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The biosynthesis of p-aminobenzoic acid: studies on the origin of the amino group
597
SHORT COMMUNICATIONS
The biosynthesis of p-aminobenzoic acid: studies on the origin of the amino group In a recent paper 1 it was shown that PABA can be formed from shikimic acid 5-phosphate and L-glutamine b y cell-free extracts of baker's yeast. Of the various amino donors studied L-glutamine was the most effective. It was therefore of interest to determine whether the amino group of PABA is derived from the amide N or the amino N of L-glutamine. This communication is concerned with the origin of the amino group of PABA as well as the effect of various glutamine analogues on this enzyme system. The preparation of the enzyme extract and the microbiological assay of PABA have been described previously 1. 16o/,moles of shikimic acid 5-phosphate, 800/*moles of L-[amide-15N~glutamine,320 mg of yeast concentrate, 16oo/~moles of Tris buffer (pH 7.4), 300 /,moles of MgCI~ and 17o ml of enzyme extract in a total volume of 320 ml were incubated for 24 h at 3 o°. After incubation, the mixture was placed in a boiling-water b a t h for IO min, cooled and the p H adjusted to 3.0 with 2 ml of 6 N H2SO ~. The precipitated protein was removed by centrifugation, and aliquots of the supernatant solution were analyzed for PABA at three different dilutions in duplicate. To 315 ml of this supernatant solution, which contained 1.22 mg of PA]3A, ioo mg of carrier PABA were added. The PABA was then isolated by continuous ether extraction and was recrystallized from water three times. I t was then analyzed for 15N by standard methods 2. L-~amideJSNlglutamine was also suitably diluted and assayed for its 15N content. The results are summarized in Table I. TABLE
I
SOURCE OF THE AMINO GROUP OF P A B A Compound analyzed
I. L - [ a m i d e - l ~ N ] g l u t a m i n e 2. P A B A
1~N (atom % excess)
Dilution factor
15N correctedfor dilv2ion (atom % excess)
1-414 1.125
67.4 83.o
95-3 93.4
The atom % excess ZSN of the isolated PABA after correction for dilution by carrier agreed very closely with the 15N content of L-glutamine. This would suggest that the amino group of PABA is derived from the amide N of L-glutamine. The requirement for glutamine in the formation of PABA would suggest the involvement of an intermediate after shikimic acid 5-phosphate with an enolic or acidic hydroxyl at C-4. This hypothesis would be in agreement with other reactions in which L-glutamine serves as an amino donor s,4. With this in view, p-hydroxybenzoic acid was investigated as a possible carbon source and was found incapable of substituting for shikimic acid 5-phosphate. These preliminary studies lead us to speculate that the intermediate m a y not only possess an enolic or acidic hydroxyl capable of amination b y glutamine amide N but also be non-aromatic in structure. Some of the reactions in which L-glutamine participates as an amino donor are Non-standard abbreviations : PABA, p-aminobenzoic acid ; DON, 6-diazo-5-oxo-L-norleucine. B i o c h i m . B i o p h y s . A c t a , 51 (1961) 5 9 7 - 5 9 9
50 N
.SHORT COMMUNICATIONS
inhibited by its structural analogs aza-L-serine and 6-diazo-5-oxo-L-norleucine *. Although the inhibition by aza-L-serine is invariably associated with a requirement for glutamine, the reverse is not true. The effect of the various gtutamine analogs on the above reaction sequence was studied in a partially purified enzyme system described below. All operations were carried out at 2 °. 5o g of baker's yeast was thoroughly stirred with 5oo ml of cold acetone in a Waring Blendor for 3o sec and filtered rapidly. The wet yeast was translerred to a desiccator over P~O 5 and dried i n vacuo. The dry acetone powder (7 g) was gently stirred with 35 ml of o.i M potassium phosphate buffer (pH 7.4) for 3 h. Centrifugation at 15 ooo × g yielded a clear grayish-yellow solution containing 23 mg protein/ml. 25 ml of the supernatant was treated with 4.3 ml of 2 °i~ protamine sulfate solution, and the precipitate was removed by centrifugation. 5.2 g of (NH4)~SO ~ were added slowly with stirring to the supernatant solution, bringing it to a level of 3o % saturation. After stirring for another 2o min, the precipitate was removed by centrifugation and discarded. The supernatant solution was then brought to 5o % (NH4)~S04 saturation by the addition of 3-7 g of (NH~)2S04. The precipitate was removed by centrifugation, dissolved in 6. 3 ml of o.o33 M potassium phosphate buffer (pH 7.4), and extensively dialyzed against the same buffer. 5.01 A
3°
i
m
fz5.0 ~a. < ~~ tn
I 1 CONCENTRATION
t
5 OF A Z A - / - S E R I N E
1 /0
(xto 4 M ) Fig. I. Effect of aza-L-serine on the synthesis of PABA.
