THE JOURNAL oy‘ BIOLOGICAL CHEMISTRY

Vol. 246, No. 12, Issue of June 25, PP. 3870-3878, 1971

Printed in U.S.A.

The Metabolism of Aromatic Compounds in Higher Plants

X. PROPERTIES OF THE CINNAMIC ACID 4-HYDROXYLASE OF PEA SEEDLINGS AND SOME ASPECTS OF ITS METABOLIC AND DEVELOPMENTAL CONTROL*

DAVID W. RUSSELLS

(Received for publication, October 6, 1970)

From the Department of Biochemistry and Biophysics, University of California, Davis, California 95616

SUMMARY

The cinnamic acid 4-hydroxylase of pea seedlings is a microsomal mixed function oxidase which requires molecular oxygen, NADPH, and Z-mercaptoethanol for activity. Opti- mal activity occurs at pH 7.5. NADH will not substitute for NADPH. Glutathione, ascorbic acid, cysteine, and dithio- threitol will not replace mercaptoethanol. The latter com- pound is required in millimolar concentrations for optimal activity but does not serve as an external reductant for the hydroxylation reaction.

The K, for cinnamic acid is about 0.02 mu. The enzyme does not catalyze the hydroxylation of either phenylalanine or 4-hydroxycinnamic acid. The product exerts a strong regula- tory effect on activity; inhibition commences at about 0.03 lll~ and increases sharply to a maximum at 0.10 mu, at which more than 90% inhibition may occur. Inhibition is noncom- petitive and shows cooperativity with increasing product con- centration.

The reaction is inhibited by carbon monoxide, and the in- hibition is reversed by light. Azide is inhibitory, but cyanide has little effect.

The activity of the hydroxylase in etiolated seedlings is in- creased S-fold by a brief irradiation about 1’2 hours before har- vest and assay. The enzyme in green seedlings is restricted to young, developing tissues. Activity is highest in the apical bud, declines sharply with the onset of maturity, and is not detected in mature leaves.

In higher plants, feeding experiments have established that the aromatic amino acids, phenylalanine and tyrosine, are primary precursors of a wide variety of natural products (1). In addi-

tion, the general pathways of biosynthesis of some secondary plant products have been outlined by feeding labeled compounds believed to be intermediates and measuring the subsequent iso- tope dilution. However, due to the paucity of enzyme studies in this area, the details of the biosynthetic pathways remain

* This work was supported in part by Grant GM-5301 awarded to E. E. Conn bv the Institute of General Medical Sciences. Na- tional Institutes of Health.

$ Present address, Department of Biochemistry, University of Otago, Dunedin, New Zealand.

largely unknown or speculative, and almost nothing is known about the biosynthetic control mechanisms and their relationship to the growth and development of the plant.

Conversion of phenylalanine to truns-cinnamic acid is cata- lyzed by phenylalanine ammonia lyase (EC 4.3.1.5), which is found in both dicotyledons and monocotyledons and whose prop- erties have been described by Koukol and Conn (2). Further studies have been carried out on the developmental control of activity (3, 4) and other characteristics of this enzyme (5). The enzyme which catalyzes the conversion of tyrosine to 4-hydroxy- cinnamic acid, tyrosine ammonia lyase, has been described by Neish (6) and is confined largely to grasses within the monocot- yledons. Thus, 4-hydroxycinnamic acid in most dicotyledons is synthesized from phenylalanine via cinnamic acid, and it is likely that this pathway is significant in monocotyledons also. Since 4-hydroxyrcinnamic acid is situated at a branch point in the bio- synthesis of many phenolic compounds, the majority of which possess a hydroxyl group in a position corresponding to carbon atom 4 of the cinnamic acid-derived moiety, the factors which control its synthesis and metabolism are, therefore, of considera- ble interest. The present work describes some properties of the cinnamic acid 4-hydroxylase of pea seedlings and some of the conditions affecting its activity. A preliminary report of part of this work has been published elsewhere (7).

Attempts in this laboratory to demonstrate cinnamic acid 4-hydroxylase activity’ and phenylalanine 4-hydroxylase activity2 under the conditions reported by Nair and Vining (8) have been unsuccessful.

EXPERIMENTAL PROCEDURE

Materials

Cinnamic acid-2-i4C was obtained from Tracerlab. Glucose- 6-P, NADP+, glucose-6-P dehydrogenase, and tetrahydrofolate were supplied by Sigma. 2-Mercaptoethanol was obtained from Eastman. Aminopterin (Lederle Laboratories) was a kind gift from Dr. R. H. Doi of this department. D,L-Phenylalauine- 2.14~ was obtained from Volk Radiochemical Company, Bur- bank, California, and 4-hydroxycinnamic acid-U-14C, generously supplied by Dr. T. Kosuge, Department of Plant Pathology of this institution, was synthesized from tyrosine-U-l% (New Eng-

1 H. G. Floss, J. R. Stoker, and E. E. Conn, unpublished obser- vations.

2 D. W. Russell, and E. E. Conn, unpublished observations.

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land Nuclear) by the action of tyrosine ammonia lyase prepared from barley seedling acetone powders (6). Polyclar AT (in- soluble polyvinyl pyrrolidone) was purchased from General Aniliue and Film Corporation, New York, New York.

