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The enzymic deamination and amide cleavage of folic acid
ARCHIVES
OF
BIOCIIEMISTRY
AND
The Enzymic
BIOPHYSICS
101,
Deamination
l-6
(1963)
and Amide
Cleavage
of Foiic Acid’
WALTER S. McNUTT2 From the Department of Pharmacology,
Tufts
University
School of Medicine,
Boston,
Massachusetts
Received January 16, 1963 Crude enzyme, prepared from cells of Alcaligenes jaecalis which had been cultured in response to xanthopterin, catalyzed the deamination of folic acid and the splitting of the amide bond of folic acid. “Deaminated folic acid,” pteroic acid, and “deaminated pteroic acid” were characterized as enzymic products derived from folic acid. Pteroic acid was deaminated by the enzyme preparation, whereas rhizopterin was not attacked at an appreciable rate. INTRODUCTION
tally
faecalis
FOLIC
ACID”
prepared specimens at pH
PTEROIC ARID
(5)
The crude product (0.5 g.), prepared as described (5), was taken up in hot water and NHdOH, and the cooled solution (pH 7.0) was added to a DEAEcellulose column 7.8 X 27 cm. The column was developed with a gradient of 12.5 1. water and 12.5 I. of 0.7 M NHHC03. Pteroic acid passed from the columns at about 0.63 M. The solution was evaporated to dryness, and the residue was taken up in 40 ml. of corm. HCI at room temperature, treated with a little Norit, and filtered. Water (750 ml.) was added to the filtrate, and the crystals which separated were washed with water, ethanol, and ether, and dried. Fifty-two milligrams of pteroic acid was obtained. Its absorption spectrum agreed with the published value (5). On a paper chromatogram (Table I) it gave a single ultraviolet-absorbing spot.
EXPERIMENTAL “DEAMINATED
and biologically
7.5 is shown in Fig. 2.
has the ability to utilize certain pteridines and purines as substrates for growth (1). Although folic acid is not effectively utilized as a nitrogen source by this organism (2), cells which have been grown in response to xanthopterin as the nitrogen-source contain enzymes which catalyze (a) the deamination of folic acid and (b) the cleavage of the amide bond of folic acid and “deaminated folic acid.” It seems likely that folic acid is not an effective substrate because the organism is unable to convert it to tetraoxypteridine, the key intermediate (3) in the metabolism of pteridines in this organism. Alcaligenes
(4)
The chemically synthesized compound melted with decomposition at 230-232” (melting point stage). The reported value (4) is 220-224’. It showed the following ultraviolet absorption characteristics: in 0.1 N HCI, l %,?‘ 16,800and in 0.1 N NaOH, e2E8!“ 25,600 and %?’ 7480. The values calculated from the published data (4) are: in 0.1 N HCI, E’::“ 17,800 and in 0.1 N NaOH, ~‘2~:s 26,300 and c3Egs 7160. A comparison of the chemi-
RHIZOPTERIN
(6)
Formylation of 85 mg. pteroic acid gave 74 mg. rhizopterin. On a paper chromatogram (Table I) it showed a single fluorescent spot. “DEAMINATED
RHIZOPTERIN”
(6)
From 50 mg. rhizopterin, 36 mg. of “deaminated rhizopterin” was obtained. This product was contaminated with “deaminated pteroic acid,” and it was used in the chemical synthesis of “deaminated pteroic acid” without further purification. The fluorescent compound (I& 0.83) cochromatographed with the chemically formylated derivative of the
1 Supported by U. S. Public Health Service Grant No. A-3675. 2 U. S. Public Health Service Career Development Awardee. 1
McNUTT TABLE I Rf VALUES OF FOLIC ACID AND RELATED COMPOUNDSIN 5y0 AMMONIUM BICARBONATE SOLUTION Compound
Rf value
10.Formyl-“deaminated folic “Deaminated rhizopterin” Rhizopterin “Deaminated folic acid” Folic acid “Deaminated pteroic acid” Pteroic acid
acid”
0.88 0.77 0.69 0.65 0.42 0.24 0.09
enzymic product CR, 0.84), and the fluorescent band from the chromatogram had an ultraviolet absorption spectrum characteristic of pure “deaminated rhizopterin” which was prepared as follows: “Deaminated pteroic acid” (58 mg.) was heated with 30 ml. of 97y0 formic acid in a boiling water bath for 2 hr. The solution was evaporated to dryness at 55”, and the residue was taken up in 10 ml. of 1 N NHaOH and filtered. The solution at 60” was acidified with acetic acid and cooled. The white needle-like crystals were filtered off, washed with water, ethanol, and ether, and dried over HZSOI. Yield: 52 mg., m.p. 325327” (dec.). The reported value (6) is 321-323” (dec.). The substance showed a single fluorescent spot on a paper chromatogram (Table I).