0
Il
I
5
I
I0
CONGENTR~TION OF 6 - DIAZO- 5 - O X O -
/ - NORLIEuGINE (X IO e~ M )
Fig. 2. Effect of 6-diazo-5-oxo-L-norleucine on the synthesis of PABA.
The incubation mixtures contained 0.4 ml of the above enzyme extract, 2/,moles of MgCI~, i mg of yeast concentrate*, 5°/~moles of Tris (pH 7.4), I ~mole of shikimic acid 5-phosphate, 5/~moles of L-glutamine plus additions, in a total volume of I ml. After incubation at 3 °0 for 4 h, the reaction was stopped by adjusting the p H to i.o with 6 N HC1. PABA was estimated microbiologically in the supernatant solution. The effect of aza-L-serine and DON on the synthesis of PABA is illustrated in Figs. I and 2. With increasing levels of aza-L-serine, a progressive decrease in the PABA synthesis was observed. Under the present conditions a concentration of * The yeast concentrate (Sigma Chemical Co., St. Louis) employed in these e x p e r i m e n t s contains a m i x t u r e of pyridine nucleotides, nucleoside m o n o p h o s p h a t e s and nucleoside d i p h o s p h a tes. Y e a s t concentrate can be replaced b y a m i x t u r e of A T P and D P N in the partially purified e n z y m e s y s t e m b u t n o t in the crude e n z y m e extract. However, the activity in the purified s y s t e m is only 6o-8o % of t h a t observed w i t h yeast concentrate as a source of co-factors. Mg °-+ was also found to be essential for the enzymic reaction.
Biochim. Biophys. Acta, 51 (~96I) 597 599
S I-tORT COMMUNICATIONS
599
1.5" IO-~ M aza-L-serine (L-glutamine = 5" IO-3 M) resulted in a 50 % inhibition. The nature of the effect of DON is extremely interesting. Addition of o.ooi/~mole to the incubation mixture raised the synthesis of PABA from 3.7 /~g to IO.O ~g. Further additions resulted in a marked inhibition of synthesis. The initial increase in the synthesis of PABA can be explained as due to the resulting greater availability of glutamine for the formation of PABA. The enzyme fraction, although partially purified, still contains the complete enzyme system for the formation of anthranilic acid, which also arises from shikimic acid 5-phosphate and L-glutamine 6. Furthermore, the formation of anthranilic acid is extremely sensitive to DON, which is at least 200 times more inhibitory than aza-L-serine 7. So, in the absence of DON, there exists a competition for glutamine by the anthranilic acid and PABA-forming enzyme systems. Addition of a trace amount of DON suppresses the more sensitive anthranilic acid system, thereby making available an increased concentration of glutamine for the formation of PABA. This m a y be responsible for the increased synthesis of PABA in the presence of o.ooi ~mole of DON. Of the other glutamine analogs studied, only L-glutamyl hydrazine and Ocarbamyl-DL-serine were inhibitory, but their capacity for inhibition was considerably less than that of aza-L-serine. Thus, apart from their inhibition of the biosynthesis of purines, aza-L-serine and DON also exert their effect in the formation of anttlranilic acid and PABA. The inhibition pattern suggests a similar mechanism of amination and perhaps a similar (or even identical) sequence of residues at the active sites of these enzymes. Further work is in progress to elucidate the intermediates in the formation of PABA from shikimic acid 5-phosphate. This investigation was supported by a grant from the U.S. Public Health Service, RG-58o 9. We are grateful to Dr. A. NEIDLE for the generous gift of L-[15N~glutamine and to Miss L. PONTICORVO for her assistance with the 15N determinations.
Department of Biochemistry, College of Physicians and Surgeon~, Columbia University, New York, N.Y. (U.S.A.)
P. R. SRINIVASAN B. WEISS
1 B. WEISS AND P. R. SRINIVASAN, Proc. Natl. Acad. Sci. U.S., 45 (1959) 1491. 2 D. RITTENBERG, in J. W. EDWARDS, Preparation and Measurement o/Isotopic Tracers, A nn Arbor, Mich., 1946 . 3 B. LEVENBERG AND J. M. BUCHANAN, J. Biol. Chem., 224 (1957) lO19. 4 R. ABRAMS AND IV[. BENTLEY, Arch. Biochem. Biophys., 79 (1959) 91. 5 B. LEVENBERG, I. ~IELNICK AND J. M. BUCHANAN, J. Biol. Chem., 225 (1957) 163. 6 p. R. SRINIVASAN, .]. Am. Chem. Soc., 81 (1959) 1772. 7 p. R. SRINIVASAN, to be p u b l i s h e d .
