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Phenylalanine Ammonia-Lyase in Poppy Plants (Papaver somniferum L.)1)
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Biochem. Physiol. Pflanzen (BPP), Bd. 163, S. 411-415 (1972) Biochemical Institute, Komensky University, Bratislava, CSSR
Phenylalanine Ammonia-Lyase in Poppy Plants (Papaver somniferum L.)1) By PAVEL NEMEC, PETER KovAcs, MIKULAS PSENAK und ANTONiN JINDRA With 4 figures (Received February 7, 1972)
Summary The enzyme phenylalanine ammonia-lyase (EC 4.3.1.5.) has been found in seedlings of Papaver somniferumL. var. Vahovecky, isolated and purified by ammonium sulphate precipitation, Sephadex G-l00 filtration and a sorption on hydroxylap~tite. The enzyme preparation with specific activity approximately 60 times higher than the original homogenate was used for determination of pH optimum, heat stability, apparent Michaelis constant and activation energy. The effects of various hydroxyderivatives of cinnamic acid on this enzyme are also described.
Introduction
The deamination of L-phenylalanine to trans-cinnamic acid catalysed by phenylalanine ammonia-lyase (PAL) is the key reaction for biosynthesis of phenolics in higher plants (ZUCKER 1965). We have discussed the importance of this reaction in our previous communication (NEMEC et al. 1972) mainly from the point of view of the regulation of phenolics biosynthesis. The determination of PAL activity in poppy plants seedlings reported in this paper underlines the role of cinnamic acid and its derivatives as potential intermediates in opium alkaloid formation, which corresponds to the scheme presented by MOTHES 1966. According to this scheme norlaudanosoline is formed by condensation of dopamine with dihydroxyphenylacetaldehyde originated from cinnamic acid. Experimental The enzyme was prepared from the seedlings of Papaver somniferum L. var. Vahovecky (seeds harvested in 1968). The seeds treated beforehand with Agronal H (2 g for 1000 g of seeds) were germinated at 25 0 in dark for three days on filter paper moistened with water. The seedlings were homogenised by grinding with sand in 0.05 11 phosphate buffer pH 7.0 and filtered through 1) Part VIII in tlie series "Enzymes in Amino Acid Metabolism"; for Part VII, see Chern. Papers, in press.
412
P. NEMEC, P. KOVAcs, M. PSENAK und A. JINDRA
the n) Ion doth. After centrifugation (10000 g for 15 min) the homogenate was salted out at 0 0 with ammonium sulphate according to PSENAKOVA et al. 1969. The fraction obtained at 20 bis 60 % of saturation was dissolved in 10 ml 0.005 M phosphate buffer pH 7.0 and dialysed for 10 hrs against 4000 ml of 0.001 M phosphate buffer pH 7.0. The solution was applied at 0 0 on the column of Sephadex G-100 (3 cm x 100 cm) and for elution 0.05 M phosphate buffer pH 7.0 was used. The rate of the flow was 6 ml/hr; the volume of individual fractions was 3-4 mi. The enzymatically active eluate from the column was adsorbed on hydroxylapatite (4 g) prepared according to KElLIN and HARTREE 1938. After centrifugation (10000 g for 20 min) the sediment was treated with successive portions (10 ml each) of phosphate buffer pH 7.0 with ascending concentration (from 0.05 M to 0.5 M). The enzyme activity was eluted at 0.45 and 0.5 M concentration. The enzyme activity was determined as follows: To 0.5 ml of enzyme preparation and 1 ml of L-phenylalanine solution (20 flmoles). Tris-HCI buffer solution (0.2 M, pH 8.8) was added to the total volume of 3 ml and the mixture incubated at 40 0 for 60 min. The concentration of cinnamie acid was determined spectrophotometric ally at 268 nm. One unit of enzyme activity (u)is the amount of enzyme which catalyses the formation of l,umole of cinnamic acid in 1 minute Lnder the (ondition described. The protein contents in enzyme preparations was determin£d according to LOWRY et al. 1951.
Results and discussion
The separation of PAL activity and protein contents on Sephadex G-100 column is visualised on fig. 1. The individual steps in enzyme purification are given in table 1. We have obtained various kinetic data using the purified enzyme. pH optimum was found at 8.8 (see fig. 2.); this value corresponds to pH optima of PAL pre para-
r-\/.~
0.8
~
w f-
o
I/ \
'""-oo E
::J
.
0.2
.
.
)
E
>f-
~~-.-.-.-./............