“DEAMINATED
PTEROIC ACID”
(6)
“Deaminated rhizopterin” (65 mg.) was hydrolyzed with HCI, giving 50 mg. of “deaminated pteroic acid” m.p. 322-325” (dec.). On a paper chromatogram (Table I) the specimen showed a trace of fluorescent material which remained close to the origin and a single uRraviolet-absorbing spot (RI 0.24). A comparison of its ultraviolet absorption spect,rum with that of the enzymic product is shown in Fig. 4.
lo-FORMYL “DEAMINATED
FOLIC
ACID”
“Deaminated folic acid” (200 mg.) was heated with 30 ml. of 977; formic acid at 80” for 1 hr. The almost colorless solution was evaporated to dryness at 55”. The residue was crystallized from hot water, washed with water, ethanol, and ether, and dried over HZSO,. Yield: 135 mg. of white cryst,als, m.p. 185187”. It moved as a single fluorescent, spot on a paper chromatogram (Table I).
PREPARATION
OF ENZYME
Crude enzyme was prepared as described previously (2) from cells which had been cultured for
24 hr. in inorganic medium supplemented with 1% sodium succinate.6HzO and 0.0032% xanthopterin.HzO or 6-oxylumazine
THE SPECTROPHOTOMETRIC ESTIMATION THE DEAMINATION OF FOLK ACID
OF
Folic acid (133 mpmoles) and enzyme from xanthopterin-grown cells (55 pg. protein) in 1 ml. of 0.005 M Tris-HCl at pH 7.5 were incubated at 23”. The spectral changes are shown in Fig. 1. The theoretical values for the conversion of folic acid to deaminated folic acid at pH 7.5 are an isosbestic point at 343 rnF (e 7320) an increase at 330 rnr (AE -t-1630), and a decrease at 360 rnp (Ae -3300). The results (Table II) are in fair agreement. with the theoretical values. In subsequent large-scale incubations which were carried out in order to isolate the enzymic product, mixtures of “deaminated folic acid” and “deaminated pteroic acid” were obtained.
THE IDENTIFICATION OF “DEAMIXATED FOLIC ACID" .4s &4PRODUCT OF ENZYMIC ATTA4CK OF FOLIC ACID Folic acid (240 mg.) and enzyme from xanthopterin-grown cells (220 mg. prot.ein) in4 1. of 0.005 M Tris-HCl were incubated as described above, and the decrease in optical density at 360 rnp was followed. Glacial acetic acid (50 ml.) was added when approximately 707, of the folic acid had been deaminated, and the solution was evaporated to dryness at 55”. The residue was t,aken up in dilute NHdOH, and the solution was evaporated to remove excess xH,OH. The solution (pH 7) was diluted to 4 1. and chromatographed on a DEAEcellulose column (7.7 X 34 cm.). The column was developed with a linear gradient of 18 1. of 0.35 A4 NH4HC03 vs. 18 1. of 0.5 M NH,HCO,. Two ultraviolet-absorbing bands passed from the column. The first peak (0.38-0.41 M) contained “deaminated folic acid” and “deaminated pteroic acid.” The second peak (0.44-0.46 M) contained folic acid. Crystalline folic acid (55 mg.) was recovered from this peak. The first peak was evaporated to dryness, taken up in dilute KHhOH, and filtered. The solution was acidified, and the yellow gelatinous product was collected and dried. The yellow solid (110 mg.) was extracted with concentrated HCl (2 ml.), whereupon a white solid (“deaminated pteroic acid,” see below) separated from the yellow solution. The yellow solution was filtered from the white solid and added to 20 ml. water. The orangeyellow solid was collected and dried: 73 mg. It was chromatographed along 371 cm. (7 sheets) of Whatman No. 3 MM filter paper in 570 NHJHCO~ solution. The upper ultraviolet-absorbing bands
ENZYMIC
ATTACK
ON FOLIC
ACID
3
(Rf 0.60) containing “deaminated folic acid” were washed with water. The washings were reduced to 10 ml. and warmed to 60”. One milliliter of 5 N HCl was added to the solution. The orange-yellow crystals, which separated upon cooling the solution, were washed with water, ethanol, and ether, and dried over H&lOh. Yield: 25 mg., m.p. 230234” dec. The substance gave a single ultravioletabsorbing spot (Rf 0.62) on a paper chromatogram (Table I) and cochromatographed with synthetic “deaminated folic acid” (R, 0.62). Its ultraviolet absorption spectrum at pH 7.5 agreed with that of the synthetic compound (Fig. 2). THE IDENTIFICATION OF (‘DEAMINATED PTEROIC ACID” AS A PRODUCT OF ENZYMIC ATTACK OF FOLIC ACID The white solid which failed to dissolve in 2 ml. of cont. HCl (see above) was dissolved in 10 ml. of cone. HCl at room temperature. Water (50 ml.) was added, and the canary yellow crystals which separated were washed with water and ethanol and dried over H2S04. Yield: 26 mg., m.p. 325’ dec.