Received April 5th, 196i Biochim. Biophys. Acta, 51 (1961) 597-599
SHORT COMMUNICATIONS
The biosynthesis of p-aminobenzoic acid: studies on the origin of the amino group In a recent paper 1 it was shown that PABA can be formed from shikimic acid 5-phosphate and L-glutamine b y cell-free extracts of baker's yeast. Of the various amino donors studied L-glutamine was the most effective. It was therefore of interest to determine whether the amino group of PABA is derived from the amide N or the amino N of L-glutamine. This communication is concerned with the origin of the amino group of PABA as well as the effect of various glutamine analogues on this enzyme system. The preparation of the enzyme extract and the microbiological assay of PABA have been described previously 1. 16o/,moles of shikimic acid 5-phosphate, 800/*moles of L-[amide-15N~glutamine,320 mg of yeast concentrate, 16oo/~moles of Tris buffer (pH 7.4), 300 /,moles of MgCI~ and 17o ml of enzyme extract in a total volume of 320 ml were incubated for 24 h at 3 o°. After incubation, the mixture was placed in a boiling-water b a t h for IO min, cooled and the p H adjusted to 3.0 with 2 ml of 6 N H2SO ~. The precipitated protein was removed by centrifugation, and aliquots of the supernatant solution were analyzed for PABA at three different dilutions in duplicate. To 315 ml of this supernatant solution, which contained 1.22 mg of PA]3A, ioo mg of carrier PABA were added. The PABA was then isolated by continuous ether extraction and was recrystallized from water three times. I t was then analyzed for 15N by standard methods 2. L-~amideJSNlglutamine was also suitably diluted and assayed for its 15N content. The results are summarized in Table I. TABLE
I
SOURCE OF THE AMINO GROUP OF P A B A Compound analyzed
I. L - [ a m i d e - l ~ N ] g l u t a m i n e 2. P A B A
1~N (atom % excess)
Dilution factor
15N correctedfor dilv2ion (atom % excess)
1-414 1.125
67.4 83.o
95-3 93.4
The atom % excess ZSN of the isolated PABA after correction for dilution by carrier agreed very closely with the 15N content of L-glutamine. This would suggest that the amino group of PABA is derived from the amide N of L-glutamine. The requirement for glutamine in the formation of PABA would suggest the involvement of an intermediate after shikimic acid 5-phosphate with an enolic or acidic hydroxyl at C-4. This hypothesis would be in agreement with other reactions in which L-glutamine serves as an amino donor s,4. With this in view, p-hydroxybenzoic acid was investigated as a possible carbon source and was found incapable of substituting for shikimic acid 5-phosphate. These preliminary studies lead us to speculate that the intermediate m a y not only possess an enolic or acidic hydroxyl capable of amination b y glutamine amide N but also be non-aromatic in structure. Some of the reactions in which L-glutamine participates as an amino donor are Non-standard abbreviations : PABA, p-aminobenzoic acid ; DON, 6-diazo-5-oxo-L-norleucine. B i o c h i m . B i o p h y s . A c t a , 51 (1961) 5 9 7 - 5 9 9
50 N
.SHORT COMMUNICATIONS
inhibited by its structural analogs aza-L-serine and 6-diazo-5-oxo-L-norleucine *. Although the inhibition by aza-L-serine is invariably associated with a requirement for glutamine, the reverse is not true. The effect of the various gtutamine analogs on the above reaction sequence was studied in a partially purified enzyme system described below. All operations were carried out at 2 °. 5o g of baker's yeast was thoroughly stirred with 5oo ml of cold acetone in a Waring Blendor for 3o sec and filtered rapidly. The wet yeast was translerred to a desiccator over P~O 5 and dried i n vacuo. The dry acetone powder (7 g) was gently stirred with 35 ml of o.i M potassium phosphate buffer (pH 7.4) for 3 h. Centrifugation at 15 ooo × g yielded a clear grayish-yellow solution containing 23 mg protein/ml. 25 ml of the supernatant was treated with 4.3 ml of 2 °i~ protamine sulfate solution, and the precipitate was removed by centrifugation. 5.2 g of (NH4)~SO ~ were added slowly with stirring to the supernatant solution, bringing it to a level of 3o % saturation. After stirring for another 2o min, the precipitate was removed by centrifugation and discarded. The supernatant solution was then brought to 5o % (NH4)~S04 saturation by the addition of 3-7 g of (NH~)2S04. The precipitate was removed by centrifugation, dissolved in 6. 3 ml of o.o33 M potassium phosphate buffer (pH 7.4), and extensively dialyzed against the same buffer. 5.01 A
3°
i
m
fz5.0 ~a. < ~~ tn
I 1 CONCENTRATION
t
5 OF A Z A - / - S E R I N E
1 /0
(xto 4 M ) Fig. I. Effect of aza-L-serine on the synthesis of PABA.
