L-kynurenine aminotransferase and L-α-aminoadipate aminotransferase. I. Evidence for identity

Vol. 62, No. 2, 19Z5

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

L-KYNURENINE AMINOTRANSFERASE AND L-~-AMINOADIPATE AMINOTRANSFERASE. I. EVIDENCE FOR IDENTITY Michael C. Tobes and Merle Mason Department of Biological Chemistry, The University of Michigan Ann Arbor, Michigan 48104 Received

November

18,1974

Summary: The following observations strongly suggest that L-kynurenine aminotransferase (KAT) and L-e-aminoadipate aminotransferase (AAT) activities are associated with the same protein: i. a similar distribution in the supernatant and mitochondrial fractions of kidney and liver of male rats, 2. a similar distribution in these fractions of kidney and liver of female rats, 3. a similar sex difference in the supernatant and mitochondrial fractions of kidney with no sex difference in liver fractions, 4. a similar pattern of inhibition with a homologous series of dicarboxylic acids with adipate being most effective, 5. competitive inhibition of KAT with D,L-a-aminoadipate (eAA), 6. substrate inhibition of KAT with e-ketoadipate (~KA), and 7. AAT activity in a highly purified preparation of KAT. A scheme for the interaction of the two activities in lysine and tryptophan metabolism is presented. L-Kynurenine aminotransferase L-kynurenine

(E.C. 2.6. i. 7) catalyzes the conversion of

(Kyn) or L-3-hydroxykynurenine to kynurenic acid or xanthurenic

acid with the concomitant conversion of ~-ketoglutarate (Glu).

(~KG) to L-glutamate

Several observations have suggested to us that it might also trans-

aminate L-eAA. Inhibition of KAT with dicarboxylic acids of varying chain length was reported; adipate was the most inhibitory of those acids in the homologous series from oxalate, C 2, to suberate, C 8 (i).

Adipate was a competitive in-

hibitor with respect to L-Kyn but not to aKG.

This maximal inhibition was

postulated to be reflective of the distance between two cationic groups at the active site of the enzyme. ferase

Similar observations with aspartate aminotrans-

(2-4) demonstrated that carboxylic acids which were analogs of the best

keto acid substrates were the strongest inhibitors. Applying this generalization to KAT, one would expect ~KA to be the best keto acid substrate.

Recently eKA has been shown to be a good substrate for

Abbreviations: KAT, L-Kynurenine Aminotransferase; AAT, L-~-Aminoadipate Aminotransferase; Kyn, Kynurenine; ~AA, ~-Aminoadipate; ~KG, a-Ketoglutarate; ~KA, a-Ketoadipate; Glu, Glutamate; PLP, Pyridoxal-5'-Phesphate.

Copyright © 1975 by Academic' Press, Ine. All rights of reproduction in any form reserved.

390

Vol. 62, No. 2, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

KAT with an apparent Km similar to that of eKG higher concentrations,

possibly because

competed with Kyn for the enzyme. stantially

(5).

it was transaminated

but the type of inhibition

(6).

The AAT of rat liver was located in the mitochondria the conversion of L-~AA to ~KA with the concomitant A substrate

to c&~A which then

It has been reported that D,L-~AA sub-

inhibits KAT from kidney mitochondria,

was not studied

It was inhibitory at

specificity

(7).

It catalyzed

conversion

of ~KG to L-GIu.

study was done with a partial purified preparation,

but Kyn was not tested. In this communication,

we present evidence that strongly

concept that the two activities

supports

the

are associated with the same protein.

MATERIALS AND METHODS Tissue Fractionation:

Adult albino rats, weighing approximately

sacrificed by guillotine.

The kidneys and livers were immediately placed in

ice and were then homogenized The supernatant

Preparation

and fractionated

and mitochondrial

used within 3 weeks.

250 gms, were

as described previously

fractions were

frozen immediately,

(8).

and were

Little activity was lost over 3 months at -15°C.

of Partially Purified Enzyme:

All procedures

were carried out at

0-4°C unless stated otherwise.