FIG. 2. The ultraviolet absorption spectrum of “deaminated folic acid.” Natural and ----synthetic “deaminated folic acid” at pH 7.5 in 0.05 M Tris-HCl. In 0.1 N NaOH its ultraviolet absorption char. acteristics were: s2gy 26,400, ~“2,:“ 7070. The substance gave a single ultraviolet-absorbing spot (RJ 0.24) on a paper chromatogram (Table I) and cochromatographed with synthetic “deaminated pteroic acid” (Rf 0.23). Its ultraviolet absorption spectrum at pH 7.5 agreed with that of the synthetic compound (Fig. 4). THE IDENTIFICATION OF “DEAMINATED PTEROIC ACID” AS A PRODUCT OF ENZYMIC ATTACK OF “DEAMINATED FOLIC ACID"
300
350 r-y)
400
450
FIG. 1. The spectral changes which accompany the deamination of folic acid. The heavy line is the absorption spectrum initially (folic acid), and the other lines are the absorption spectra taken at intervals over a period of 24 hr. At the end of this time about 68% of the folic acid had been deaminated.
“Deaminated folic acid” (165 mg.) and enzyme from 6-oxylumazine-grown cells (570 mg. protein) in 3 1. of 0.005 1MTris-HCl at pH 7.5 were incubated at 21” for 24 hr. The solution was poured into 3 1. of boiling ethanol, and the mixture was brought to boiling. The precipitated protein was removed, and the filtrate was reduced to 2 1. The solution was chromatographed on a DEAE-cellulose column (7.7 X 17 cm.) and developed with a linear gradient of 10 1. of 0.3 M NH~HCOI vs. 10 1. of 0.5 M NH4HCOa. The residue from the peak which passed from the column at 0.37 M was worked up as described above. “Deaminated pteroid acid”
4
McNUTT
It (58 mg.; m.p. 32&325” dec.) was obtained. showed the characteristic ultraviolet absorption spectrum at pH 7.5 and the R, value of “deaminated pteroic acid.” TABLE
II
THE SPECTROPHOTOMETRIC DETERMINATION OF FOLIC ACID DEAMINASE In this assay the decrease in optical density at 360 rnp is taken as a measure of the disappearance of folic acid, and the increase in optical density at 330 rnp is taken as a measure of the formation of “deaminated folic acid.” Folk acid present initially
Observed A O.D. at:
360m,,
330mp
m~moles 133 -0.310
+0.144
FoE;li;;id nated “DeamiI f&c disappeared frrizd
mpmoles m/.lmoles 94 88
FIG. 4. The ultraviolet absorption spectrum of “deaminated pteroic acid.” Natural and ----synthetic “deaminated pteroic acid” at pH 7.5 in 0.05 M Tris-HCl.
THE ENZYMIC
FIG. 3. Spectral changes associated with the conversion of folic acid to “deaminated pteroic acid.” Folic acid (96 mpmoles) and enzyme from xanthopterin-grown cells (260 pg. protein) in 1 ml. of 0.02 M Tris-HCl at pH 7.5 were incubated at 19”. The absorption spectrum of the incubation mixture initially (folic acid) and ----- after 20 hr. at which time 65% of the folic acid had been deaminated
CONVERSION OF FOLIC ACID TO PTEROIC ACID
Folic acid (2 pmoles) and enzyme from xanthopterin-grown cells (5 mg. protein) in 20 ml. of 0.02 M Tris-HCl at pH 7.5 were incubated at 19” for 22 hr. The solution was poured into an equal volume of boiling alcohol, and the precipitated protein was removed. The filtrate was placed along 20 cm. of Whatman No. 3 MM and chromatographed in 5% NH,HC03 solution. Only two ultraviolet-absorbing bands were detected: a principal band of “deaminated pteroic acid” (RI 0.25) and pteroic acid (Rf 0.09). The substances were eluted from the paper with 0.1 N NaOH, and the amounts present were estimated spectrophotometrically. The results are shown in Table III.
THE ENZYMIC DEAMINATION PTEROIC ACID
OF
Pteroic acid (2 pmoles) was incubated as described in the preceding section, and the mixture bands was similarly chromatographed. Two appeared on the chromatogram. The substance eluted from the principal band (R, 0.24) showed
ENZYMIC
ATTACK
ON FOLIC ACID an Rf value of 0.38, it would have appeared on the chromatogram as a fluorescent band above rhizopterin, had it been formed. RESULTS
AND DISCUSSION
Crude enzyme preparations from cells of Alcaligenes jaecalis, which had been cultured in responseto xanthopterin or 6-oxylumazine TABLE
III
THE
CONVERSION OF FOLK ACID TO PTEROIC ACID AND “DEAMINATED PTEROIC ACID”
Folk acid present initially
Pteroic acid recovered
“Deaminated pteroic acid” recovered
FIG. 5. Spectral changes which accompany the deamination of pteroic acid. Pteroic acid (0.1 #mole) and enzyme from xanthopterin-grown cells (260 pg. protein) in 1 ml. of Tris-HCI at pH 7.5 were incubated at, 19”. The heavy line is the absorption spectrum of the incubation mixture initially (pteroic acid), and the other lines are the absorption spectra taken at intervals over a period of 22 hr. at which time about 7Oyo of the pteroic acid had been deaminated.