0
Il
I
5
I
I0
CONGENTR~TION OF 6 - DIAZO- 5 - O X O -
/ - NORLIEuGINE (X IO e~ M )
Fig. 2. Effect of 6-diazo-5-oxo-L-norleucine on the synthesis of PABA.
The incubation mixtures contained 0.4 ml of the above enzyme extract, 2/,moles of MgCI~, i mg of yeast concentrate*, 5°/~moles of Tris (pH 7.4), I ~mole of shikimic acid 5-phosphate, 5/~moles of L-glutamine plus additions, in a total volume of I ml. After incubation at 3 °0 for 4 h, the reaction was stopped by adjusting the p H to i.o with 6 N HC1. PABA was estimated microbiologically in the supernatant solution. The effect of aza-L-serine and DON on the synthesis of PABA is illustrated in Figs. I and 2. With increasing levels of aza-L-serine, a progressive decrease in the PABA synthesis was observed. Under the present conditions a concentration of * The yeast concentrate (Sigma Chemical Co., St. Louis) employed in these e x p e r i m e n t s contains a m i x t u r e of pyridine nucleotides, nucleoside m o n o p h o s p h a t e s and nucleoside d i p h o s p h a tes. Y e a s t concentrate can be replaced b y a m i x t u r e of A T P and D P N in the partially purified e n z y m e s y s t e m b u t n o t in the crude e n z y m e extract. However, the activity in the purified s y s t e m is only 6o-8o % of t h a t observed w i t h yeast concentrate as a source of co-factors. Mg °-+ was also found to be essential for the enzymic reaction.
Biochim. Biophys. Acta, 51 (~96I) 597 599
S I-tORT COMMUNICATIONS
599
1.5" IO-~ M aza-L-serine (L-glutamine = 5" IO-3 M) resulted in a 50 % inhibition. The nature of the effect of DON is extremely interesting. Addition of o.ooi/~mole to the incubation mixture raised the synthesis of PABA from 3.7 /~g to IO.O ~g. Further additions resulted in a marked inhibition of synthesis. The initial increase in the synthesis of PABA can be explained as due to the resulting greater availability of glutamine for the formation of PABA. The enzyme fraction, although partially purified, still contains the complete enzyme system for the formation of anthranilic acid, which also arises from shikimic acid 5-phosphate and L-glutamine 6. Furthermore, the formation of anthranilic acid is extremely sensitive to DON, which is at least 200 times more inhibitory than aza-L-serine 7. So, in the absence of DON, there exists a competition for glutamine by the anthranilic acid and PABA-forming enzyme systems. Addition of a trace amount of DON suppresses the more sensitive anthranilic acid system, thereby making available an increased concentration of glutamine for the formation of PABA. This m a y be responsible for the increased synthesis of PABA in the presence of o.ooi ~mole of DON. Of the other glutamine analogs studied, only L-glutamyl hydrazine and Ocarbamyl-DL-serine were inhibitory, but their capacity for inhibition was considerably less than that of aza-L-serine. Thus, apart from their inhibition of the biosynthesis of purines, aza-L-serine and DON also exert their effect in the formation of anttlranilic acid and PABA. The inhibition pattern suggests a similar mechanism of amination and perhaps a similar (or even identical) sequence of residues at the active sites of these enzymes. Further work is in progress to elucidate the intermediates in the formation of PABA from shikimic acid 5-phosphate. This investigation was supported by a grant from the U.S. Public Health Service, RG-58o 9. We are grateful to Dr. A. NEIDLE for the generous gift of L-[15N~glutamine and to Miss L. PONTICORVO for her assistance with the 15N determinations.
Department of Biochemistry, College of Physicians and Surgeon~, Columbia University, New York, N.Y. (U.S.A.)
P. R. SRINIVASAN B. WEISS
1 B. WEISS AND P. R. SRINIVASAN, Proc. Natl. Acad. Sci. U.S., 45 (1959) 1491. 2 D. RITTENBERG, in J. W. EDWARDS, Preparation and Measurement o/Isotopic Tracers, A nn Arbor, Mich., 1946 . 3 B. LEVENBERG AND J. M. BUCHANAN, J. Biol. Chem., 224 (1957) lO19. 4 R. ABRAMS AND IV[. BENTLEY, Arch. Biochem. Biophys., 79 (1959) 91. 5 B. LEVENBERG, I. ~IELNICK AND J. M. BUCHANAN, J. Biol. Chem., 225 (1957) 163. 6 p. R. SRINIVASAN, .]. Am. Chem. Soc., 81 (1959) 1772. 7 p. R. SRINIVASAN, to be p u b l i s h e d .
Received April 5th, 196i Biochim. Biophys. Acta, 51 (1961) 597-599