Methods

Etiolated Plants-Pea seeds, variety Alaska, (Asgrow, Inc., Tracy, California) were soaked overnight, planted in trays of moist vermiculite, and set in a dark cupboard at room tempera- ture. On Day 6 or 7, at the third internode stage, seedlings were exposed to fluorescent light for about 10 min, returned to dark- ness, and harvested at least 6 hours later. Apical buds were plucked off and used as the source of enzyme; no attempt was made to remove any subjacent internode tissues which remained attached to the bud.

Green Plants-Pea seeds (Alaska) were soaked and sown in flats in a peat-humus mixture in a greenhouse. Seedlings were harvested after about 2 weeks, when they were 18 to 26 cm high. Apical buds and enclosing stipules and the youngest leaf (if less than half expanded) were selected and used as the enzyme source.

Preparation of Subcellular Fractions-Plant tissues (5 to 15 g fresh weight) were ground in a cold mortar with 0.05 M phosphate buffer, pH 7.5, together with Polyclar AT (0.1 X fresh weight of tissues). Where green tissues were used, sand wasadded to assist grinding. Tissues were ground for about 3 min with enough buffer to give a workable slurry, which was squeezed through four layers of cheesecloth. The filtrate was centrifuged at 500 x g for 10 min, the residue discarded, and the supernatant sol-

ution retained for further fractionation. To determine the subcellular localization of hydroxylase ac-

tivity, the following fractionation procedure was carried out. The crude homogenate was centrifuged at 12,000 X g for 10 min to obtain a particulate fraction (12,000 x g residue), designated as mitochondria. The mitochondria were washed twice by resuspension in buffer and resedimentation. The 12,000 X g supernatant solution was further centrifuged at 105,000 X g for 1 hour to obtain the final supernatant fraction (105,000 X g super- natant) and a high speed particulate fraction (105,000 X g residue), hereafter referred to as microsomes. The final super- natant and the particulate fractions, resuspended in buffer, were assayed for enzyme activity.

In subsequent studies the microsomal fraction, which con- tained all the enzyme activity, was routinely obtained from the cheescloth filtrate as follows. The filtrate was centrifuged at 12,000 x g as described above to remove the mitochondria, which were discarded. The microsomes in the supernatant were sedi- mented at 105,000 x g for 1 hour. The final supernatant was discarded and the microsomes resuspended in 0.075 M phosphate buffer, pH 7.5, made to a given volume and retained as the en- zyme source. When testing the effect of pH on activity, micro- somes were resuspended in 0.025 M buffer.

Enzyme activity was readily detectable in crude homogenates. E$ect of Extraction and Storage Procedures on Enzyme Activity-

The omission of Polyclar AT during the grinding of etiolated tis- sues yielded preparations with 10% less activity than homoge- nates made in the presence of Polyclar. The effect on green tis- sues was not examined.

Homogenization of the tissue in the presence of 2-mercapto- ethanol did not give higher activity than those experiments in which mercaptoethanol was absent during extraction but was added to the reaction mixture prior to assay. The latter proce-

TABLE I Effect of storage conditions on slability of cinnamic acid

.J-hydroxylase in microsomal preparations

Microsomes, isolated from apical buds of green pea seedlings, were resuspended in 0.075 M phosphate buffer, pH 7.5 (16 mg per ml of protein) and immediately assayed by incubating 7.2 mg of microsomal protein in 3.0-ml reaction volumes for 2 hours at 30”. The remaining suspension was made to 3.6 mM mercapto- ethanol and divided into 4 equal volumes; to 2 of these 10% glyc- erol (v/v) was added. The four tubes were assayed after storage for 1 week under the conditions indicated.

Conditions Activity as per cent of fresh microsomes

4O 4’ + 10% glycerol (v/v)

-20° -20” + loci0 glycerol (v/v)

% 42 18 6

110

dure offered the advantage of precise control of the concentration of mercaptoethanol in the reaction mixture.

The enzyme was unstable; 64% and 70y0 of the activity in a crude homogenate was lost during storage for 24 hours at 4” and -2O”, respectively. Microsomal preparations were also un- stable and, when stored for 2 weeks at 4” or -20” in the presence or absence of 6 mM mercaptoethanol, lost 88 to 96% of the original activity. However, microsomal preparations stored in a mixture composed of 0.075 M phosphate buffer, pH 7.5, 3.6 mM mercapto- ethanol, and 10 to 15% glycerol (v/v) at a protein concentration of 15 to 20 mg per ml were relatively stable when stored at -20”. Such preparations showed no apparent loss in activity after storage for 1 week (Table I); they retained 54’$& of their original activity after storage for 2 weeks and 38% after 3 weeks. One cycle of thawing and refreezing caused a 54% loss in activity. The presence of mercaptoethanol was not essential for the stabil- ity of microsomal preparations when stored frozen, in the presence of glycerol.

The microsomal fraction derived from a given weight of tissue was more active than the same quantity of microsomes present in the crude homogenate or in the 12,000 x g supernatant fraction. The activity of isolated microsomes was 150% of the activity of the same quantity of microsomes in an aliquot of crude homoge- nate and 140% of the activity in the 12,000 x g supernatant frac- tion (based on extract equivalent to 1.0 g fresh weight of tissues).

Enzyme Assays-Two standard assay procedures were used, one employing a 3.0.ml reaction volume and the other a volume of 0.1 ml. Variations from the standard procedures are noted in the text.