the ultraviolet absorption spectrum characteristic of “deaminated pteroic acid.” The lower band (RI 0.09) contained unchanged pteroic acid. THE INABILITY OF THE ENZYME PREPARATION TO CATALYZE TRANSFORMYLATION BETWEEN RHIZOPTERIN AND “DEAMINATED FOLK ACID” Rhizopterin (2 pmoles), “deaminated folic acid” (10 pmoles), and enzyme from xanthopteringrown cells (5 mg. protein) were incubated in 0.02 M Tris-HCl (pH 7.5) at 19” for 7 hr. The incubation mixture was poured into boiling alcohol, and the solution was chromatographed as described above. The chromatogram showed ultraviolet-absorbing bands of “deaminated folic acid” (I& 0.61) and “deaminated pteroic acid” (R, 0.27) and a single fluorescent band of rhizopterin (Rf 0.66). As IO-formyl “deaminated folic acid” has
FIG. 6. The inability of the enzyme to attack rhizopterin. Rhizopterin (0.1 pmole) and enzyme from xanthopterin-grown cells (260 pg. protein) in 1 ml. of 0.02 M Tris-HCl (pH 7.5) were incubated at 19”. The heavy line is the absorption spectrum of the incubation mixture initially (rhizopterin), and the thin line is the absorption spectrum of the incubation mixture at the end of 24 hr. The absorption spectra of equimolar amounts of pteroic acid (-----) and “deaminated rhizopterin” (-a-e-*-) at pH 7.5 are shown.
6
McNUTT
as the source of nitrogen, catalyzed two chemical alterations of folic acid which could be followed spectrophotometrically. One change was associated with an increase in optical density at 330 rnp and a decrease in optical density at 360 rnti at pH 7.5 (Fig. l), as one would predict for the conversion of folic acid to “deaminated folic acid” (I). “Deaminated folic acid” was isolated and characterized as one of the products of enzymic attack of folic acid (Fig. 2). The other change was associated with the appearance of an absorption maximum at 335 mp at pH 7.5. This spectral shift (Fig. 3) is due to the appearance of “deaminated pteroic acid” (II). “Deaminated pteroic acid” was isolated and characterized as one of the products of enzymic attack of folic acid (Fig. 4). The relative activities of the deaminase and the amidase, in different enzyme preparations, varied, and in some of the incubation mixtures little “deaminated folic acid” accumulated. It is surprising that the splitting of the amide bond of “deaminated folic acid” (I), thereby releasing a free carboxyl group in conjugation with a benzene ring (II), causes a pronounced shift in the absorption spectrum in the region 330-340 rnp (compare Figs. 2 and 4). This effect on the absorption spectrum at the longer wavelengths may be related to the resonance forms assumed by the N-alkyl-p-aminobenzoic acid portion of the molecule (7, 8).
i-d”,“, H
CH COOH **
The amidase splits folic acid to pteroic acid (Table III), and the deaminase converts pteroic acid to “deaminated pteroic acid” (Fig. 5). Rhizopterin was not deaminated or deformylated at an appreciable rate (Fig. S), nor did the enzyme preparation catalyze trans-formylation between rhizopterin and “deaminated folic acid.” There was no evidence that the enzyme preparation catalyzed the further attack of “deaminated pteroic acid.” The inability of the organism to effectively utilize folic acid as a nitrogen source may be due to its inability to convert “deaminated pteroic acid” to tetraoxypteridine, the key intermediate in the metabolism of simple pteridines by this organism (3). Folic acid deaminase and folic acid amidase are probably of no general biological importance. ACKNOWLEDGMENT I would like to thank Mrs. Roberta for technical help in this work.
McDuffie
REFERENCES 1. LEVY, C. C., AND MCNUTT, W. S., Biochemistry 1, 1161 (1962). 2. MCNUTT, W. S., J. Biol. C/wan., in press. 3. MCNUTT, W. S., Intern. Symp. Chem. Biol. Pteridines, 1962. 4. ANGIER, R. B., BOOTHE, J. H., MOWAT, J. H., WALLER, C. W., AND SEMB, J., J. Am. Chem. sot. 74, 408 (1952). 5. WALLER, C. W., HUTCHINGS, B. L., MOWAT, J. H., STOKSTAD, E. L. It., BOOTHE, J. H., ANGIER, R. B., SEMB, J , SUBBAROW, Y., COSULICH, D. B., FAHRENBACH, M. J., HULTQUIST, M. E., KUH, E., NORTHEY, E. H., SEEGER, D. R., SICKELS, J. P., AND SMITH, J. M., JR., J. Am. Chem. Sot. 70, 19 (1948). 6. WOLF, D. E., ANDERSON, R. C., KACZKA, E. A., HARRIS, S. A., ARTH, G. E., SOTJTHWICK, P. L., MOZINGO, R., AND FOLKERS, K., J. Am. Chem. Sot. 69, 2753 (1947). 7. KUMLER, W. D., AND STRAIT, L. A., J. Am. Chem. Sot. 66, 2349 (1943). 8. KUMLER, W. D., J. Am Chem. Sot. 68, 1184 (1946).