Kidney supernatant

were thawed to obtain 50.0 ml.

The solution was made 0.01 m M with respect

pyridoxal-5'-phosphate

(PLP).

at 30,000g for i0 min.

to pH 6.5 with 1.0 N NaOH. was 35% saturated.

Solid

stirring.

The pooled supernatant (NH4)2SO 4 was added

respect to PLP.

fluid was adjusted

(9) until the solution the pH at 6.5, the

While constantly

the suspension was brought to 55 ~ in a boiling water bath. it was brought back to 4°C by stirring in an ice bath. 30,000g for 20 min.

to

The suspension was

After stirring for 1 hr and maintaining

suspension was made 0.05 m M w i t h

from male rats

It was titrated to pH 5.3 with 1.0 N CH3COOH

and allowed to stand for 5 min with occasional centrifuged

fractions

After 5 min at 55 ° ,

It was centrifuged

While stirring for 1-2 hrs and maintaining

391

stirring,

at

at pH 6.5, the

Vol. 62, No. 2, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

pooled supernatant

fluid was made 65% saturated with additional

The suspension was centrifuged dissolved

at 30,000g for 20 min.

The precipitates

in a minimum volume of i0 m M potassium phosphate

which contained

i0 m M 2-mercaptoethanol.

pooled and dialyzed overnight

(NH 4) 2S04.

buffer,

were

pH 7.0,

The dissolved precipitates

were

against 2.1 liters of the buffer with one change

of the buffer. L-Kynurenine m M PLP,

Aminotransferase

5.00 m M ~KG,

Assay:

The incubation

3.26 m ~ L-Kyn sulfate,

6.5, and 0 to 0.i0 ml enzyme solution

mixture

contained 0.20

i00 m M imidazole-HCl

buffer,

in a total volume of 0.60 ml.

pH

The in-

cubation mixture was held for I0 min at room temperature

before the addition

of L-Kyn,

The reaction was

and it was then incubated

for 30 min at 37°C.

stopped with the addition of i0 ml of 1% boric acid in 95% ethanol. tubes were mixed well, 5000g for 5 min.

and the precipitate

Kynurenic

was removed by centrifugation

acid formation was estimated

to increase the mitochondrial

activity but not that of the supernatant

Aminotransferase

preparations

Assay:

The buffer was at pH 7.0.

frac-

(6,8).

The assay system was a modification

of the assay with L-GIu and eKA as substrates (7).

Crude mitochondrial

of 1.42 m M CaCI 2 which has been reported

tions or of the disrupted mitochondrial L-e-Aminoadipate

at

spectrophotometrically

(i0) using a reaction blank containing everything but ~KG. fractions were assayed in the presence

The

described by Nakatani

et al.

After stopping the reaction with 0.i0 ml of

1.0 N HCI, 0.i0 ml of i.i N KOH was added to raise the pH to 7.5. cipitates were removed by centrifugation blank contained everything but eKA.

at 5000g for 5 min.

The pre-

A reaction

Assays of crude mitochondrial

fractions

were done in the presence of 2.00 m M CaCl 2 which was found to increase mitochondrial

activity but not that of the supernatant

effect on glutamate dehydrogenase Protein Determination: bovine

and to have no

(ii).

Protein was measured according to Lowry et al. with

serum albumin as the standard

Chemicals:

fractions

L-Kyn sulfate,

(12).

~KG, ~KA, D,L-~AA,

392

PLP, the dicarboxylic

acids, and

Vol. 62, No. 2, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I. D i s t r i b u t i o n of L - K y n u r e n i n e A m i n o t r a n s f e r a s e A m i n o t r a n s f e r a s e in the K i d n e y and Liver of M a l e Rats.