>
e«
,~\
•
-'
•....... B
«
"-
~·-r--
10
20
30
40
50
60
70
NUMBER OF FRACTION
Fig. 1. Gel filtration on Sephadex G-100 A, Phenylalanine ammonia-lyase activity; B, Absorbance at 280 nm
413
Phenylalanine Ammonia-Lyase in Poppy Plants etc. Table 1
Scheme of PAL purification Protein [mg]
Specific activity
Increase of specific activity n-fold
Level of purity
Total mU volume lml]
Crude homogenate Dialysed enzyme Sephadex G-ll 0 gel filtration Adsorption on hydroxylapatite
70 9
226,8 42.7
1121 194
0.202 0.220
1 1.09
65
61.5
36
1,694
9.38
19
58.4
4.9
11.8
58.50
tions from other sources as was described by KOUKOL and CONN 1961, YOUNG and NEISH 1966 and by O'NEAL and KELLER 1970. The temperature influence is to be seen from fig. 3. The apparent Michaelis constant for L-phenylalanine was found 5.10-3M (according to LINEWEAVER and BURK 1934) at pH 8.8, concentration of substrate being from 3.3.1O-4M to 2.10-2M. The activation energy for L-phenylalanine (35-45°) is 10120 cal. mol-i. The value of QlO was 1.69. The value of the apparent Michaelis constant given above corresponds to values found for PAL isolated from other sources: Zea mays (4.10- 3M) reported by NEMEC et al. 1972 and Hordeum vulgare (1.4.10-3 M) described by KOUKOL and CONN 1961. Tyrosine and 3,4-dihydroxyphenylalanine are precursors in the biosynthesis of opium alkaloids (BATTERSBY and MCCALDIN 1962). Phenylalanine and tyrosine might be transformed in higher plants into cinnamic acid and its hydroxyderivatives, which in turn are supposed to be transformed to flavonoids, coumarins, lignin and
>-
0.2
>-
> i= ~ 0,1
7,5
8,0
8,5
9,0
pH
Fig. 2. Effect of pH on phenylalanine ammonia-lyase activity
414
rI
P. NEMEC, P. KOVAcs, M. PSENAK und A. JINDRA Table 2
Effect of inhibitors
Inhibitor
concentration [M]
Relative activity
Control p-Coumaric acid Caffeic acid
10-4 10- 4
100 68 24
%
benzoic acid derivatives respectively. According to MOTHES 1966, cinnamic acid might be used in poppy plants as prescursor of opium alkaloids. We have studied, therefore, the effect of p-coumaric acid and caffeic acid on PAL activity from Papaver somniferum. These results are given in table 2. and fig. 4. The enzyme activity was inhibited by both acids. Ki for p-coumaric acid was 1,21.10-4 M, for caffeic acid 3,04.10-5M. We can assume from these data, in accordance with other reports (NEMEC et al. 1972; O'NEAL and KELLER 1970) that phenolic derivatives of cinnamic acid function in poppy plants as regulators of PAL activity and of the intensity of opium alkaloid formation as well. c ~
(),3
w
.... aat: "-
E :::>
0,2
E
/
>-
.... >
....
u
«
/.-
0,1
./.
/\\
1
V 100
.
/' /
.//' .~.
:_.-.---
.
.,,/
~
«
"-
30
40
50
60
--'
~.
-A
100
70
TE M PERATURE ("I:)
4
3
Fig. 3. Effect of temperature on phenylalanine ammonia-lyase activity Fig. 4. LINEWEAVER and BURR plots in the presence and absence of inhibitors A, Control; B, p-Coumaric acid; C, Caffeic acid
Literature BATTERSBY, A. R., MCCALDIN, D. J. M., Proc. Chern. Soc. (London) 365 (1962). KElLIN, D., HARTREE, E. F., Proc. Roy. Soc. B 124, 397 (1928). KOUKOI, J., CONN, j • E., J. 1 iol. Chern. 236, 2692 (1961). LINEWEAVER, H., BURK, D., J. Am. Chern. Soc. 56, 563 (1934).
Ii.
; £S£±
Phenylalanine Ammonia-Lyase in Poppy Plants etc.
415
LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., RANDALL,R. J.:, J. BioI. Chem.193, 265 (1951). MOTHES, K., Lloydia 29, 156 (1966). O'NEAL, D., KELLER, C. J. Phytochem. 9, 1373 (1970). NEMEC, P., PSENAK, M., KOVAcs, P., JINDRA, A., Chern. Papers 26 (1972). PSENAKOVA, T., KovAcs, P., PSENAK, M., Phytochem. 8, 2277 (1969). YOUNG, M. R., NEISH, A. C., Phytochem. 5, 1121 (1966). ZUCKER, M., Plant Physiol. 40, 779 (1965). Author's address: Dipl. Ing. P. NEMEC, Doc. Dr. P. KOVACS, Dr. M. PSENAK and Prof. Dr. A. JINDRA, Biochemical Institute, Komensky University, Kalinciakova 8, Bratislava (CSSR).
