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A POSSIBLE MECHANISM FOR KYNURENINASE ACTION

BY J. B. LONGENECKER AND ESMOND E. SNELL

(From the Bioc hem ical Institute and the Department of Chem istry, Th e University

of Texas, and the Clayton Foundation for Research, Austin, Texas)

(Received for publication, August 12, 1954)

The cleavage of kynurenine to anthranilic acid and alanine is catalyzed

by a single pyridoxal phosphate-dependent enzyme, kynureninase, and

does not involve a preliminary transamination reaction (l-3). Appar-ently, the same or a closely related enzyme cleaves 3-hydroxykynurenine

to 3-hydroxyanthranilic acid and alanine (3).

ANTHRANILIC

ACID

ALAiiNE

These reactions can be formulated as occurring by a simple cleavage

reaction involving addition of the elements of water between the p- and 7..

carbon atoms of the substrate. Such a formulation is difficult to reconcile

with the general mechanism recently proposed from this laboratory (4) or

the similar mechanism proposed independently from Braunstein’s labora-

tory (5) for pyridoxal-catalyzed reactions.

An alternative scheme involving an o( ,P elimination reaction can be

formulated in which kynureninase would fall into the same category as

serine dehydrase, tryptophanase, and cysteine desulfhydrase (Fig. 1). In

this scheme, the Schiff base (I) formed between the pyridoxal phosphateenzyme and kynurenine eliminates a proton from the a-carbon and the

anion of the o-aminobenzoyl radical (II) from the P-carbon, yielding the

Schiff base of a-aminoacrylic acid (III). Intermediate II (as its hydrate)

either before or after stabilization as o-aminobenzaldehyde then undergoes

an oxidation-reduction reaction with III to yield anthranilic acid and the

Schiff base of alanine, which hydrolyzes to alanine with regeneration of the

phosphopyridoxal enzyme. This scheme is similar to that proposed by

Braunstein and Shemyakin (5), but differs from it in proposing that pelimination occurs before rather than after the oxidation-reduction step,

thus making unnecessary the assumption that this reaction differs in princi-

ple from cleavage of other p substituted amirro acids (e.g. serine, cysteine,

tryptophan, cystathionine).

Intermediate III is the same as that formulated (4) as arising in serine

229

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230 KYNURENINASE ACTION

dehydration, tryptophan cleavage, and similar reactions, in which it hy-

drolyzes to yield pyridoxal, pyruvate, and ammonia. Support for the

feasibility of the proposed mechanism (Fig. 1) would appear if reduction ofintermediate III, formed in one of these latter reactions, by o-amino-

benzaldehyde or other reductant could bedemonstrated. It is shown below

that, in accordance with the proposal of Fig. 1, alanine and anthranilic acid

appear in increased amounts when serine, pyridoxal, and metal salts (used

as a source of III) are heated with o-aminobenzaldehyde. A much greater

PYRlbOXAL ‘CH2=y---C=O

~,y~‘M;d +

FIG. 1. A poss ible mecha nism for the cleavage of kynurenine by kynureninase.

For simp licity, the reactions are formulated with pyridoxal in place of a phospho-

pyridoxal enzyme.

formation of alanine occurs when thioglycolate replaces o-aminobenaalde-

hyde as the reducing agent.

EXPERIMENTAL

Analytical Techniques-The general technique and certain of the analyt-ical procedures have been described (4, 6, 7). o-Aminobenzaldehyde was

prepared by reduction of o-nitrobenzaldehyde (8). L-Kynurenine sulfate,

isolated from the urine of tryptophan-fed rabbits, and kynurenic acid were

gifts from Dr. R. P. Wagner.

Pyridoxamine, alanine, and kynurenine were located on paper chromato-

grams with ninhydrin; anthranilic acid, kynurenic acid, and pyridoxamine

by their characteristic fluorescence under ultraviolet light.

