A summary of the little that is known of the metabolism of lysine in animals is as follows: it is indispensable in the diet, its α-amino group does not participate in reversible transamination reaction in vivo (2), neither the L nor D form is attacked by the appropriate amino acid oxidase, certain ε-nitrogen-substituted derivatives can replace lysine in the diet and their α-amino groups are oxidized by amino acid oxidases (3, 4), no α-nitrogen-substituted derivatives yet prepared can substitute for lysine in the diet (4-6)

Borsook, Henry

Deasy, Clara L.

Haagen-Smit, A. J.

Keighley, Geoffrey

Lowy, Peter H.

English

Caltech Authors - Main

The degradation of L-lysine in guinea pig liver homogenate: formation of alpha-aminoadipic acid

In continuation of our study of the metabolism of L-lysine, α-aminoadipic acid, which is formed from lysine in guinea pig liver homogenate (1), was synthesized with C14 in the ε-position. The metabolism of the latter compound was followed by search for the radioactive tracer among the probable metabolic products. Two have been identified, α-ketoadipic and glutaric acids

THE DEGRADATION OF (r-AMINOADIPIC ACID IN GUINEA 
PIG LIVER HOMOGENATE* 
BY HENRY BORSOOK, CLARA L. DEASY, A. J. HAAGEN-SMIT, GEOFFREY 
KEIGHLEY, AND PETER H. LOWY 
(Frona the Kerckho$ Laboratories of Biology, Calijornfa Institute of Technology, 
Pasadena) 
(Received for publication, June 21, 1948) 
In continuation of our study of the metabolism of L-lysine, ar-amino- 
adipic acid, which is formed from lysine in guinea pig liver homogenate (1)) 
was synthesized with Cl4 in the e-position. The metabolism of the latter 
compound was followed by search for the radioactive tracer among the 
probable metabolic products. Two have been identified, a-ketoadipic and 
glutaric acids. 
The accompanying diagram (I) indicates one of the pathways of the 
catabolism of lysine. The asterisk indicates the position of the labeled 
carbon. 
H& *-NH2 
I 
CH2 
I 
CH2 - 
I 
C& 
I 
HC-NH2 
I 
COOH 
Lysine 
C*OOH C*OOH 
I I 
CH2 CHz 
-* CH2 __) CHz __) 
CH2 CHz 
HC-NH2 c=o 
I I 
COOH COOH 
~Aminoadipic acid a-Ketoadipic acid 
(1) 
Preparations 
C*OOH 
I 
CH2 
CHz 
CHz 
C*OOH 
Glutaric acid 
A summary of the synthesis and resolution of a-aminoadipic acid labeled 
with Cl4 in the E position is shown in diagram (II). The radioactive carbons 
are marked with an asterisk. 
* This work is part of that done under contract with the Office of Naval Research, 
United States Navy Department. It was reported at the meeting of the American 
Society of Biological Chemists, March 1.519,1948. 
The CY used in this investigation was supplied by the Monsanto Chemical Com- 
pany, Clinton Laboratories, Oak Ridge, Tennessee, and obtained on allocation from 
the United States Atomic Energy Commission. 
1395 
1396 DEGRADATION OF a-AMINOADIPIC ACID 
COO’&Ha 
BaC*03 + C*02 + KC*N -+ NC*.CH2.CH,.CHz+Cl+ NC*.(CH&&COOC& 
r-Chlorobutyronitrile ii . . 
o=c c=o 
//--A 
\/ 
Diethyl (3-cyanopropylphthalimido) malonate 
+ HOOC*. CHI. CH2. CHI. CH(NHzJ . COOH 
cy-Aminoadipic acid 
(II) 
The details of the preparation have been described in a previous com- 
munication (1). 
89.3 mg. of BaU40z containing 0.625 millicurie of Cl4 yielded 0.127 gm. 
of pure L-ar-aminoadipic acid, giving 14,450 counts (corrected) per mg. per 
minute. 
a-Ketoadi@c A&&-Diagram (III) is a summary of the synthesis of 
a-ketoadipic acid. 
HOOC.(CH&.COOH--+ C~Hr.OOC.(CH2)3.COO.C2H5~ 
Glutaric acid Diethyl glutarate 
4 C~Hs~COO~CO~CH~(CH~)a~COO~CzH~--,HOOC~(CH~)~~CO~COOH 
COO+CzHs 
Triethyl 2-oxaloglutarate 
(III) 
or-Ketoadipic acid 
Diethyl glutarate was prepared by the method of Locquin and Elghozy 
(2), and triethyl 2-oxaloglutarate by the method of Gault (3). The latter 
compound was decarboxylated with HCI. After removal of the HCl 
in vacua spontaneous crystallization set in. The crude product was puri- 
fied by solution in warm ether and reprecipitation by the addition of an 
equal volume of petroleum ether. The light orange crystals had the fol- 
lowing composition. 
CsHa06 (160.06). M.p. 124”. Calculated, C 44.98, H 5.04; Found, C 45.12, II 5.04 
