ARTICLE: THE ENZYMATIC CONVERSION OF HISTIDINE TO GLUTAMIC ACID Herbert Tabor and Osamu Hayaishi

J. Biol. Chem. 1952, 194:171-175.

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THE

ENZYMATIC

CONVERSION OF HISTIDINE GLUTAMIC ACID

TABOR AND OSAMU HAYAISHI* Diseases, Bethesda, 18, 1951) National

TO

BY HERBERT (From the National of Health,

Institute of Arthritis United States Public (Received for

and Metabolic Health Service, August

Institutes

Maryland)

publication,

Certain discrepancies exist in the reported evidence concerning the mechanism of the enzymatic degradation of histidine. Edlbacher and his collaborators (1,2) showed that liver preparations converted L-histidine to ammonia and an unidentified labile intermediate from which L-glutamic and formic acids could be obtained by acid hydrolysis. Sera and Yada (3) and Takeuchi (4), on the other hand, demonstrated that histidine was converted to urocanic acid, which was then degraded to optically inactive isoglutamine. An intermediate product of this reaction, proisoglutamine, isolated by Sera and Aihara (5), was levorotatory, while Oyamada (6) obtained a product which he identified as racemic formylisoglutamine. Oyamada could not demonstrate an enzymatic hydrolysis of fOrIT@-DLisoglutamine, although he could convert it oxidatively to CO2 and optically inactive isoglutamine with a mixture of defibrinated blood and liver homogenate. Although glutamic and formic acids could be obtained by the acid hydrolysis of the reaction products of histidine metabolism, no experimental evidence has been presented for the direct enzymatic production of Lglutamic and formic acids (2, 7). In fact, recent papers have questioned the formation of L-glutamic acid from histidine in mammalian systems both ;In vitro and in viva (7-9). In this paper we shall describe the preparation of a cell-free extract from Pseudomonas jihorescens which catalyzes the quantitative conversion of L-histidine to L-glutamic acid, formic acid, and 2 moles of ammonia. r,-Glutamic acid hydrochloride has been isolated in crystalline form from the incubation mixture.
* Special Research Fellow. 1 Miyahara and Suda (personal communication) of Osaka University have recently shown that glutamic acid is formed from either histidine or urocanic acid by resting cell suspensions or acetone-dried cells of histidine-adapted Pseudomonas. Glutamic acid was measured by glutamic decarboxylase of Escherichia coli. Neither formyl-nn-isoglutamine nor nn-isoglutamine was decomposed by their Pseudomonas preparations. 171

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172

HISTIDINE

METABOLISM

Methods and Materials Histidine was determined by a modified Pauly diazotization reaction, as previously described; n-butanol was used to extract the final color (10, 11). Ammonia was distilled from the incubation mixture (made alkaline with 0.5 volume of saturated potassium carbonate) into 0.1 N sulfuric acid by a stream of air for 40 minutes at 28. Tributyl citrate (Commercial Solvents Corporation) was used as an antifoaming agent. The ammonia was then determined in the distillate by nesslerization. L-Glutamic acid was determined in the crude incubation mixture by the COZ evolved upon treatment with a resting cell suspension of Clostridium welchii, strain SR 12,2 in the presence of cetyl trimethylammonium bromide (12,13), as measured in conventional Warburg manometers. Glutamic acid was crystallized and identified as described below. Formic acid was determined by the ceric sulfate-palladium method of Pickett, Ley, and Zygmuntowicz (14). The formic acid was sublimed from the frozen incubation mixture in vacua (15) after acidification (to Congo red) with phosphoric acid. Paper chromatography of amino acids was performed by an ascending method, the spots being located by reaction with ninhydrin. C, H, and N analyses were performed by the Microanalytical Laboratory of the National Institutes of Health, under the direction of Dr. William C. Alford. L-Hi&dine Monohydrochloride, L-Glutamic Acid Hydrochloride, and LGlutamine were commercial preparations (Eastman Kodak Company and Nutritional Biochemicals Corporation). Dowex-50, a cation exchange resin, was obtained from The Dow Chemical Company and washed repeatedly with 4 N HCl and then with water. Chromatography columns (height 12.5 cm.; diameter 1 cm.) were prepared from 200 to 500 mesh material. Preparation of Cell-Free Extract-P. jkorescens, strain 6, was grown on a medium consisting of 0.15 per cent KEHPOJ, 0.05 per cent KHZPOJ, 0.02 per cent MgS04, 0.2 per cent L-histidine monohydrochloride, and 0.1 per cent Bacto yeast extract in distilled water. After being shaken mechanically in air for 16 hours at 28, the cells were harvested in a Sharples centrifuge, washed with a 0.5 per cent NaCl-0.5 per cent KC1 mixture, and stored at - 10. 0.8 gm. of wet cells was obtained per liter of culture. To prepare the cell-free extract, 5 gm. of frozen cells were ground with 10 gm. of alumina (Alcoa A-301) in a chilled mortar for 5 minutes, extracted with 25 cc. of a 0.01 M phosphate buffer, pH 7.0, and centrifuged at 18,000 r.p.m. in the high speed angle head of the International refrigerThe residue was extracted with ated centrifuge at 0 for 10 minutes. 20 cc. of the same buffer, centrifuged, and the clear supernatant fluids
* Kindly supplied by Dr. Alton Meister.