OF
BIOCIIEMISTRY
AND
The Enzymic
BIOPHYSICS
101,
Deamination
l-6
(1963)
and Amide
Cleavage
of Foiic Acid’
WALTER S. McNUTT2 From the Department of Pharmacology,
Tufts
University
School of Medicine,
Boston,
Massachusetts
Received January 16, 1963 Crude enzyme, prepared from cells of Alcaligenes jaecalis which had been cultured in response to xanthopterin, catalyzed the deamination of folic acid and the splitting of the amide bond of folic acid. “Deaminated folic acid,” pteroic acid, and “deaminated pteroic acid” were characterized as enzymic products derived from folic acid. Pteroic acid was deaminated by the enzyme preparation, whereas rhizopterin was not attacked at an appreciable rate. INTRODUCTION
tally
faecalis
FOLIC
ACID”
prepared specimens at pH
PTEROIC ARID
(5)
The crude product (0.5 g.), prepared as described (5), was taken up in hot water and NHdOH, and the cooled solution (pH 7.0) was added to a DEAEcellulose column 7.8 X 27 cm. The column was developed with a gradient of 12.5 1. water and 12.5 I. of 0.7 M NHHC03. Pteroic acid passed from the columns at about 0.63 M. The solution was evaporated to dryness, and the residue was taken up in 40 ml. of corm. HCI at room temperature, treated with a little Norit, and filtered. Water (750 ml.) was added to the filtrate, and the crystals which separated were washed with water, ethanol, and ether, and dried. Fifty-two milligrams of pteroic acid was obtained. Its absorption spectrum agreed with the published value (5). On a paper chromatogram (Table I) it gave a single ultraviolet-absorbing spot.
EXPERIMENTAL “DEAMINATED
and biologically
7.5 is shown in Fig. 2.
has the ability to utilize certain pteridines and purines as substrates for growth (1). Although folic acid is not effectively utilized as a nitrogen source by this organism (2), cells which have been grown in response to xanthopterin as the nitrogen-source contain enzymes which catalyze (a) the deamination of folic acid and (b) the cleavage of the amide bond of folic acid and “deaminated folic acid.” It seems likely that folic acid is not an effective substrate because the organism is unable to convert it to tetraoxypteridine, the key intermediate (3) in the metabolism of pteridines in this organism. Alcaligenes
(4)
The chemically synthesized compound melted with decomposition at 230-232” (melting point stage). The reported value (4) is 220-224’. It showed the following ultraviolet absorption characteristics: in 0.1 N HCI, l %,?‘ 16,800and in 0.1 N NaOH, e2E8!“ 25,600 and %?’ 7480. The values calculated from the published data (4) are: in 0.1 N HCI, E’::“ 17,800 and in 0.1 N NaOH, ~‘2~:s 26,300 and c3Egs 7160. A comparison of the chemi-
RHIZOPTERIN
(6)
Formylation of 85 mg. pteroic acid gave 74 mg. rhizopterin. On a paper chromatogram (Table I) it showed a single fluorescent spot. “DEAMINATED
RHIZOPTERIN”
(6)
From 50 mg. rhizopterin, 36 mg. of “deaminated rhizopterin” was obtained. This product was contaminated with “deaminated pteroic acid,” and it was used in the chemical synthesis of “deaminated pteroic acid” without further purification. The fluorescent compound (I& 0.83) cochromatographed with the chemically formylated derivative of the
1 Supported by U. S. Public Health Service Grant No. A-3675. 2 U. S. Public Health Service Career Development Awardee. 1
McNUTT TABLE I Rf VALUES OF FOLIC ACID AND RELATED COMPOUNDSIN 5y0 AMMONIUM BICARBONATE SOLUTION Compound
Rf value
10.Formyl-“deaminated folic “Deaminated rhizopterin” Rhizopterin “Deaminated folic acid” Folic acid “Deaminated pteroic acid” Pteroic acid
acid”
0.88 0.77 0.69 0.65 0.42 0.24 0.09
enzymic product CR, 0.84), and the fluorescent band from the chromatogram had an ultraviolet absorption spectrum characteristic of pure “deaminated rhizopterin” which was prepared as follows: “Deaminated pteroic acid” (58 mg.) was heated with 30 ml. of 97y0 formic acid in a boiling water bath for 2 hr. The solution was evaporated to dryness at 55”, and the residue was taken up in 10 ml. of 1 N NHaOH and filtered. The solution at 60” was acidified with acetic acid and cooled. The white needle-like crystals were filtered off, washed with water, ethanol, and ether, and dried over HZSOI. Yield: 52 mg., m.p. 325327” (dec.). The reported value (6) is 321-323” (dec.). The substance showed a single fluorescent spot on a paper chromatogram (Table I).