L-Kynurenine Aminotransferase

and L - ~ - A m i n o a d i p a t e

L-~-Aminoadipate Aminotransferase

Total Units per O r g a n a

% Activity b

Total Units per O r g a n a

% Activit~

Mitochondrial

1520 ± 341

46.9 ± 4.1

11900 ± 3220

42.4 ± 5.1

Supernatant

1700 ± 219

53.1 ± 4.1

15800 ± 2050

57.6 ± 5.1

2790 ± 626

81.4 ± 2.5

31500 ± 6 9 4 0

76.3 ± 1.6

633 ± 152

18.6 ± 2.5

9690 ± 1480

K i d n e y fraction:

Liver fraction: Mitochondrial Supernatant

23.7

± 1.6

Four m a l e rats w e r e used: body wt., 251.9 ± 4.1 gms; k i d n e y wt., 1.97 ± 0.19 gms; liver wt., 9.89 ± 0.50 gms. E a c h f r a c t i o n was a n a l y z e d in duplicate. The m e a n and standard d e v i a t i o n are presented. A s s a y c o n d i t i o n s are those d e s c r i b e d in the text with the f o l l o w i n g m o d i f i c a t i o n s : KAT assay - 0.17 ~ 4 PLP, 4.29 m M ~KG, 2.80 m M L-Kyn, 85.7 m M i m i d a z o l e - H C l buffer, p H 6.5, and a r e a c t i o n v o l u m e of 0.70 ml; and AAT a s s a y - 5 m i n i n c u b a t i o n of k i d n e y fractions. Linearity was m a i n t a i n e d in all r a t e determinations. a Units are d e f i n e d as m~moles/min. b

A c t i v i t y is b a s e d on the a c t i v i t y r e c o v e r e d in the two fractions. T h e two f r a c t i o n s a c c o u n t e d for 80 to 90% of the two a c t i v i t i e s in the k i d n e y homog e n a t e and for 60 to 70% of the two a c t i v i t i e s in the liver homogenate.

glutamate dehydrogenase icals were o b t a i n e d

were purchased

f r o m Sigma C h e m i c a l

f r o m other r e p u t a b l e

Co.

A l l other chem-

firms.

RESULTS A N D D I S C U S S I O N The d i s t r i b u t i o n m£tochondrial

of hepatic and renal AAT b e t w e e n the s u p e r n a t a n t

fractions of m a l e rats was

The k i d n e y s u p e r n a t a n t mitochondrial

fraction,

the m i t o c h o n d r i a l

similar to that of KAT

and

(Table I).

fraction had s l i g h t l y m o r e of b o t h a c t i v i t i e s

t h a n the

w h e r e a s m o s t of b o t h a c t i v i t i e s of the liver was in

fraction.

Distribution

of KAT has been r e p o r t e d

earlier

(13,14). Table II shows the d i s t r i b u t i o n chondrial

of AAT b e t w e e n the s u p e r n a t a n t

f r a c t i o n s of female rats to be similar to that of KAT.

393

and m i t o -

Both activity

Vol. 62, No. 2, 1975

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I I . D i s t r i b u t i o n of L - K y n u r e n i n e A m i n o t r a n s f e r a s e A m i n o t r a n s f e r a s e in the K i d n e y and Liver of F e m a l e Rats.

L-Kynurenine Aminotransferase Total Units per O r g a n a Kidney

L-~-Aminoadipate Aminotransferase

% Activit~ b

Total Units per Organ a

% Activity b

fraction:

Mitochondrial

682 ±

Supernatant Liver

and L - e - A m i n o a d i p a t e

35

37.4 ± 1.9

1150 ± 1 3 4

2760 i 761 ±

6460 i

156

36.3 ± 3.0

62.6 ± 1.9

11400 i 1290

63.7 ± 3.0

68

78.4 ± 1.9

26600 ± 1900

72.6 ± 2.9

93

21.6 ± 1.9

i0000 i 1170

27.4 ± 2.9

fraction:

Mitochondrial Supernatant

Four female rats were used: body wt., 249.8 ± 1.6 gms; k i d n e y wt., 1.72 ± 0.09 gms; liver wt., 9.20 ± 0.84 gms. E a c h fraction was analyzed in duplicate. The m e a n and standard d e v i a t i o n are presented. Assay conditions and footnotes are p r e s e n t e d in Table I.