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J. B. LONGENECKER AND E. E. SNELL 231

Optimal separation on paper chromatograms of the various products

from interfering materials was effected with the following solvents and

procedures: for alanine and kynurenic acid, water-saturated phenol and

the descending technique; for anthranilic acid and pyridoxamine, butanol-

acetic acid-water (80: 20: 20 parts by volume) and the ascending technique.

For quantitative determination, alanine was separated from other com-

ponents of the reaction mixtures by passage through a 1 X 40 cm. column

of Dowex 50 (300 to 500 mesh, Hf form) and elution with 1.5 N hydrochlo-

ric acid. Preliminary test runs established that quantitative recoveries

of added alanine were obtained by collecting the acid effluent fraction from

60 to 120 ml. This fraction was evaporated to dryness, dissolved in water

and assayed for alanine with Leuconostoc citrovorum 8081 (9, 10).Trial runs established that quantitative extraction of anthranilic acid

from the reaction mixtures was effected by extracting twice with an equal

volume of ethyl ether. Anthranilic acid in the extract was determined

microbiologically with Lactobacillus arabinosus 8014, which utilizes an-

thranilic acid in place of tryptophan (11). Spectrophotometric deter-

minations of this substance failed because of interfering amounts of o-

aminobenzaldehyde; the latter substance in the amounts present neither

increases nor inhibits the response of L. arabinosus to anthranilic acid.Kynurenic acid and pyridoxamine were separated from reaction mixtures

by paper chromatography, followed by elution of the appropriate zones into

0.05 M phosphate buffer, and spectrophotometric estimation at 332 and 324

rnp, respectively.

Pyridoxal-Catalyzed Formation of Alanine and Anthranilic Acid from

Serine and o-Aminobenxaldehyde-Buffered reaction mixtures (Table I)

containing o-aminobenzaldehyde, serine, pyridoxal, and potassium alum

were heated at 100” for 30 minutes.Appropriate analyses (Table I) showed

a considerably enhanced alanine production in the presence of o-amino-

benzaldehyde at pH 3.5 and 4.0, but not at pH 5.0 and 6.0. The alanine

formed in the absence of o-aminobenzaldehyde undoubtedly arises in part

by transamination of pyruvate (formed from serine via the dehydration

reaction under these conditions (12)) with pyridoxamine (formed by

transamination of pyridoxal with serine under these conditions (12)). The

pH optimum for this alum-catalyzed transamination reaction is near 5.0

(7). Additional alanine may arise in the absence of o-aminobenzaldehyde

by reduction of III (Fig. 1) by formaldehyde formed under these conditions

by the pyridoxal-catalyzed cleavage of serine to formaldehyde and glycine

(6).Corresponding to the “extra” production of alanine at pH 3.5 and 4.0

induced by the presence of o-aminobenzaldehyde is an increased production

of anthranilic acid (Table I). In the absence of side reactions, the amounts

of alanine and anthranilic acid shown in the fourth and seventh columns

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232 KYNURENINASE ACTION

should be equimolar; experimentally, anthranilic acid is found in much

lower amounts. Separate tests showed, however, that added anthranilic

acid was partially destroyed under the reaction conditions. For example,

10 y of added anthranilic acid were recovered quantitatively from the un-

heated reaction mixture, pH 3.5. After 30 minutes of heating, only 8 y

(in place of the 14.1 y expected from that added and that formed (Table I))

were found.

Similar results were obtained repeatedly in both qualitative and quanti-

tative tests, and pyridoxal was required for the reaction. Thus, the ana-

TABLE I

Formation oj Alanin e and Anthra nilic Ac id in Reaction Mixtures Containing

o-Aminobenzaldehyde, Serine, Pyridoxal, and Metal Ions

Values in micrograms per ml

DH*

3.5

4.0

5.0

6.0

Compound omitted fromreaction mixturet

Formed byreduction

with 0.