“DEAMINATED
PTEROIC ACID”
(6)
“Deaminated rhizopterin” (65 mg.) was hydrolyzed with HCI, giving 50 mg. of “deaminated pteroic acid” m.p. 322-325” (dec.). On a paper chromatogram (Table I) the specimen showed a trace of fluorescent material which remained close to the origin and a single uRraviolet-absorbing spot (RI 0.24). A comparison of its ultraviolet absorption spect,rum with that of the enzymic product is shown in Fig. 4.
lo-FORMYL “DEAMINATED
FOLIC
ACID”
“Deaminated folic acid” (200 mg.) was heated with 30 ml. of 977; formic acid at 80” for 1 hr. The almost colorless solution was evaporated to dryness at 55”. The residue was crystallized from hot water, washed with water, ethanol, and ether, and dried over HZSO,. Yield: 135 mg. of white cryst,als, m.p. 185187”. It moved as a single fluorescent, spot on a paper chromatogram (Table I).
PREPARATION
OF ENZYME
Crude enzyme was prepared as described previously (2) from cells which had been cultured for
24 hr. in inorganic medium supplemented with 1% sodium succinate.6HzO and 0.0032% xanthopterin.HzO or 6-oxylumazine
THE SPECTROPHOTOMETRIC ESTIMATION THE DEAMINATION OF FOLK ACID
OF
Folic acid (133 mpmoles) and enzyme from xanthopterin-grown cells (55 pg. protein) in 1 ml. of 0.005 M Tris-HCl at pH 7.5 were incubated at 23”. The spectral changes are shown in Fig. 1. The theoretical values for the conversion of folic acid to deaminated folic acid at pH 7.5 are an isosbestic point at 343 rnF (e 7320) an increase at 330 rnr (AE -t-1630), and a decrease at 360 rnp (Ae -3300). The results (Table II) are in fair agreement. with the theoretical values. In subsequent large-scale incubations which were carried out in order to isolate the enzymic product, mixtures of “deaminated folic acid” and “deaminated pteroic acid” were obtained.
THE IDENTIFICATION OF “DEAMIXATED FOLIC ACID" .4s &4PRODUCT OF ENZYMIC ATTA4CK OF FOLIC ACID Folic acid (240 mg.) and enzyme from xanthopterin-grown cells (220 mg. prot.ein) in4 1. of 0.005 M Tris-HCl were incubated as described above, and the decrease in optical density at 360 rnp was followed. Glacial acetic acid (50 ml.) was added when approximately 707, of the folic acid had been deaminated, and the solution was evaporated to dryness at 55”. The residue was t,aken up in dilute NHdOH, and the solution was evaporated to remove excess xH,OH. The solution (pH 7) was diluted to 4 1. and chromatographed on a DEAEcellulose column (7.7 X 34 cm.). The column was developed with a linear gradient of 18 1. of 0.35 A4 NH4HC03 vs. 18 1. of 0.5 M NH,HCO,. Two ultraviolet-absorbing bands passed from the column. The first peak (0.38-0.41 M) contained “deaminated folic acid” and “deaminated pteroic acid.” The second peak (0.44-0.46 M) contained folic acid. Crystalline folic acid (55 mg.) was recovered from this peak. The first peak was evaporated to dryness, taken up in dilute KHhOH, and filtered. The solution was acidified, and the yellow gelatinous product was collected and dried. The yellow solid (110 mg.) was extracted with concentrated HCl (2 ml.), whereupon a white solid (“deaminated pteroic acid,” see below) separated from the yellow solution. The yellow solution was filtered from the white solid and added to 20 ml. water. The orangeyellow solid was collected and dried: 73 mg. It was chromatographed along 371 cm. (7 sheets) of Whatman No. 3 MM filter paper in 570 NHJHCO~ solution. The upper ultraviolet-absorbing bands
ENZYMIC
ATTACK
ON FOLIC
ACID
3
(Rf 0.60) containing “deaminated folic acid” were washed with water. The washings were reduced to 10 ml. and warmed to 60”. One milliliter of 5 N HCl was added to the solution. The orange-yellow crystals, which separated upon cooling the solution, were washed with water, ethanol, and ether, and dried over H&lOh. Yield: 25 mg., m.p. 230234” dec. The substance gave a single ultravioletabsorbing spot (Rf 0.62) on a paper chromatogram (Table I) and cochromatographed with synthetic “deaminated folic acid” (R, 0.62). Its ultraviolet absorption spectrum at pH 7.5 agreed with that of the synthetic compound (Fig. 2). THE IDENTIFICATION OF (‘DEAMINATED PTEROIC ACID” AS A PRODUCT OF ENZYMIC ATTACK OF FOLIC ACID The white solid which failed to dissolve in 2 ml. of cont. HCl (see above) was dissolved in 10 ml. of cone. HCl at room temperature. Water (50 ml.) was added, and the canary yellow crystals which separated were washed with water and ethanol and dried over H2S04. Yield: 26 mg., m.p. 325’ dec.
