3-pyruvate and cysteine were found to be efficient inhibitors for hKAT-I Based on our data, it is reasonable to propose that hKAT-I might be an important player in KYNA synthesis under physiological conditions in the human brain However, much more research is needed to fully understand its overall physiological role in vivo Neverthe-less, this study provides rather comprehensive biochemical characteristics of this important enzyme, which should be highly useful towards elucidating the accurate role hKAT-I plays in brain KYNA synthesis and towards controlling the

levels of endogenous kynurenic acid in the human brain

through modulating hKAT-I activity

Acknowledgements

We are grateful to Prof Bruce M Christensen, Department of

Animal Health and Biomedical Sciences, University of Wisconsin

(Madison, WI, USA) and Dr Menico Rizzi (Department of Genetics,

University of Pavia, Italy) for their critical reading of the manuscript.

This study was supported by the National Institutes of Health Grant

AI 44399.

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