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H.

TABOR

AND

0.

HAYAISHI

173

were combined. 0.1 cc. of this extract (2 mg. of protein) in a volume of 1 cc. at 28 at pH 7.4 catalyzed the destruction of 3 PM of histidine in 40 minutes. Results 600 PM of L-histidine droxide) were incubated
Assays of Histidine
Assay

monohydrochloride (neutralized with sodium hywith 39 cc. of cell-free extract and 161 cc. of water
TABLE

Incubation

I Mixture

after 110 Minutes

Experimental PM

at 98
Control*

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PM

Ammoniat.. . . . . .. . Formic acidI. . . . . . . . . . . . . Glutamic acids. . . . . . . . . Histidine disappearance. . . O2 consumption// . .. CO2 production11 . .

...... ...... ...... ...... ... ... .. ...

...... ...... ...... ...... .. ... .

1200 462 584 6007 0 60

40 0 8 0 0 40

* Incubation mixture identical with the experimental mixture, except for the omission of histidine. t Under conditions of the ammonia assay, essentially no ammonia (<0.5 per cent) is released from glutamine. $ Similar results were obtained by the calorimetric method of Grant (15). 8 The C. welchii preparation decarboxylates n-glutamic acid and n-glutamine, but does not attack the D isomers or isoglutamine (12, 13). Further evidence for the presence of n-glutamic acid in the reaction mixture was indicated by the reduction of diphosphopyridine nucleotide upon addition of the crystalline glutamic dehydrogenase of Olson and Anfinsen. We wish to thank Dr. Olson and Dr. Anfinsen for supplying us with this enzyme, which is to be described in a forthcoming publication. 11 Measured in conventional Warburg apparatus on aliquots of similar incubation mixtures. % No histidine disappeared in a similar reaction mixture if the bacterial extract had been previously heated to 100 for 5 minutes.

at 28 for 110 minutes. At this time, 20 cc. of the incubation mixture were removed for analysis and for paper chromatography. As indicated in Tables I and II, all of the added histidine had disappeared with the formation of 462 PM of formic acid, 576 PM of L-glutamic acid, and 1160 I*M of ammonia. To isolate glutamic acid, 180 cc. of the solution (representing 540 FM of histidine) were treated with 27 cc. of 36 per cent trichloroacetic acid and filtered. The precipitate was washed with 20 cc. of 5 per cent trichloroacetic acid. The combined filtrates were extracted three times with equal volumes of ether, and the aqueous solution was passed through a