FIG. 2. The ultraviolet absorption spectrum of “deaminated folic acid.” Natural and ----synthetic “deaminated folic acid” at pH 7.5 in 0.05 M Tris-HCl. In 0.1 N NaOH its ultraviolet absorption char. acteristics were: s2gy 26,400, ~“2,:“ 7070. The substance gave a single ultraviolet-absorbing spot (RJ 0.24) on a paper chromatogram (Table I) and cochromatographed with synthetic “deaminated pteroic acid” (Rf 0.23). Its ultraviolet absorption spectrum at pH 7.5 agreed with that of the synthetic compound (Fig. 4). THE IDENTIFICATION OF “DEAMINATED PTEROIC ACID” AS A PRODUCT OF ENZYMIC ATTACK OF “DEAMINATED FOLIC ACID"
300
350 r-y)
400
450
FIG. 1. The spectral changes which accompany the deamination of folic acid. The heavy line is the absorption spectrum initially (folic acid), and the other lines are the absorption spectra taken at intervals over a period of 24 hr. At the end of this time about 68% of the folic acid had been deaminated.
“Deaminated folic acid” (165 mg.) and enzyme from 6-oxylumazine-grown cells (570 mg. protein) in 3 1. of 0.005 1MTris-HCl at pH 7.5 were incubated at 21” for 24 hr. The solution was poured into 3 1. of boiling ethanol, and the mixture was brought to boiling. The precipitated protein was removed, and the filtrate was reduced to 2 1. The solution was chromatographed on a DEAE-cellulose column (7.7 X 17 cm.) and developed with a linear gradient of 10 1. of 0.3 M NH~HCOI vs. 10 1. of 0.5 M NH4HCOa. The residue from the peak which passed from the column at 0.37 M was worked up as described above. “Deaminated pteroid acid”
4
McNUTT
It (58 mg.; m.p. 32&325” dec.) was obtained. showed the characteristic ultraviolet absorption spectrum at pH 7.5 and the R, value of “deaminated pteroic acid.” TABLE
II
THE SPECTROPHOTOMETRIC DETERMINATION OF FOLIC ACID DEAMINASE In this assay the decrease in optical density at 360 rnp is taken as a measure of the disappearance of folic acid, and the increase in optical density at 330 rnp is taken as a measure of the formation of “deaminated folic acid.” Folk acid present initially
Observed A O.D. at:
360m,,
330mp
m~moles 133 -0.310
+0.144
FoE;li;;id nated “DeamiI f&c disappeared frrizd
mpmoles m/.lmoles 94 88
FIG. 4. The ultraviolet absorption spectrum of “deaminated pteroic acid.” Natural and ----synthetic “deaminated pteroic acid” at pH 7.5 in 0.05 M Tris-HCl.
THE ENZYMIC
FIG. 3. Spectral changes associated with the conversion of folic acid to “deaminated pteroic acid.” Folic acid (96 mpmoles) and enzyme from xanthopterin-grown cells (260 pg. protein) in 1 ml. of 0.02 M Tris-HCl at pH 7.5 were incubated at 19”. The absorption spectrum of the incubation mixture initially (folic acid) and ----- after 20 hr. at which time 65% of the folic acid had been deaminated
CONVERSION OF FOLIC ACID TO PTEROIC ACID
Folic acid (2 pmoles) and enzyme from xanthopterin-grown cells (5 mg. protein) in 20 ml. of 0.02 M Tris-HCl at pH 7.5 were incubated at 19” for 22 hr. The solution was poured into an equal volume of boiling alcohol, and the precipitated protein was removed. The filtrate was placed along 20 cm. of Whatman No. 3 MM and chromatographed in 5% NH,HC03 solution. Only two ultraviolet-absorbing bands were detected: a principal band of “deaminated pteroic acid” (RI 0.25) and pteroic acid (Rf 0.09). The substances were eluted from the paper with 0.1 N NaOH, and the amounts present were estimated spectrophotometrically. The results are shown in Table III.
THE ENZYMIC DEAMINATION PTEROIC ACID
OF
Pteroic acid (2 pmoles) was incubated as described in the preceding section, and the mixture bands was similarly chromatographed. Two appeared on the chromatogram. The substance eluted from the principal band (R, 0.24) showed
ENZYMIC
ATTACK
ON FOLIC ACID an Rf value of 0.38, it would have appeared on the chromatogram as a fluorescent band above rhizopterin, had it been formed. RESULTS
AND DISCUSSION
Crude enzyme preparations from cells of Alcaligenes jaecalis, which had been cultured in responseto xanthopterin or 6-oxylumazine TABLE
III
THE
CONVERSION OF FOLK ACID TO PTEROIC ACID AND “DEAMINATED PTEROIC ACID”
Folk acid present initially
Pteroic acid recovered
“Deaminated pteroic acid” recovered
FIG. 5. Spectral changes which accompany the deamination of pteroic acid. Pteroic acid (0.1 #mole) and enzyme from xanthopterin-grown cells (260 pg. protein) in 1 ml. of Tris-HCI at pH 7.5 were incubated at, 19”. The heavy line is the absorption spectrum of the incubation mixture initially (pteroic acid), and the other lines are the absorption spectra taken at intervals over a period of 22 hr. at which time about 7Oyo of the pteroic acid had been deaminated.