Alanine form ed Anthranilic acid forme d

23 13 10

18

I

12

:

6

I6 16 0

4.1 2.7 1.4

4.0 3.4 1.5

5.0 4.6 0 .4

4.1 3.7 0.4

T

Compound omitted fromreaction mixturet

Formed by--- reaction with

serine

None Scrine

* Formate buffer with ion ic strength of 0.1 was used for p1-E 3.5 and acetate buffer

with ionic strength of 0.1 for all other pH values.

t The reaction mixtures 0.04 AX in o-aminobenzaldehyde, 0.02 nr in se&e, 0.01 11

in pyridoxal, and 0.002 M in IIAl(S04)2.HzO at the indicated pH values were heated

at 100” for 30 minutes.

lytical values support the supposition that reduction of intermediate III

(Fig. 1) can occur at the expense of o-aminobenzaldehyde, which is thereby

oxidized to the anthranilic acid, and argue in favor of the proposed mechan-

ism of Fig. 1 for kynureninase action. The low yield of products under

these conditions is to be expected from the many other reactions that

serine undergoes under these conditions and the transitory existence of

intermediate III. At an enzyme surface, where II and III would coexistin close juxtaposition at the moment of their formation in a specifically

directed reaction, no such impediments to the oxidation-reduction would

exist.

Pyridoxal-Catatyxed Formation of Alanine from Serine and Sodium Thio-

glycolate--As added evidence that, intermediat)e III (Fig. I) can be reduced,

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5. B. LONGENECKER AND E. E. SNELL 233

and to throw additional light on the observed formation of alanine from

cysteine by a cysteine desulfhydrase in the presence of excess substrate

(13), sodium thioglycolate was substituted for o-aminobenzaldehyde as the

reducing agent in t,he above reaction mixtures. The results (Table II)

indicate that thioglycolate is more efficient than o-aminobenzaldehyde in

reduction of intermediate III to yield alanine. Significantly, the pH opti-

mum for the reduction is the same in t,he two cases. Again, pyridoxal and

alum were required for the reaction. The result confirms the mechanism

suggested for alanine production from cysteine by Ohigashi et al. (13) with

the modification that the Schiff base of cysteine with pyridoxal (or the

TABLE II

Production of Alan ine by Reaction of Serine with Sodium Thioglycolate in Presence

of Pyridoxal and Alum

PHI

Compound omitted from reaction m ixturetFormed by reduction

with sodium

None 1 ~odium thiogl~~~ thiog’ycolate

Alanine formed, y per ml.

3.5 G2 13 49

4.0 52 12 40

5.0 39 16 23

6.0 19 9 10

* The same buffers listed in Tab le I were use d in these reaction mixtures.

t The reaction mixtures 0.04 M in sodium thioglycolate, 0.02 M in serine, 0.01 M

in pyridoxal, and 0.002 M in KAI(SOa)t;HeO at the indicated pH values were heated

at 100” for 30 minutes.

pyridoxal phosphate-activated cysteine desulfhydrase) replaces the free

amino acid. By providing additional evidence for the type of oxidation-

reductions indicated in Fig. 1, the result also supports the proposed mecha-

nism for kynureninase action, It may be noted here that intermediate III

has also been “trapped” by reaction with indole to form tryptophan in a

pyridoxal-catalyzed reaction (4).

Reaction of Kynurenine with Pyridoxal in Presence of Alum-It was hoped

that a direct demonstration of the reactions of Fig. 1 might be achieved in

model systems containing kynurenine, pyridoxal, and metal ions. How-

ever, in the limited number of experiments permitted by supplies of kynure-

nine, neither alanine nor anthranilic acid could be detected in reaction

mixtures heated at pH 3.5 or 5.0. Instead, kynurenic acid, presumably

formed by spontaneous ring closure of o-aminobenzoylpyruvic acid pro-

duced from kynurenine by transamination with pyridoxal, was formed in

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234 KYNURENINASE ACTION

equimolar amount with pyridoxamine. For example, a reaction mixture

0.04 M in kynurenine, 0.01 M in pyridoxal, 0.002 M in potassium alum, and

containing acetate buffer of 0.1 ionic strength, pH 5.0, was heated at 100”for 30 minutes. Analysis of the mixture showed that 10 per cent of the