o

60--

10c

50

4O 1-

00

30 z 20

I0

I

0

I

2

3

4 N

~

5

6

7

I 8

Fig. 1. Inhibition of a p a r t i a l l y purified p r e p a r a t i o n of L - k y n u r e n i n e amino£ransferase (- o -) and L - ~ - a m i n o a d i p a t e a m i n o t r a n s f e r a s e (- • -) with dicarboxylic acids. T h e p r e p a r a t i o n had a specific activity of 41.2 m ~ m o l e s / m i n / m g protein. Assay conditions are those described in the text with these m®difications: KAT assay--0.19 r~4 PLP, 6.00 m M ~KG, 3.92 n ~ L-Kyn, i00 m M p o t a s s i u m phosphate, pH 7.0, 0.50 ml r e a c t i o n volume, and 20 m i n incubation; and AAT a s s a y - - 6 . 0 0 n ~ eKA, 4.00 m M L-GIu, and 7 m i n incubation. No inhibition of g l u t a m a t e d e h y d r o g e n a s e was o b s e r v e d w i t h a n y o f the dicarboxylic acids at the conditions described. L i n e a r i t y was maintained. D i c a r b o x y l i c acids were 6.00 n ~ w h e n present. All assays were done in quadruplicate.

394

Vol. 62, No. 2, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0"24t 0.20~

~.~

--

S

e.o. 2,o~_Eu

.//

/e

0.12-- ~ ~ . ~ . 0 . 0

mM

0.08

0.04 I 01

0

I 0.2

I I 0.3 0.4 ~Kyn E mM 3

I 0.5

Fig. 2. I n h i b i t i o n of a p a r t i a l l y p u r i f i e d p r e p a r a t i o n of L - k y n u r e n i n e aminotransferase with D , L - ~ - a m i n o a d i p a t e at the c o n c e n t r a t i o n s indicated. The p r e p a r a t i o n had a specific a c t i v i t y of 59.4 m ~ m o l e s / m i n / m g protein. Assay conditions were those described in the text except that L - K y n c o n c e n t r a t i o n s w e r e varied. A l l assays were done in triplicate.

levels were fractions in liver

substantially

of females fractions.

distribution compared

lower in the supernatant

than those of males b u t w e r e Sex differences

of the two activities

to males,

and m i t o c h o n d r i a l similar

of KAT were r e p o r t e d

was different

but no sex difference

kidney

to those of the m a l e s earlier

(15).

The

in the k i d n e y of females

in d i s t r i b u t i o n

was observed

as

in the

liver. Partially-purified homologous

two activities These results

are identical, suggest

expected

later

The p a t t e r n s

and the i n h i b i t i o n

that the active

competition

kidney

by a

length f r o m oxalate,

of inhibition

w i t h adipate

of the

was maximal.

sites for K y n and ~AA are either

between

supernatant

if both substrates

tial inhibition

i.

chain

inhibited

identi-

similar.

2 indicates purified

KAT and AAT w e r e

acids of increasing

C10 , as shown in Fig.

cal or r e m a r k a b l y

partially

supernatant

series of dicarboxylic

C2, to sebacate,

Fig.

kidney

D,L-~AA

fraction.

are t r a n s a m i n a t e d

and L - K y n for the KAT of a This c o m p e t i t i o n

by the same protein.

of AAT with L-Kyn has been o b s e r v e d

(ll).

395

would be Substan-

also but w i l l be r e p o r t e d

Vol. 62, No. 2, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

l

~

ANTHRANILATE + ALANINE

~

f -KYNUiDEN~ / / /

~

3- HYDROXYKYI~URENINE / X A ~ "~IHDURE NIG

~ r CO2÷ H20

- 5-HYDROXYANTHRANILATE + ALANINE

~-.~,.o-~-c...o~.uco~,c

SEMIA,LDEHYDE l ~ ' ~ QUINOLINATE

(7-AMINOMUCONATE

SACGHAROPINE

I Fig. 3. Abbreviated scheme of potential interactions of tryptophan and lysine catabolism, based on the view that kynurenine aminotransferase and ~-aminoadipate aminotransferase are identical.