the ultraviolet absorption spectrum characteristic of “deaminated pteroic acid.” The lower band (RI 0.09) contained unchanged pteroic acid. THE INABILITY OF THE ENZYME PREPARATION TO CATALYZE TRANSFORMYLATION BETWEEN RHIZOPTERIN AND “DEAMINATED FOLK ACID” Rhizopterin (2 pmoles), “deaminated folic acid” (10 pmoles), and enzyme from xanthopteringrown cells (5 mg. protein) were incubated in 0.02 M Tris-HCl (pH 7.5) at 19” for 7 hr. The incubation mixture was poured into boiling alcohol, and the solution was chromatographed as described above. The chromatogram showed ultraviolet-absorbing bands of “deaminated folic acid” (I& 0.61) and “deaminated pteroic acid” (R, 0.27) and a single fluorescent band of rhizopterin (Rf 0.66). As IO-formyl “deaminated folic acid” has
FIG. 6. The inability of the enzyme to attack rhizopterin. Rhizopterin (0.1 pmole) and enzyme from xanthopterin-grown cells (260 pg. protein) in 1 ml. of 0.02 M Tris-HCl (pH 7.5) were incubated at 19”. The heavy line is the absorption spectrum of the incubation mixture initially (rhizopterin), and the thin line is the absorption spectrum of the incubation mixture at the end of 24 hr. The absorption spectra of equimolar amounts of pteroic acid (-----) and “deaminated rhizopterin” (-a-e-*-) at pH 7.5 are shown.
6
McNUTT
as the source of nitrogen, catalyzed two chemical alterations of folic acid which could be followed spectrophotometrically. One change was associated with an increase in optical density at 330 rnp and a decrease in optical density at 360 rnti at pH 7.5 (Fig. l), as one would predict for the conversion of folic acid to “deaminated folic acid” (I). “Deaminated folic acid” was isolated and characterized as one of the products of enzymic attack of folic acid (Fig. 2). The other change was associated with the appearance of an absorption maximum at 335 mp at pH 7.5. This spectral shift (Fig. 3) is due to the appearance of “deaminated pteroic acid” (II). “Deaminated pteroic acid” was isolated and characterized as one of the products of enzymic attack of folic acid (Fig. 4). The relative activities of the deaminase and the amidase, in different enzyme preparations, varied, and in some of the incubation mixtures little “deaminated folic acid” accumulated. It is surprising that the splitting of the amide bond of “deaminated folic acid” (I), thereby releasing a free carboxyl group in conjugation with a benzene ring (II), causes a pronounced shift in the absorption spectrum in the region 330-340 rnp (compare Figs. 2 and 4). This effect on the absorption spectrum at the longer wavelengths may be related to the resonance forms assumed by the N-alkyl-p-aminobenzoic acid portion of the molecule (7, 8).
i-d”,“, H
CH COOH **
The amidase splits folic acid to pteroic acid (Table III), and the deaminase converts pteroic acid to “deaminated pteroic acid” (Fig. 5). Rhizopterin was not deaminated or deformylated at an appreciable rate (Fig. S), nor did the enzyme preparation catalyze trans-formylation between rhizopterin and “deaminated folic acid.” There was no evidence that the enzyme preparation catalyzed the further attack of “deaminated pteroic acid.” The inability of the organism to effectively utilize folic acid as a nitrogen source may be due to its inability to convert “deaminated pteroic acid” to tetraoxypteridine, the key intermediate in the metabolism of simple pteridines by this organism (3). Folic acid deaminase and folic acid amidase are probably of no general biological importance. ACKNOWLEDGMENT I would like to thank Mrs. Roberta for technical help in this work.
McDuffie
REFERENCES 1. LEVY, C. C., AND MCNUTT, W. S., Biochemistry 1, 1161 (1962). 2. MCNUTT, W. S., J. Biol. C/wan., in press. 3. MCNUTT, W. S., Intern. Symp. Chem. Biol. Pteridines, 1962. 4. ANGIER, R. B., BOOTHE, J. H., MOWAT, J. H., WALLER, C. W., AND SEMB, J., J. Am. Chem. sot. 74, 408 (1952). 5. WALLER, C. W., HUTCHINGS, B. L., MOWAT, J. H., STOKSTAD, E. L. It., BOOTHE, J. H., ANGIER, R. B., SEMB, J , SUBBAROW, Y., COSULICH, D. B., FAHRENBACH, M. J., HULTQUIST, M. E., KUH, E., NORTHEY, E. H., SEEGER, D. R., SICKELS, J. P., AND SMITH, J. M., JR., J. Am. Chem. Sot. 70, 19 (1948). 6. WOLF, D. E., ANDERSON, R. C., KACZKA, E. A., HARRIS, S. A., ARTH, G. E., SOTJTHWICK, P. L., MOZINGO, R., AND FOLKERS, K., J. Am. Chem. Sot. 69, 2753 (1947). 7. KUMLER, W. D., AND STRAIT, L. A., J. Am. Chem. Sot. 66, 2349 (1943). 8. KUMLER, W. D., J. Am Chem. Sot. 68, 1184 (1946).