Method I-Reaction volume of 3.0 ml: The reaction mixture consisted of trans.cinnamic acid-2-14C (280,000 dpm in 0.074 /*mole) ; trans.cinnamic acid, 2 pmoles; phosphate buffer, pH 7.5, 150 pmoles; glucose-6-P dehydrogenase, 0.4 unit (1 unit reduces 1.0 pmole of NADP+ per min at 25” at pH 7.4 in the presence of glucose-6-P); NADP+, 2 pmoles; glucose-6-P, 5 pmoles; 2-mer- captoethanol, 3 pmoles; and the enzyme preparation (extracts from tissues not exceeding 2.0 g fresh weight or microsomes not exceeding 12.0 mg of protein).

Method II-Reaction volume of 0.1 ml: The reaction mixture contained trans.cinnamic acid-2-‘*C (280,000 dpm in 0.074 pmole); phosphate buffer, pH 7.5, 4.5 pmoles; glucose-6-P dehy- drogenase, 0.02 unit; NADP+, 0.2 pmole; glucose-6-P, 0.2 pmole;

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3872 Cinnamic Acid ,J-Hydroxylaase of Pea Seedlings Vol. 246, No. 12

Z-merceptoethanol, 0.1 pmole; and enzyme preparation not exceeding 60 mg fresh weight of tissues or 0.4 mg of microsomal protein.

The reaction mixture with all components except the NADPH- generating system was incubated for 5 min at 30”; the NADPH- generating system was incubated separately for 5 min at, 30”. The reaction was started by adding the generating system. The systems for generating the reduced pyridine nucleotides were tested before use, and the quantities of substrates used and condi- tions of the assay assured the presence of excess reduced pyridine nucleotide in the reaction mixture.

Incubations were carried out with shaking at 30” for 30 min. In Method I, the reaction was stopped by addition of 0.1 ml of 6 N HCl. The protein was centrifuged down, washed once, and the supernatant and wash solutions were combined, acidified to pH 1, and extracted twice with 1 volume of ethyl acetate. The ethyl acetate extract was evaporated to dryness, the residue dis- solved in 0.2 ml of ethanol, and 0.1 ml of the ethanolic solution spotted onto Whatman No. 1 chromatogram strips (4 X 56 cm). In Method II, the reaction was stopped by heating for 2 min on a boiling water bath, the protein was centrifuged down, and 50 or 75 ~1 of the supernatant solution was directly spotted onto What- man No. 1 chromatogram strips for chromatography.

The papers were equilibrated over solvent and developed with benzene-acetic acid-water, 2 : 2 : 1 (v/v/v). The strips were then scanned with a Vanguard strip scanner and the radioactive peak corresponding to 4-hydroxycinnamic acid cut out and counted in Bray’s solution in a liquid scintillation counter (Packard Tri- Garb) ; a background strip was also counted. The results are expressed as mbmoles of 4-hydroxycinnamic acid produced per hour per g fresh weight of tissue or per mg of microsomal protein. Under the conditions of the assay, the reaction was linear with time up to 30 min, showed a slight deviation at 1 hour, considera- bly more at 2 hours, and declined sharply after this time. The reaction was also linear with the amount of enzyme used (up to 2 g fresh weight of tissue extracted, or 12.0 mg of microsomal

TABLE II

Subcellular localization of enzyme activity

Homogenates of apical buds were centrifuged at 500 X g for 10 min to remove debris. The mitochondrial fraction was isolated by centrifugation at 12,000 X g for 10 min, and the 12,000 X g supernatant solution sedimented at 105,000 X g for 1 hour to yield microsomes (sedimented) and the 105,000 X g supernatant solution. The mitochondrial fraction was washed two times by resuspension and resedimentation. Final mitochondrial and microsomal fractions were resuspended in 0.1 M phosphate buffer, ~1% 7.5. Assays were carried out immediately in reaction volumes of 3.0 ml. Aliquots equivalent to 1 g fresh weight from green tissues and 4 g fresh weight from etiolated tissues were incubated 2 hours at 30”.

4-Hydroxycinnamic acid produced

Subcellular fraction Etiolated Gi-eC?n

tissues tissues

mpmoles/hour/g fresh weight

Supernatant fraction (105,000 X g). 0.0 0.0 Microsomalfraction......................... 58.9 250.0 Mitochondrial fraction. 2.2 2.1

protein, in a 3.0-ml reaction volume and proportionally less in a reaction volume of 0.1 ml).

Identification of the product as 4-hydroxycinnamic acid has been described in a previous publication (7).

In experiments to test the inhibition of the enzyme reaction by carbon monoxide (CO), the gas mixtures employed were prepared by displacing known volumes of water from large brown bottles initially filled with deaerated water. The hydroxylation reac- tions were carried out in Warburg vessels with two side arms, each with a stoppered access port,. The vessels containing the reaction mixture, with the substrate in one of the side arms, were gassed with about, 60 volumes of the appropriate gas mixture and stoppered. The reaction commenced in darkness or light, by addition of substrate. Incubation in the light was carried out under normal laboratory lighting, together with a 60 watt incan- descent lamp at about 45 cm from the Warburg vessel in the incubator-shaker.

