The synthesis of hippuric acid from benaoic acid and glycine resembles in several respects the synthesis of a peptide bond. A CONH group is formed, it is a to a carboxyl group, and the free energy of its formation is of the same order of magnitude. The synthesis was demonstrated in liver and kidney slices of a number of animals; it is inhibited by 0.001 M KCN, which is in accord with the view that the necessary free energy is derived from an oxidation.(1

Borsook, Henry

Dubnoff, Jacob W.

English

Caltech Authors - Main

SYNTHESIS OF HIPPURIC ACID IN LIVER HOMOGENATE* 
Sk.% 
The synthesis of hippuric acid from benaoic acid and glycine resembles in 
several respects the synthesis of a peptide bond. A CONH group is formed, 
it is a to a carboxyl group, and the free energy of its formation is of the 
same order of magnitude. The synthesis was demonstrated in liver and 
kidney slices of a number of animals; it is inhibited by 0.001 M KCN, which 
is in accord with the view that the necessary free energy is derived from an 
oxidation’ 
It has not been possible with tissue slices to elucidate further the energy- 
coupling reaction. The cell walls evidently retarded or blocked entry into 
the cells of possible free energy donors. Consequently we have sought 
experimental conditions which permit the reaction to proceed in tissue 
extracts in which the cells were disrupted. 
We have now obtained synthesis of hippuric acid from benzoic acid and 
glycine in homogenized guinea pig liver suspended in a phosphate-saline 
solution. To obtain more than traces of synthesis it was necessary to 
homogenize the liver in the presence of the substrate, i.e. of benzoic acid 
and glycine. The yield of hippuric acid was nearly doubled when adenylic 
acid and cr-ketoglutaric acid were added. Evidently the oxidation of the 
latter substance provided ATP2 which in turn furnished the free energy for 
the synthesis. This surmise is based on experiments on the methylation 
of guanidoacetic acid by methionine, a reaction which requires ATP.3 
The reaction mixture contained in 500 ml. 10 gm. of homogenized guinea 
pig liver, 0.02 M glycine, 0.01 M benzoic acid, 0.001 M adenylic acid, and 
0.01 M cr-ketoglutaric acid in a phosphate-saline solution.4 The reaction 
was carried out under oxygen at 38”. The same results were obtained at 
pH 7.0 and 7.5. Hippuric acid was determined by isolation and identified 
by crystal form, melting point, and elementary analysis. 
Some characteristic findings appear in the table. 
Thermodynamic data give the equilibrium constant for the hydrolysis 
of hippuric acid under our experimental conditions as 211;’ accordingly the 
amount of hippuric acid to be expected from 0.02 M glycine and 0.01 M 
benzoic acid by mass action alone is 0.026 mg. The isolation of 30 mg. 
* Carried out under Contract N6-ORI-102, Task Order No. 2, United States 
Navy Department. 
1 Borsook, H., and Dubnoff, J. W., J. Biol. Chem., 132, 307 (1940). 
2 Ochoa, S., J. Biol. Chem., 166, 87 (1944). 
8 These findings will be reported shortly. 
4 Cohen, P. P., and Hayano, M., J. Biol. Chem., 166,251 (1946). 
397 
398 LETTERS TO THE EDITORS 
Hippuric acid isolated 
Liver alone 
“ homogenized first and complete reaction mixture 
added subsequently 
0 
o-3 
Liver homogenized with benzoic acid and glycine 
“ ‘I “ “ “ glycine, adenylic 
acid, and cu-ketoglutaric acid, 0 time 
Liver homogenized with benzoic acid, glycine, adenylic 
acid, and cu-ketoglutaric acid, 4 hrs. incubation 
17 
0 
30 
indicates that the synthesis had occurred through coupling with an energy- 
yielding reaction, presumably ATP. Under our experimental conditions 
the isolation is not quantitative; at least 10 mg. are not recovered. 
Experiments on the methylation of guanidoacetic acid indicated that 
guinea pig liver homogenate provides some ATP. This probably accounts 
for the yield of 17 mg. of hippuric acid when no adenylic acid and a-keto- 
glutaric acid were added to the reaction mixture. 
William G. Kerckhofl Laboratories of the Biological Sciences 
California Institute of Technology 
Pasadena 
HENRY BORSOOK 
JACOB W. DUBNOFF 
Received for publication, March 8, 1947 


Synthesis of hippuric acid in liver homogenate

https://authors.library.caltech.edu/11777/1/BORjbc47a.pdf

Synthesis of hippuric acid in liver homogenate

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