A preparation of KAT, which was 900-fold purified from the kidney homogenate activity and which appeared homogeneous by polyacrylamide gel electrophoresis, exhibited strong AAT activity. published elsewhere.

The method of purification will be

Using this preparation, a substrate specificity study

was conducted, using imidazole.HCl buffer, pH 6.5, at 37°C for 2 hrs.

The

products were separated using thin layer chromatography and detected with the ninhydrin reaction or UV light.

The only two activities detected were that of

KAT using L-Kyn and ~KG or ~KA as substrates and that of AAT assayed in either direction.

All common s-amino acids but valine, threonine, methionine,

asparagine and glutamine were tested.

These results demonstrate copurification

of the two activities. The evidence presented is strongly suggestive of the identity of KAT and AAT and we are unable to present a reasonable alternative interpretation of the data.

Additional evidence for identity will be presented elsewhere in

publications concerning the purification and purity of KAT. The view that KAT and AAT are identical suggests a scheme, shown in Fig.

396

Vol. 62, No. 2, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3, where lysine and tryptophan catabolism can interact via this transaminase. If limiting amounts of KAT(AAT) are shared with the two sets of substrates in lysine and tryptophan catabolism, one can predict that feeding increased amounts

of lysine will competitively interfere with the formation of

kynurenic and xanthurenic acids during tryptophan catabolism. have been observed with vitamin B 6 deficient rats

Such inhibitions

(16).

Acknowledgements: This work was supported in part by The University of Michigan Horace H. Rackham School of Graduate Studies (Faculty Research Grant), The University of Michigan Cancer Research Institute (Pilot Project 24), a Public Health Service research grant (AM 02294), and a Public Health Service training grant (GM 00187). It was also financed in part with Federal funds from the Environmental Protection Agency under grant number R-800637. The contents do not necessarily reflect the views and policies of the Environmental Protection Agency, nor does mention of trade n~nes or commercial products constitute endorsement or recommendation for use. REFERENCES i. 2. 3.

4.

5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15.

16.

Mason, M. (1959) J. Biol. Chem. 234, 2770-2773. Jenkins, W. T., Yphantis, D. A., and Sizer, I. W. (1959) J. Biol. Chem. 234, 51-57. Jenkins, W. T., and D'Ari, L. (1968) Pyridoxal Catalysis: Enzymes and Model Systems (E.E. Snell, et al., eds.), pp. 317-324, Interscience Publishers. Sizer, I. W., and Jenkins, W. T. (1963) Chemical and Biological Aspects of Pyridoxal Catalysis (E.E. Snell, et al., eds.), pp. 123-137, The MacMillan Co. Manning, B. (1972) Ph.D. Thesis, The University of Michigan, Ann Arbor, Michigan. Maso n , M., Tobes, M. C., and Manning, B. (1974) Acta Vitaminol. Enzymol., submitted for publication. Nakatani, Y., Fujioka, M., and Hagashino, K. (1970) Biochem. Biophys. Acta 198, 219-228. Mason, M. (1974) Biochem. Biophys. Res. Commun. 60, 64-69. Dixon, M., and Webb, E. C. (1964) Enzymes, ed. 2, p. 40, Academic Press, New York. Mason, M. (1957) J. Biol. Chem. 227, 61-68. Tobes, M. C., unpublished observations. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Ogasawara, N., Hagino, Y., and Kotake, Y. (1962) J. Biochem. 52, 162-166. Ueda, T. (1967) Nagoya J. Med. Sci. 30, 259-268. Mason, M., Ford, J., and Wu, H. L. C. (1969) Vitamin B 6 in Metabolism of the Nervous System (M.A. Kelsall, ed.), Annals N.Y. Acad. Sci. 166, 170-183. Bianchi-Donnasibilla, L., and Curro, L. (1969) Acta Vitaminol. Enzymol. 23, 165-168.

397