Procedure 
The preparation of the guinea pig liver homogenate and the saline solu- 
tion are described in a previous communication (1). 
In a representative experiment 2 ml. of homogenate containing 0.66 gm. 
of liver (wet weight) and 2 ml. of saline solution containing a mixture of 
5 mg. of L-ar-aminoadipic acid (14,450) counts (corrected) per minute per 
mg.) and 5 mg. of non-radioactive L-a-aminoadipic acid were added to 
BOBSOOK, DEASY, HAAGEN-SMIT, KEIGHLEY, AND LOWY 1397 
each of four 20 ml. beakers. Two were immediately brought to pH 5.0 
and boiled for 10 minutes, the coagulated protein extracted with water, 
and the non-protein filtrates and washings combined. The other two 
beakers were incubated at pH 7.4 under oxygen at 38” for 6 hours, then 
deproteinized by boiling at pH 5.0, and their non-protein filtrates combined. 
Results 
The non-protein filtrates were cleared by boiling with charcoal, acidified 
with hydrochloric acid to a concentration of 0.1 N, and then extracted with 
ether. After the ether was evaporated, the residue was taken up in 3 m1. 
of water; 100 mg. of non-radioactive cy-ketoadipic acid and 91 mg. of 
phenylhydrazine hydrochloride dissolved in 2 ml. of water were added. 
The oil, cr-ketoadipic acid phenylhydrazone, which settled out first, crys- 
tallized in 2 hours. The recrystallization procedure was as follows: 2 ml. 
TABLE I 
Specific Radioactivity of cy-Ketoadipic Acid Phenylhydrazone 
after Successive Recrystahations 
No. of crystallizations 
T- Counts (corrected) per mg. per min. of phenylhydrazone from - 
Reaction mixture at zero time Reaction mixture after 6 hrs. incubation 
1.8 10.1 
1.0 3.1 
0 2.7 
0 2.6 
0 2.7 
0 2.8 
of water were added to the crystals, and the suspension brought to boil and 
then treated with ethanol dropwise until all the crystals dissolved. The 
solution was then cooled, the crystals collected, and their specific radio- 
activity determined. 
Table I gives the specific radioactivities of the phenylhydrazone sampIes 
obtained from the reaction mixtures at zero time and after 6 hours incuba- 
tion. The figures show that by the second recrystallization all the radio- 
activity had been removed from the phenylhydrazone obtained from the 
zero time reaction mixture, and that constant specific activity had been 
attained in the case of the phenylhydrazone obtained after 6 hours incuba- 
tion. The counts in the first and second crystallizations probably arose 
from ar-aminoadipic acid, and, in the case of the 6 hour reaction mixture, 
also from glutaric acid arising from degradation of the cr-ketoadipic acid 
formed from the added a-aminoadipic acid. 
1398 DEGRADATION OF a-AMINOADIPIC ACID 
The melting point of the radioactive phenylhydrazone after the fifth 
crystallization was 141-142”, and of a mixture with an authentic sample 
141-142’. Gault (3) reported the melting point as 141’. After the final 
recrystallization, 15 mg. of the phenylhydrazone remained. 90 per cent 
of the added cr-ketoadipic acid was, therefore, left in the mother liquors. 
These data prove that the or-aminoadipic acid was oxidatively deaminized 
to a-ketoadipic acid. 
The rate of formation of oc-ketoadipic acid from a-aminoadipic acid can 
be calculated from the value of the constant specific activity of the 
cr-ketoadipic acid phenylhydrazone obtained from the reaction mixture 
incubated for 6 hours. The specific activity of the phenylhydrazone was 
2.7 counts (corrected) per mg. per minute. 100 mg. of a-ketoadipic acid, 
equivalent to 156.3 mg. of its phenylhydrazone, were added as a carrier. 
The weight of the radioactive form being neglected, the tot’al a-ketoadipic 
TABLE II 
Specijk Radioacitivit~ of Barium Glutarate (Dried at 100’) after 
Successive Reprecipitations 
Counts (corrected) per mg. per min. of barium glutarate from 
No. of precipitations 
Reaction mixture at zero time 
0 
0 
Reaction mixture after 6 hrs. 
incubation 
- 
1.50 
1.33 
1.30 
0.95 
1.01 
1.01 
acid in the solution contained 156.3 X 2.7 = 422 counts (corrected). 
144,500 counts (corrected) were added originally as 20 mg. of ol-aminoadipic 