When 4-hydroxycinnamic acid was tested as a substrate, the reaction mixture, after incubation, was chromatographed on paper with benzene-dioxane-acetic acid, 10:3: 1 (v/v/v) as the developing solvent. In this solvent, the RF for 4-hydroxycin- namic acid was 0.68 and for 3,4-dihydroxycinnamic acid (caffeic acid) was 0.37. When phenylalanine was examined as a sub- strate, chromatographic separation was carried out with propa- nol-2 N HCl, 7:3 (v/v), in which phenylalanine showed an RF of 0.82 and tyrosine 0.67.

Protein was assayed by the biuret method; the procedure was modified to include solubilization of the particulate material by 1.0% sodium deoxycholate (w/v). Bovine serum albumin was used as a standard.

RESULTS

The 4-hydroxylation of cinnamic acid can be observed in a crude homogenate, as reported previously (7). In a typical ex- periment, 4-hydroxycinnamic acid was synthesized at the rate of 72 mpmoles per hour when cinnamic acid (2.56 pmoles) was incu- bated with extract from 1.0 g fresh weight of etiolated tissues. The identity of the product has been confirmed by chromatog- raphy, by its ultraviolet absorption spectrum, and by three re- crystallizations with authentic 4-hydroxgcinnamic acid with no decline in specific activity.

Cell fractionation studies have shown that enzyme activity is confined to the microsomal fraction (Table II). The different cell fractions were obtained as described under “Methods.” Results are presented for experiments with preparations from both green and etiolated tissues. No activity could be detected in the 105,000 X g supernatant fraction, and the mitochondrial fraction contained negligible activity compared with that present in the microsomes.

The variation of enzyme activity in the microsomes with pI1 is shown in Fig. 1. Optimal activity is observed at pH 7.5, with a small decrease in activity at pH 7.0 and 8.0, but a marked decline above pH 8.0 and a sharp decrease at pH 6.0. No activity was detected at pH 5.0.

Previously reported studies on the cofactor requirements (7) showed that there is an absolute and specific requirement for NADPH produced by a NADPH-generating system. The data also indicated that the reaction did not proceed in the absence of tetrahydrofolate, and it was concluded that tetrahydrofolate is required. However, when aminopterin at 3 X lOA4 M was subse-

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quently demonstrated to have no inhibitory effect on the reac- tion, this conclusion became suspect. Further work has indi- cated that the apparent requirement for tetrahydrofolate is due to the presence of mercaptoethanol, which was added to the tetra- hydrofolate solution as an antioxidant, and that tetrahgdrofolate itself is not required. The data show (Table III) that NADPH alone will not support the reaction, but that NADPH together with 0.03 mM mercaptoethanol (Experiment. 2) supports low ac- tivity corresponding to about 10% of the maximum obtainable. The addition of tetrahydrofolate did not increase the yield (Experiment 3, Table III). It can be concluded, therefore, that mercaptoethanol does not function catalytically in the presence of NADPH to reduce the enzyme system, nor does tetrahydro- folate exhibit a catalytic role in this regard. That mercapto- ethanol itself does not satisfy the reducing function provided by NADPH is shown by the failure of substrate quantities of mer- captoethanol alone to sustain a reaction (Experiment 4, Table III), but when NADPH is present in addition to mercaptoetha- nol, good activity is obtained, and the further addition of tetra- hydrofolate does not increase the activity. These results suggest that mercaptoethanol functions by maintaining certain groups on the enzyme complex in the reduced state and that, although these are necessary for enzyme integrity, they do not themselves participate in the reaction mechanism. It appears that NADPH itself satisfies the reducing requirement for the actual reaction.

The specificity of the mercaptoethanol effect has also been examined. The data (Table IV) show that, whereas the addition of 0.3 or 1 mM mercaptoethanol gives good activity, addition of comparable quantities of reduced glutathione, ascorbic acid, dithiothreitol, or cysteine in the absence of mercaptoethanol re- sulted in little or no reaction.

Mercaptoethanol shows an optimum concentration effect in the range 0.6 to 1.7 mM, and marked inhibition occurs at concen- trations higher than 2 to 3 mM (Fig. 2). At 6.6 mM, the reaction was inhibited 41’5& relative to activity at the optimum concen- tration.

That molecular oxygen is required was shown by measuring the amount of hydroxylation in the absence of air. The reaction vessel was evacuated and nitrogen admitted three times before starting the reaction. Under these conditions, an inhibition of 93 y0 was observed.

The effects of CO and light on the activity of the enzyme have also been examined. Incubations were carried out in darkness under CO-O2 atmospheres with CO and 02 in a ratio of 1: 1 and at various concentrations. The results (Table V) show that CO inhibits the reaction and that the extent of the inhibition issimilar at a constant CO :02 ratio, regardless of the percentage composi- tion of CO. A mixture of CO and 02 in the ratio of 1: 1 caused more than 70% inhibition, a greater inhibition than is found in animal systems (9). It was also found that light reversed the inhibition by these gas mixtures. Whereas in darkness a mixture of 12% CO and 12% OS caused a 74yc inhibition relative to the 12y0 O2 controls, an inhibition of 217, was observed when the reaction vessel was illuminated. This corresponds to reversal by light of more than 70% of the inhibition produced by CO.