28
Biochem. Physio!. Pflanzen Bd.
16~.
Biochem. Physiol. Pflanzen (BPP), Bd. 163, S. 411-415 (1972) Biochemical Institute, Komensky University, Bratislava, CSSR
Phenylalanine Ammonia-Lyase in Poppy Plants (Papaver somniferum L.)1) By PAVEL NEMEC, PETER KovAcs, MIKULAS PSENAK und ANTONiN JINDRA With 4 figures (Received February 7, 1972)
Summary The enzyme phenylalanine ammonia-lyase (EC 4.3.1.5.) has been found in seedlings of Papaver somniferumL. var. Vahovecky, isolated and purified by ammonium sulphate precipitation, Sephadex G-l00 filtration and a sorption on hydroxylap~tite. The enzyme preparation with specific activity approximately 60 times higher than the original homogenate was used for determination of pH optimum, heat stability, apparent Michaelis constant and activation energy. The effects of various hydroxyderivatives of cinnamic acid on this enzyme are also described.
Introduction
The deamination of L-phenylalanine to trans-cinnamic acid catalysed by phenylalanine ammonia-lyase (PAL) is the key reaction for biosynthesis of phenolics in higher plants (ZUCKER 1965). We have discussed the importance of this reaction in our previous communication (NEMEC et al. 1972) mainly from the point of view of the regulation of phenolics biosynthesis. The determination of PAL activity in poppy plants seedlings reported in this paper underlines the role of cinnamic acid and its derivatives as potential intermediates in opium alkaloid formation, which corresponds to the scheme presented by MOTHES 1966. According to this scheme norlaudanosoline is formed by condensation of dopamine with dihydroxyphenylacetaldehyde originated from cinnamic acid. Experimental The enzyme was prepared from the seedlings of Papaver somniferum L. var. Vahovecky (seeds harvested in 1968). The seeds treated beforehand with Agronal H (2 g for 1000 g of seeds) were germinated at 25 0 in dark for three days on filter paper moistened with water. The seedlings were homogenised by grinding with sand in 0.05 11 phosphate buffer pH 7.0 and filtered through 1) Part VIII in tlie series "Enzymes in Amino Acid Metabolism"; for Part VII, see Chern. Papers, in press.
412
P. NEMEC, P. KOVAcs, M. PSENAK und A. JINDRA
the n) Ion doth. After centrifugation (10000 g for 15 min) the homogenate was salted out at 0 0 with ammonium sulphate according to PSENAKOVA et al. 1969. The fraction obtained at 20 bis 60 % of saturation was dissolved in 10 ml 0.005 M phosphate buffer pH 7.0 and dialysed for 10 hrs against 4000 ml of 0.001 M phosphate buffer pH 7.0. The solution was applied at 0 0 on the column of Sephadex G-100 (3 cm x 100 cm) and for elution 0.05 M phosphate buffer pH 7.0 was used. The rate of the flow was 6 ml/hr; the volume of individual fractions was 3-4 mi. The enzymatically active eluate from the column was adsorbed on hydroxylapatite (4 g) prepared according to KElLIN and HARTREE 1938. After centrifugation (10000 g for 20 min) the sediment was treated with successive portions (10 ml each) of phosphate buffer pH 7.0 with ascending concentration (from 0.05 M to 0.5 M). The enzyme activity was eluted at 0.45 and 0.5 M concentration. The enzyme activity was determined as follows: To 0.5 ml of enzyme preparation and 1 ml of L-phenylalanine solution (20 flmoles). Tris-HCI buffer solution (0.2 M, pH 8.8) was added to the total volume of 3 ml and the mixture incubated at 40 0 for 60 min. The concentration of cinnamie acid was determined spectrophotometric ally at 268 nm. One unit of enzyme activity (u)is the amount of enzyme which catalyses the formation of l,umole of cinnamic acid in 1 minute Lnder the (ondition described. The protein contents in enzyme preparations was determin£d according to LOWRY et al. 1951.
Results and discussion
The separation of PAL activity and protein contents on Sephadex G-100 column is visualised on fig. 1. The individual steps in enzyme purification are given in table 1. We have obtained various kinetic data using the purified enzyme. pH optimum was found at 8.8 (see fig. 2.); this value corresponds to pH optima of PAL pre para-
r-\/.~
0.8
~
w f-
o
I/ \
'""-oo E
::J
.
0.2
.
.
)
E
>f-
~~-.-.-.-./............