174

HISTIDINE

METABOLISM

Dowex-50 column. The column was washed with water and eluted with 1 N HCI. The fractions containing the amino acid (as determined by a ninhydrin spot test on paper) were evaporated to dryness in vacua over KOH and HzS04. The residue was dissolved in 1 cc. of water, treated with 2 cc. of 0.5 M calcium chloride, and then NaOH was added until the reaction was alkaline to phenol red. 10 volumes of absolute ethanol were added, the solution stored at 0 overnight, and the precipitate collected by centrifugation. The precipitate was dissolved in water, adsorbed on a fresh Dowex-50 column, washed with water, and eluted with 1 N HCl. The fractions giving a ninhydrin reaction were evaporated to dryness in vacua over KOH.
TABLE

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II

Paper
Solvent No

Chromatography
Compound RF

Ethanol Water Tertiary butanol Formic acid Water Lutidine Ethanol Water Diethylamine

per cent 77 23 70 15 15 55 20 24 1

Incubation Authentic Incubation Authentic Incubation Authentic

II

III

mixture glutamic glutamine mixture glutamic glutamine mixture glutamic glutamine

acid

acid

acid

0.46 0.43 0.37 0.57 0.56 0.39 0.34 0.38 0.45

* 16 hours at 26-28. Filter paper, Schleicher

and

Schtill

No.

598.

The white crystalline residue weighed 75 mg. (equivalent to 409 pM or 74 per cent of theory). Melting Point (Corrected)-204-210 with decomposition. Authentic glutamic acid hydrochloride melted at 209-216 with decomposition; mixed melting point, 208-214 with decomposition. Optical Rotation--[ac]~ = i-31.9 (0.1 M solution in 5 N HCl) ((16) [# = +31.9y.
Analysis-CsHpOrN.HCl. Calculated. Found.
SUMMARY

C 32.71, 32.92,

H 5.49, 5.32,

N 7.63 7.45

A cell-free extract of Pseudomonas jluorescens, grown on a histidinecontaining medium, has been shown to catalyze the reaction, L-histidine + 4Hz0 -+ L-glutamic acid + HCOOH + 2NH3.

H.

TABOR

AND

0.

HAYAISHI

175

BIBLIOGRAPHY

1. Edlbacher, S., 2. physiol. Chem., 157, 106 (1926); Edlbacher, S., and Kraus, J., 2. physiol. Chem., 191, 225 (1927); 195, 267 (1931). 2. Leuthardt, F., in Sumner, J. B., and Myrbtick, K., The enzymes, New York, 1, pt. 2, 1156 (1951). 3. Sera, K., and Yada, S., Mitt. med. Ges. Osaka, 38, 1107 (1939). Cited by Kotake, Y., 2. physiol. Chem., 270, 38 (1941). 4. Takeuchi, M., J. Biochem., Japan, 34, 1 (1941). 5. Sera, II., and Aihara, D., Mitt. med. Ges. Osaka, 41, 745 (1942). 6. Oyamada, Y., J. Biochem., Japan, 36, 227 (1944). 7. Parshin, A. N., and Goryukhina, T. A., Biokhimiya, 15, 499 (1950). 8. Tesar, C., and Rittenberg, D., J. BioZ. Chem., 170, 35 (1947). 9. DIorio, A., and Bouthillier, L. P., Rev. canad. biol., 9, 338 (1950). 10. Gebauer-Fulnegg, E., 2. physiol. Chem., 191, 222 (1930). 11. Rosenthal, S. M., and Tabor, H., J. Phurmucol. and Exp. lherap., 92,425 (1948). 12. Gale, E. F., Biochem., J., 39, 46 (1945). 13. Meister, A., Sober, H. A., and Tice, S. V., J. BioZ. Chem., 189, 577 (1951). 14. Pickett, M. J., Ley, H. L., and Zygmuntowicz, N. S., J. Biol. Chem., 156, 303

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(1944). 15. Grant, W. M., Ind. and Eng. Chem., Anal. Ed., 18, 729 (1946); Anal. Chem., 19, 206 (1947). 16. Levintow, L., Greenstein, J. P., and Kingsley, R. B., Arch. Biochem. and Biophys., 31, 77 (1951).