The effects of different inhibitors on the reaction are shown in Table VI. A concentration of 10 ml1 cyanide caused a 23% inhibition, but there was no detectable inhibition at concentra- tions of 1.0 mM or lower. However, the effects of azide were more pronounced; 78% inhibition was obtained at 10 m&f, and

I

BUFFERS

x Citrate

0 Phosphate P Borate

6.0 ZO BO 9.0 PH

FIG. 1. The effect of pH on cinnamic 4-hydroxylase activity Microsomes from buds (1 g fresh weight) of etiolated seedlings were incubated for 2 hours at 30” in 3-ml reaction mixtures, con- taining buffers of different pH.

TABLE III Mercaptoethanol requirement and absence of tefrahydrofolate

requirement

Microsomes (8 mg of protein) from buds of green seedlings were incubated for 2 hours at 30” in reaction volumes of 3 ml. Fresh solutions of mercaptoethanol and tetrahydrofolate were used, and the absorbance of the latter (A? = 2.2 X 104) was checked before assay to ensure that it was not oxidized or otherwise de- graded. In Experiments 2 and 3, 0.1 rmole of mercaptoethanol was added, and Experiments 4 to 6 contained 10.0 pmoles. When tetrahydrofolate was tested, 0.1 pmole was added. NADPH was supplied by a generating system described under “Methods.”

Experiment

1 2 3

4 5 6

7

Reductant added 4-Hydroxy-

cinnamic acid produced

NADPH NADPH + 0.03 mM mercaptoethanol NADPH + 0.03 mM mercaptoethanol +

0.03 mM HI-folate 3.0 mM mercaptoethanol 3.0 mM mercaptoethanol + NADPH 3.0 mM mercaptoethanol + NADPH +

0.03 mM Ha-folate 3.0 mM mercaptoethanol + NADPH

(heat-inactivated enzyme)

m&e?mles

0.0 11.2

7.0 0.0

118.3

115.5

0.0

an inhibition of 2270 was observed at 1.0 mM. EDTA (1.0 mM) had no detectable effect, but a,ol-dipyridyl at the same concen- tration caused a 27% inhibition.

The specificity of the enzyme for the 4-hydroxylation of cin-

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3874 Cinnamic Acid J-Hydroxylase of Pea Seedlings Vol. 246, No. 12

TABLE IV Specificity of mercapfoethanol requirement

Microsomes from buds (1 g fresh weight) of green seedlings were incubated for 2 hours at 30” in 3-ml reaction mixtures, with mer- captoethanol and other reductants as indicated.

Reductant Conversion as percentage of control

Experiment 1 0.3 rnM mercaptoethanol.. . . 0.3 mM glutathione.. 0.2 mM ascorbic acid

Experiment 2 1.0 rnM mercaptoethanol.. 1.0 mna dithiothreitol.. . l.OmM cysteine........................

100.0 0.0 2.2

100.0 1.3 0.3

2-MERCAPTOETHANOL (mM)’

FIG. 2. The effect of mercaptoethanol on cinnamic 4-hydroxyl- ase activity. Microsomes (8.6 mg of protein) from apical buds of green seedlings were incubated 2 hours at 30” in 3-ml reaction mixtures with various concentrations of mercaptoethanol.

namic acid has been examined by testing phenylalanine and 4-hydroxycinnamic acid for possible activity as substrates. Although the enzyme preparation was active in the 4-hydroxyla- tion of cinnamic acid, no detectable hydroxylation activity was exhibited toward either phenylalanine or 4-hydroxycinna,mic acid when these compounds were tested at the same concentration (0.75 mM). The enzyme thus appears to be quite specific for cinnamic acid.

The effect of substrate concentration on velocity of the reaction has been examined. The curve obtained by plotting the velocity against substrate concentration shows that the kinetic data are typically hyperbolic at concentrations up to 1 mM; concentrations of substrate in excess of 1 mM are inhibitory. The enzyme is saturated at a concentration of 0.3 to 0.4 mM cinnamic acid. It is apparent that the enzyme has relatively high affinity for cinnamic acid, and the K,, obtained from the reciprocal plot (Fig. 3) is I.7 X lo-+ M.

The possibility of product inhibition has also been examined. When 4-hydroxycinnamic acid was added to incubation mix- tures, it was found (Table VII) that concentrations of 5 x 10e6 M

TABLE V

Inhibition by carbon monoxide and reversal by light

Microsomes from buds (1 g fresh weight) of green seedlings were incubated in 3-ml reaction mixtures in Warburg flasks with 2 side arms. After flushing with the appropriate gas mixture, the ves- sels were sealed and the reaction started by addition of substrate from a side arm. Incubations were carried out at 30” for 2 hours in darkness unless otherwise indicated. Assays were run under mixtures of CO and 02 in the ratio 1:l and with the percentage compositions shown; the balance was made up with Nz. In the controls, the CO was replaced with Nz. The difference between CO treatments and 02 controls is expressed as a percentage of the 02 control (i.e. percent of inhibition).

Gas mixture

co 02

% %

20 20 12 12 12 12 + light

4 14

-

.

Inhibition Light reversal of CO inhibition

% % 76.0 74.2 21.3 71.3 66.2

TABLE VI

Effects of inhibitors

In Experiment 1, microsomes from buds (1 g fresh weight) of etiolated seedlings were assayed in 3-ml reaction mixtures with addition of cyanide or azide as indicat’ed. Reactions were run for 1 hour at 30”. In Experiment 2, microsomes from buds (1 g fresh weight) of green seedlings were incubated for 2 hours at 30” in 3-ml reaction mixtures. The chelating agents were previously incubated for 10 min with the reaction mixture and the reaction started by addition of substrate. Controls were incubated with- out inhibitor. The difference between controls and inhibitor treatment is expressed as a percentage of the control (percentage of inhibition).