>
e«
,~\
•
-'
•....... B
«
"-
~·-r--
10
20
30
40
50
60
70
NUMBER OF FRACTION
Fig. 1. Gel filtration on Sephadex G-100 A, Phenylalanine ammonia-lyase activity; B, Absorbance at 280 nm
413
Phenylalanine Ammonia-Lyase in Poppy Plants etc. Table 1
Scheme of PAL purification Protein [mg]
Specific activity
Increase of specific activity n-fold
Level of purity
Total mU volume lml]
Crude homogenate Dialysed enzyme Sephadex G-ll 0 gel filtration Adsorption on hydroxylapatite
70 9
226,8 42.7
1121 194
0.202 0.220
1 1.09
65
61.5
36
1,694
9.38
19
58.4
4.9
11.8
58.50
tions from other sources as was described by KOUKOL and CONN 1961, YOUNG and NEISH 1966 and by O'NEAL and KELLER 1970. The temperature influence is to be seen from fig. 3. The apparent Michaelis constant for L-phenylalanine was found 5.10-3M (according to LINEWEAVER and BURK 1934) at pH 8.8, concentration of substrate being from 3.3.1O-4M to 2.10-2M. The activation energy for L-phenylalanine (35-45°) is 10120 cal. mol-i. The value of QlO was 1.69. The value of the apparent Michaelis constant given above corresponds to values found for PAL isolated from other sources: Zea mays (4.10- 3M) reported by NEMEC et al. 1972 and Hordeum vulgare (1.4.10-3 M) described by KOUKOL and CONN 1961. Tyrosine and 3,4-dihydroxyphenylalanine are precursors in the biosynthesis of opium alkaloids (BATTERSBY and MCCALDIN 1962). Phenylalanine and tyrosine might be transformed in higher plants into cinnamic acid and its hydroxyderivatives, which in turn are supposed to be transformed to flavonoids, coumarins, lignin and
>-
0.2
>-
> i= ~ 0,1
7,5
8,0
8,5
9,0
pH
Fig. 2. Effect of pH on phenylalanine ammonia-lyase activity
414
rI
P. NEMEC, P. KOVAcs, M. PSENAK und A. JINDRA Table 2
Effect of inhibitors
Inhibitor
concentration [M]
Relative activity
Control p-Coumaric acid Caffeic acid
10-4 10- 4
100 68 24
%
benzoic acid derivatives respectively. According to MOTHES 1966, cinnamic acid might be used in poppy plants as prescursor of opium alkaloids. We have studied, therefore, the effect of p-coumaric acid and caffeic acid on PAL activity from Papaver somniferum. These results are given in table 2. and fig. 4. The enzyme activity was inhibited by both acids. Ki for p-coumaric acid was 1,21.10-4 M, for caffeic acid 3,04.10-5M. We can assume from these data, in accordance with other reports (NEMEC et al. 1972; O'NEAL and KELLER 1970) that phenolic derivatives of cinnamic acid function in poppy plants as regulators of PAL activity and of the intensity of opium alkaloid formation as well. c ~
(),3
w
.... aat: "-
E :::>
0,2
E
/
>-
.... >
....
u
«
/.-
0,1
./.
/\\
1
V 100
.
/' /
.//' .~.
:_.-.---
.
.,,/
~
«
"-
30
40
50
60
--'
~.
-A
100
70
TE M PERATURE ("I:)
4
3
Fig. 3. Effect of temperature on phenylalanine ammonia-lyase activity Fig. 4. LINEWEAVER and BURR plots in the presence and absence of inhibitors A, Control; B, p-Coumaric acid; C, Caffeic acid
Literature BATTERSBY, A. R., MCCALDIN, D. J. M., Proc. Chern. Soc. (London) 365 (1962). KElLIN, D., HARTREE, E. F., Proc. Roy. Soc. B 124, 397 (1928). KOUKOI, J., CONN, j • E., J. 1 iol. Chern. 236, 2692 (1961). LINEWEAVER, H., BURK, D., J. Am. Chern. Soc. 56, 563 (1934).
Ii.
; £S£±
Phenylalanine Ammonia-Lyase in Poppy Plants etc.
415
LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., RANDALL,R. J.:, J. BioI. Chem.193, 265 (1951). MOTHES, K., Lloydia 29, 156 (1966). O'NEAL, D., KELLER, C. J. Phytochem. 9, 1373 (1970). NEMEC, P., PSENAK, M., KOVAcs, P., JINDRA, A., Chern. Papers 26 (1972). PSENAKOVA, T., KovAcs, P., PSENAK, M., Phytochem. 8, 2277 (1969). YOUNG, M. R., NEISH, A. C., Phytochem. 5, 1121 (1966). ZUCKER, M., Plant Physiol. 40, 779 (1965). Author's address: Dipl. Ing. P. NEMEC, Doc. Dr. P. KOVACS, Dr. M. PSENAK and Prof. Dr. A. JINDRA, Biochemical Institute, Komensky University, Kalinciakova 8, Bratislava (CSSR).
28
Biochem. Physio!. Pflanzen Bd.
16~.