acid. 0.29 per cent of the latter was found as the keto acid, or 0.058 mg. 
Expressed as a & value,’ this rate is 0.005. It is about one-twelfth that of 
the formation of a-aminoadipic acid from L-lysine (1). 
In another experiment similar to the preceding, the non-protein filtrate 
was examined for evidence of formation of glutaric acid. The procedure 
was the same as before to the stage after evaporation of the ethereal extract 
of the non-protein filtrate. 100 mg. of non-radioactive glutaric acid were 
added as a carrier in the subsequent crystallization. The mixture was 
taken up in 3 ml. of water and treated with saturated barium hydroxide 
1 & is the rate of change of the substance in question expressed as if it were a gas in 
c.mm., at standard temperature and pressure, per mg. of dry weight of tissue used per 
hour. 
BORSOOK, DEASY, HAAGEN-SMIT, KEIGHLEY, AND LOWY 1399 
solution to pH 9.0; the final volume was 5 ml. No precipitation occurred. 
10 ml. of 95 per cent ethanol were then added. The precipitate of barium 
glutarate pentahydrate was filtered off. 96 per cent of the water of hydra- 
tion was driven off by drying at loo”, and the specific radioactivity of the 
compound was determined. 
The solution in water and precipitation with ethanol were repeated five 
times. Table II gives the specific radioactivities of the barium glutarate 
samples obtained as above from the reaction mixtures at zero time and after 
6 hours incubation. After the final reprecipitation 30 mg. of barium salt 
(as pentahydrate) remained, corresponding to 11 per cent of the original 
glutaric acid added. 
Analysis of the radioactive barium salt after the last precipitation gave, 
on an anhydrous basis, 51.3 per cent of barium (calculated, 51.4 per cent). 
These data prove that glutaric acid was one of the products formed in 
the liver homogenate from the added ar-aminoadipic acid. There can be 
little doubt that cr-ketoadipic acid was formed first, and that the glutaric 
acid arose by its oxidative decarboxylation. 
The calculation of the rate of formation of glutaric acid from the added 
ac-aminoadipic acid is as follows: the final constant specific activity of the 
barium glutarate was 1.0 count (corrected) per mg. per minute; 100 mg. of 
glutaric acid, equivalent to 202.5 mg. of anhydrous barium glutarate, were 
added as carrier; the glutaric acid formed from the added ar-aminoadipic 
acid contained, therefore, 202 counts (corrected) per minute; 144,500 counts 
were contained in the a-aminoadipic acid; 0.14 per cent of the latter was, 
therefore, found as glutaric acid. The & value is 0.0024. 
Of the two successive oxidative steps in the degradation of cr-aminoadipic 
acid, deamination followed by decarboxylation, the latter is probably the 
faster. 0.43 per cent of the added cr-aminoadipic acid was found in the 
above two degradation products. This figure represents the rate of its 
deamination. The corresponding rate of decarboxylation was 0.14/0.43 
or 33 per cent. 
SUMMARY 
1. The synthesis of cr-ketoadipic acid is described. 
2. In guinea pig liver homogenate cu-aminoadipic acid is oxidatively 
deaminized to a-ketoadipic acid and the latter is oxidatively decarboxylated 
to glutaric acid. 
3. The deamination of a-aminoadipic acid is much slower than its forma- 
tion from L-lysine. The decarboxylation of ar-ketoadipic acid is faster than 
the deamination of a-aminoadipic acid. 
4. The foregoing evidence was obtained with the use of lysine and of 
cr-aminoadipic acid labeled with Cl4 in the E position. 
1400 DEGRADATION OF a-AMINOADIPIC ACID 
The elementary analyses were carried out by Dr. G. Oppenheimer. 
The authors were assisted in this work by A. A. Dvorsky, D. Eggarter, 
H. E. Jeffery, and A. Tollestrup. 
BIBLIOGRAPHY 
1. Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G, and Lowy, P. %I., 
J. Biol. Chem., 173,423 (1948); 176,1383 (1948). 
2. Locquin, R., and Elghozy, F., Bull. Sot. chim., 41,445 (1927). 
3. Gault, H., Bull. Sot. chim,, 11,382 (1912). 


The degradation of L-lysine in guinea pig liver homogenate: formation of alpha-aminoadipic acid

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