Addition to reaction mixture I Inhibition

Experiment 1 Cyanide

0.1 rnM 1.0 rnM

10.0 rnM Azide

0.1 rnM 1.0 rnM

10.0 rnM Experiment 2

EDTA (1.0 miv) a,ol-Dipyridyl (1.0 mM)

I %

0.0 0.0

23.0

0.0 22.0 77.6

0.0 27.0

or less had little or no effect on activity. At 5 X lOA M 4-hy- droxycinnamic acid, 70% inhibition occurs, and concentrations of lo-* M or higher are strongly inhibitory, causing more than 909’, inhibition.

A reciprocal plot of velocity against substrate concentration in the presence and absence of product (Fig. 3) shows that the inhibition exerted by 4-hydroxycinnamic acid is of the noncom- petitive type. When the vertical intercepts of similar plots for a range of different concentrations of inhibitor (4.hydroxycinnamic acid) are replotted against inhibitor concentration (Fig. 4), it is seen that the relationship between inhibition and inhibitor con-

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I I I I 1 I 20 40 60 80 100

l/El (mM)

FIG. 3. Reciprocal plot of velocity against substrate concentration in the presence and absence of product, 4-hydroxycinnamic acid Microsomes (0.2 mg of protein) from buds of green seedlings were incubated in O.l-ml reaction volumes for 30 min at 30’ with a range of substrate concentrations and the following amount of 4-hydroxycinnamic acid: X, zero; 0,5 X 10eB M; q ,5 X 10e6 M.

TABLE VII

Znhibition by &hydroxycinnamic acid

In Experiment 1, microsomes (0.2 mg of protein) from buds of green seedlings were incubated in O.l-ml reaction mixtures for 30 min at 30”. In Experiment 2, microsomes from buds (0.7 g fresh weight) of etiolated seedlings were incubated in 3-ml reaction mixtures for 1 hour at 30”. 4-Hydroxycinnamic acid was added as indicated, and the difference between each experimental treat- ment and the control with no added product is expressed as a percentage of the controls.

4-Hydroxycinnamic acid Inhibition

% Experiment 1

5X10-‘M 5 x lo-6 M 5 x lo-’ M

Experiment 2 1o-4 M lo+ M

5.0 6.6

70.4

96.8 97.8

centration is nonlinear. At very low concentrations of 4-hy- droxycinnamic acid, there is virtually no inhibition, but between 3 x 1OW M and 10m4 M (Fig. 4; also cj. Table VII) inhibition in- creases sharply. The data thus indicate that inhibition by 4-hy- droxycinnamic acid is noncompetitive and that the product exerts a stringent control over the activity of the enzyme which catalyzes its synthesis. The kinetic data indicate (Fig. 4) that the mechanism involves a cooperativity effect with increasing concentration of product over the effective concentration range.

In the initial studies aimed at demonstrating activity of cin- namic acid 4-hydroxylase in cell-free preparations, apical portions

l-

8-

6-

4-

P

2-

0- 0 0.02 0.04 a06 0.08 a10

4-WfDROXYCINNAMIC ACID (ti)

FIG. 4. Relationship between product concentration and in- hibition. Microsomes (0.2 mg of protein) from buds of green seedlings were incubated in 0.1~ml reaction volumes for 30 min at 30” with a range of substrate concentrations (9.3 PM, 18.5 pM, 46.3 pM, and 0.46 mM) in combination with the range of product concentrations above. Double reciprocal plots were obtained at each concentration of the product 4-hydroxycinnamic acid and the figure shows the relationship between the vertical intercepts (1/V,) of those plots and product concentration.

of etiolated seedlings were used as the source of enzyme because previous work has defined the conditions which stimulate active synthesis in these tissues of derivatives of the flavonoids, kaemp- feral and quercetin (10, 11). Thus, when etiolated seedlings are

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3876 Cinnamic Acid &Hydroxylase of Pea Seedlings Vol. 246, No. 12

TABLE VIII

Light-induced increase in hydroxylase activity in etiolated seedlings

Incubations were carried out with crude homogenates from apical buds (1 g fresh weight) of totally etiolated seedlings (dark control) and similar seedlings which had been illuminated for 10 min with fluorescent light 12 hours before harvest and assay. Reactions were carried out in 3 ml for 2 hours at 30”

Source of homogenate 4-Hydroxycinnamic acid produced

?npmoles/haur/g fresh weight

Totally dark-grown seedlings.. 23.5 Irradiated seedlings. 112.5

TABLE IX

Varialion in hydroxylase activity with stage of development

Homogenates were prepared from 1 g fresh weight of apical buds, leaves half to almost fully expanded (Leaf No. I), and ma- ture leaves (Leaf No. 2). The 12,000 X g supernatant solutions from these homogenates were then divided into two parts; micro- somes were isolated from one part and assayed (Experiment l), and the other was retained as such and assayed (Experiment 2). Assays were carried out in 3-ml reaction mixtures for 1 hour at 30”.