kynurenine had been transformed to kynurenic acid, with formation of an

equimolar amount (3.9 pmoles per ml.) of pyridoxamine. Either the

kynureninase reaction does not occur in model systems under these condi-

tions, or it occurs relatively slowly.

SUMMARY

The feasibility of a mechanism for the splitting of kynurenine by kynur-

eninase that permits visualization of this reaction in terms of the general

mechanism previously proposed (4) for vitamin Bs-catalyzed reactions

was tested in model reactions. This mechanism involves an cr , p elimina-

tion of the elements of o-aminobenzaldehyde from the Schiff base of kynur-

enine with kynureninase, with the formation of a Schiff base of the pyri-

doxal phosphate enzyme with a-aminoacrylic acid. Oxidation-reduction

on the enzyme surface between the latter Schiff base and o-aminobenzalde-

hyde, followed by hydrolysis, yields alanine and anthranilic acid.

The proposed Schiff base of a-aminoacrylic acid and pyridoxal wasgenerated in model systems by heating serine with pyridoxal and aluminum

ions. It was shown that alanine formation under these conditions was

enhanced by addition of o-aminobenzaldehyde, with the simultaneous

formation of anthranilic acid. Substitution of sodium thioglycolate for

o-aminobenzaldehyde increased alanine formation even more, thus empha-

sizing the possibility of the proposed oxidation-reduction step in kynure-

ninase action and providing experimental evidence in support of the pro-

posed explanation (4) for the observed (13) formation of alanine duringenzymatic desulfhydration of cysteine.

When kynurenine, pyridoxal, and aluminum ions are heated at pH 3.5

or 5.0, kynurenic acid and pyridoxamine are formed in equimolar amounts.

Kynurenic acid must arise by spontaneous ring closure of o-aminobenzoyl-

pyruvic acid formed by transamination between kynurenine and pyridoxal.

BIBLIOGRAPHY

1. Braunstein, A. E., Goryachenkova, E. V., and Paskhina, T. S., Biokhimiya, 14,

163 (1949).

2. Miller, I. L., and Adelbe rg, E. A., J. Biol. Chem., 206, 691 (1953).

3. Jakoby, W. B., and Bonner, D. M., J. Biol. Chem., 206, 699 (1953).4. Meteler, D. E., Ikawa, M., an d Sn ell, El. E., J. Am . Chem. Sot., 76, 648 (1954).

5. Brau nstein, A. E., and Shemyakin, M. M., Biokhimiya, 16, 393 (1953).

6. Metaler, D. E:., Longenecke r, J. B., and Sn ell, E. E., J. Am . Chewe. Sot., 76, 639(1954).

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J. B. LONGENECKER AND E. E. SNELL 235

7. Metzler, D. E., and Sn ell, E. E., J. Am. Chem. Sot., 74,979 (1952).

8. Sm ith, L. I., and Opie, J. W., Org. Synth eses, 28, 11 (1948).

9. Steele, B. F., Saub erlich, H. E., Reynolds, M. S., and Baumann , C. A., J. Bio l.

Chem ., 177, 533 (1949).10. Saub erlich, H. E., and Baumann, C. A., J. BioZ. Chem ., 177, 545 (1949).

11. Sne ll, E. E., Arch. Biochem., 2, 389 (1943).

12. Metzler, D. E., and Sn ell, E. E., J. Biol. Chem ., 198,353 (1952).

13. Ohigashi, K., Tsun etoshi, A., Uchida, M., and Ichihara, I., J. Bioche m., Japan,

39, 211 (1952).

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