Tissues used as enzyme source

Experiment 1

I-Hydroxycinnamic acid produced

m~moles/hour/g fresh weight

Apical bud......................... Leaf No. 1. Leaf No. 2.

Experiment 2

302.0 7.0 0.0

Apical bud.. 214.2 Leaf No. l......................... 26.2 Leaf No. 2. 0.0

briefly irradiated and returned to darkness, after an initial lag period of 2 to 3 hours, the synthesis of flavonoid derivatives is stimulated, and their concentration increases 3- to 4-fold over the succeeding 20 hours. This response is phytochrome-mediated (10, 11). Activities of cinnamic acid 4-hydroxylase were, there- fore, compared in totally etiolated seedlings and in similar seed- lings which were exposed to fluorescent light for 10 min, returned to darkness, and harvested 12 hours later. The data (Table VIII) show that the brief exposure to fluorescent light caused a dramatic increase in hydroxylase activity in the tissues. Ac- tivity in the homogenates prepared from seedlings maintained entirely in the dark was low. However, homogenates prepared from the seedlings exposed to light for only 10 min showed a 5-fold increase in activity. These results support and extend previously demonstrated effects of light on growth and development and on metabolitesand enzymesin this pathway in pea seedlings (W-13).

Examination of apical buds and leaves of different ages from green plants demonstrates that there is a striking localization of hydroxylase activity in young tissues (Table IX). Assay of 12,000 x g supernatant fractions showed that apical buds con- tained very high activity, whereas in semimature leaves (Leaf No. l), activity had dropped approximately IO-fold, and no activity was detected in mature leaves (Leaf No. 2). Similar results were obtained when the activities of microsomal preparations from the different tissues were compared. The distribution of activity

is, therefore, interestingly correlated with the developmental stage of the tissues and is highest in tissues which are in rapid developmental transition.

DISCUSSION

Data have been presented on the properties of cinnamic acid 4-hydroxylase, the second enzyme in the pathway of biosynthesis of phenolic compounds from phenylalanine. The enzyme re- quires molecular oxygen and NADPH, supplied by a NADPH- generating system, for activity; NADH is inactive. Enzymatic activity is associated with the microsomal fraction, and the en- zyme requires 2-mercaptoethanol for functional integrity. This requirement is not satisfied by reduced glutathione, ascorbic acid, dithiothreitol, or cysteine. In contrast to our earlier report (7), it has now been conclusively shown that tetrahydrofolate is not required. Optimum activity occurs at pH 7.5, and the enzyme appears to be specific for cinnamic acid. The K, for cinnamic acid is 1.7 X 10F5 M.

The data on intracellular localization and the requirement for NADPH and molecular oxygen classify this enzyme as a micro- somal mixed function oxidase or monooxygenase. The proper- ties of the cinnamic acid hydroxylase described in the present work are remarkably similar to those of some mixed function oxidases from animal sources. The available evidence from animal systems indicates that these oxidases consist of a multi- enzyme complex in which electrons from the reduced pyridine nucleotide are transferred via one or more intermediary proteins to cytochrome P-450. Reduced cytochrome P-450 is the com- ponent responsible for the binding and activation of molecular oxygen and carries out the actual hydroxylation reaction (14). The evidence for the catalytic function of cytochrome P-450 is based on the following. (a) Th e action spectrum for the light reversal of CO-induced inhibition of enzyme activity corresponds with the absorption (and difference) spectrum of the reduced P-450-CO complex, and (b) the binding of CO is competitive with that of 02, as indicated by data on the inhibition of enzyme activity by mixtures of CO and O2 containing different percent- ages of CO and various ratios of CO :02 (9). In the present study, cinnamic acid 4-hydroxylase also was inhibited by CO, and this inhibition was reversed by light. Furthermore, the degree of inhibition was independent of the percentage of CO in the gas mixtures when a constant ratio of CO :02 was used. The CO inhibition and its reversal by light suggest participation of a P-450 type of cytochrome in the hydroxylation reaction. How- ever, this tentative conclusion requires confirmation by studies of the action spectrum for light reversal of the CO inhibition and also by more detailed studies on the competitive binding of CO and OZ. It is of interest that action spectrum studies have been carried out on the light reversal of CO inhibition of two oxidative reactions in the biosynthesis of gibberellins in higher plants (15). These studies have shown that conversion of kaurene to kaurenol and of kaurenal to kaurenoic acid both require NADPH and 02. Both are inhibited by CO, and the inhibition is reversed by light. Furthermore, action spectra for the light reversal showed peaks at 450 nm, confirming the participation of a P-450 type of cyto- chrome in these reactions.

In regard to the mechanism of the reaction, it has been shown (16) that when 4.Wcinnamic acid is used as a substrate, as much as 93% of the tritium is retained in the meta position in the hy- droxylated product. The extent of migration and retention of tritium thus indicates that the mechanism is similar to that pro-

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Issue of June 25, 1971 D. W. Russell 3877

posed for those aromatic hydroxylases from animal sources where similar tritium migration and retention values have been ob- tained (17).

The cinnamic acid hydroxylase appears to be quite specific for cinnamic acid; when phenylalanine or 4-hydroxycinnamic acid were tested as possible substrates, there was no detectable con- version to their respective hydroxylated derivatives, tyrosine and 3,4-dihydroxycinnamic acid (caffeic acid). This suggests that the plant enzyme is much more specific than many steroid and drug hydroxylases in animals (18) and raises the possibility of additional enzymes with specific hydroxylase functions in plants. Vaughan and Eutt (19) have recently described some properties of an enzyme, purified (lOOO-fold) from the leaves of spinach beet, which catalyzes the conversion of ii-hydroxy- cinnamic acid to 3,4-dihydroxycinnamic acid. The enzyme appears to be a phenolase and also catalyzes the 3’.hydroxylation of a number of 4’-hydroxyflavonoids (20). The existing data on substrate specificity engender some reservations about the role of this enzyme in phenolic biosynthesis, and further information in the properties of the enzyme is awaited with interest.

Kinetic studies have shown that the cinnamic acid 4-hydrox- ylase has a high affinity for cinnamic acid (K, = 1.7 X 10M5 M) and that it is inhibited noncompetitively by low concentrations of the product. Studies of the kinetics demonstrated that the relationship between inhibition and inhibitor (product) concen- tration is nonlinear. Inhibition commenced at about 3 x 10e5 M and rapidly rose to maximum levels at about 1O-4 M. A plot of l/V, against inhibitor (product) concentration yielded a sigmoid curve, indicating that the product exerts a stringent control over the activity of the enzyme which catalyzes its synthesis.

The inhibitor kinetics suggests that the control mechanism involves increasing cooperativity in inhibition with increasing concentration of the product. The high affinity of the enzyme for its substrate suggests on one hand that cinnamic acid is probably present in low concentrations in the cell; on the other hand, the nature and degree of inhibition by the product indicate that the concentration of 4-hydroxycinnamic acid in the tissues is under stringent self-regulation, that the product concentration is regulated at a rather low level and that enzymes responsible for the further metabolism of 4-hydroxycinnamic acid are, there- fore, likely to have a high affinity for their substrate. It is also of some interest to note that the kinetic properties of the enzyme, which is present mainly in young, growing tissues, ensure not only that 4-hydroxycinnamic acid concentration in the cell is regulated within narrow limits, but also that significant levels of product are likely to be present even at very low concentrations of cinnamic acid. This conclusion assumes added interest in view of the evidence for the action of monohydric phenols as cofactors (21) for the peroxidase-catalyzed degradation of the plant growth hormone, indoleacetic acid (22, 23), the first step in the further metabolism (24, 25) of this hormone.

Although product inhibition and inhibition by 4-hydroxy- cinnamic acid have been noted also for phenylalanine ammonia lyase (a), the effects appear to be small when compared with the product inhibition of cimramic acid 4-hydroxylase observed in the present study. This, in conjunction with the noted high affinity of the hydroxylase for its substrate, suggests that the regulation of cinnamic acid hydroxylase activity is an important control point in phenolic biosynthesis and that inhibition of phenylalanine ammonia lyase by its product probably represents a secondary control point. It is therefore suggested that product

repression effects noted for phenylalanine ammonia lyase (3) are likely to originate, in the organism, with product inhibition of cinnamic acid 4-hydroxylase. It should also be noted that the specificity of the product inhibition effects on cinnamic acid 4-hydroxylase have not been examined, though the effective con- centration range suggests that the effects are physiologically significant.

Previous work with seedlings of etiolated peas has shown that a brief irradiation with red light (660 nm) stimulates the synthe- sis of flavonoid derivatives in the apical tissues. This is a phyto- chrome-mediated response since the red light effect is typically reversed by immediate irradiation with far red light (730 nm) (10, 11). It has also been shown that red light induces an in- crease in the activity of phenylalanine ammonia lyase in these tissues (12, 26) and that this effect also is phytochrome-mediated (12). Light-induced, phytochrome-mediated growth responses have also been examined, and the onset of the responses occurs about the same time (2 to 3 hours) as the biochemical changes (10, 11). The biochemical changes may thus relate to the growth-regulating mechanism. It was, therefore, of consider- able interest to note that cinnamic acid hydroxylase activity increases dramatically in response to a brief illumination. Ir- radiation of etiolated seedlings for 10 min induced a 5-fold in- crease in activity obtained from apical tissues harvested about 12 hours after returning the seedlings to darkness. It is sug- gested, therefore, that, in addition to the metabolic regulation of activity, the total amount of enzyme in the tissues is under epi- genetic control.

The amount of enzyme in green tissues showed large variations according to the age of the tissues. Activity was highest in the apical bud and youngest leaf, but only 5 to 10% of this activity was found in the first leaf (half to almost fully expanded) below the apical bud, and no activity was detected in the second leaf. Thus, total activity of the enzyme is correlated in green seedlings also, with tissues undergoing extensive and rapid growth and developmental changes, and declines sharply with the onset of maturity. This correlation may be of significance in regard to processes controlling growth, lignification, and differentiation in developing tissues.

Acknowkdgment- I wish to acknowledge the encouragement and support of Dr. E. E. Conn, in whose laboratory the research was conducted.

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David W. RussellASPECTS OF ITS METABOLIC AND DEVELOPMENTAL CONTROL

THE CINNAMIC ACID 4-HYDROXYLASE OF PEA SEEDLINGS AND SOME The Metabolism of Aromatic Compounds in Higher Plants: X. PROPERTIES OF

1971, 246:3870-3878.J. Biol. Chem. 

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