The Hydroxylation of Phenylalanine and Tyrosine by ... · THE JOURNAL OF BIOLOGICAL CHEMISTRV Vol....

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THE JOURNAL OF BIOLOGICAL CHEMISTRV Vol. 266, No. 24, Issue of August 25, pp. 16207-16211,1991 Printed in U.S.A. The Hydroxylation of Phenylalanine and Tyrosine by Tyrosine Hydroxylase from Cultured Pheochromocytoma Cells* (Received for publication, February 22, 1991) Paula RibeiroS, Dominique Pigeong, and Seymour Kaufman From the Laboratory of Neurochemistry, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892 Pheochromocytoma tyrosine hydroxylase was re- portedtohaveunusualcatalyticproperties, which might be unique to the tumor enzyme (Dix, T. A., Kuhn, D. M., and Benkovic, S. J. (1987) Biochemistry 24, 3354-3361). Two such properties, namely the appar- ent inability to hydroxylate phenylalanine and an un- precedented reactivity with hydrogen peroxide were investigated further in thepresent study. Tyrosine hydroxylasewaspurifiedto apparent homogeneity from culturedpheochromocytoma PC12 cells. The pu- rified tumor enzyme was entirely dependent on tetra- hydrobiopterin (BH,) for the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine and hydrogen peroxide could not substitute for the natural cofactor. Indeed, in the presence of BH4, increasing concentrations of hy- drogen peroxide completely inhibited enzyme activity. The PC12 hydroxylase exhibited typical kinetics of tyrosine hydroxylation, both as a function of tyrosine (so.8 Tyr = 15 MM) and BH4 (apparent K, BH, = 210 PM). In addition, the enzyme catalyzed the hydroxyl- ation of substantial amountsof phenylalanine to tyro- sine and 3,4-dihydroxyphenylalanine (apparent K,,, Phe = 100 PM). Phenylalanine did not inhibit the en- zyme in the concentrations tested, whereas tyrosine showed typical substrate inhibition at concentrations 250 MM. At higher substrate concentrations, the rate of phenylalanine hydroxylation was equal to or ex- ceeded that of tyrosine. Essentially identical results were obtained with purified tyrosine hydroxylase from pheochromocytoma PC18 cells. The data suggest that the tumor enzyme has the same substrate specificity and sensitivity to hydrogen peroxide as tyrosine hy- droxylase from other tissues. Tyrosine hydroxylase, the rate-limiting enzyme in the bio- synthesis of catecholamines, is known to catalyze the hydrox- ylation of both phenylalanine and tyrosine. Ikeda and co- workers (1) reported that crude preparations of rat striatalor adrenal tyrosine hydroxylase catalyzed the hydroxylation of small amounts of phenylalanine. These authors furtherdem- onstrated that the reaction was not mediated by a liver type of phenylalanine hydroxylase but was catalyzed by a tyrosine hydroxylase which converted phenylalanine to Dopa’ (2). * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “aduertisemerzt” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should he addressed. § Present address: Laboratoire de Recherches Dermatologiques, Facult6 de Medecine, Avenue de Valombrose, 06034 Nice, France. The abbreviations used are: Dopa, ~-3,4-&hydroxyphenylalanine; BH,, (6R,6S)-5,6,7,8-tetrahydrobiopterin; 6-MPH,, 6-methyltetra- hydropterin; SDS, sodium dodecyl sulfate. Subsequently, using highly purified tyrosine hydroxylase from bovine adrenal medulla, Shiman et al. (3) showed that the activity of the enzyme towards phenylalanine was very much increased in thepresence of the natural cofactor BH,. When a synthetic cofactor analogue was used, the rate of phenylal- anine hydroxylation was less than 10% that of tyrosine, whereas in the presence of BH, the rates of hydroxylation of the two amino acids were approximately the same (3). Similar results were obtained by Katz et al. (4) with partially purified tyrosine hydroxylase from rat striatum and by Fukami et al. (5) with pure tyrosine hydroxylase from bovine adrenal me- dulla. Other authors reported that phenylalanine is converted to tyrosine and Dopa in rat brain in uiuo (6, 7), as well as in suspensions of intact striatal synaptosomes (8) and in cultures of adrenal chromaffin cells (5). These observations reinforced the generalized notion that tyrosine hydroxylase shares many of the properties of the more extensively studied phenylalanine hydroxylase of liver. In addition to recognizing the same substrate, the two en- zymes have substantial sequence homology (9, lo), and share mechanistic elements of oxygen activation and substrate hy- droxylation (11). However, according to a report by Dix et al. (12), the tyrosine hydroxylase purified from cultured pheo- chromocytoma PC12 cells has unusual properties which have not been found either with phenylalanine hydroxylase or tyrosine hydroxylase from other tissues. One such unique property of the PC12 enzyme was its apparent lack of phen- ylalanine hydroxylating activity. In contrast to tyrosine, which showed typical kinetic properties (13, 14), phenylala- nine was reported to be inactive as a substrate for the PC12 enzyme, even in the presence of BH, (12). An even more unexpected finding was that tyrosine hydroxylase from PC12 cells could use hydrogen peroxide in place of the pterin cofactor in the hydroxylation of tyrosine (12). Such reactivity with hydrogen peroxide is unprecedented among the aromatic. amino acid hydroxylases, which have a strict specificity for tetrahydrobiopterin or a structurally related pyrimidine (15), and are markedly inhibited by hydrogen peroxide (3, 11). The findings of Dix and co-workers (12) raise the interest- ing possibility that tyrosine hydroxylase from tumor cells has unique characteristics of substrate specificity and cofactor utilization, presumably reflecting a change in the structure of the enzyme. We have therefore decided to re-examine these properties of pheochromocytoma tyrosine hydroxylase puri- fied from PC12 cells. Our present results do not conform to the earlier findings of Dix et al. (12), but rather support the conclusion that the PC12 hydroxylase has the same kinetic properties as tyrosine hydroxylase from other tissues. EXPERIMENTAL PROCEDURES Materials-Phenylalanine, L-tyrosine, ~-3,4-dihydroxyphenyl- alanine, NADH, and dihydropteridine reductase were obtained from 16207

Transcript of The Hydroxylation of Phenylalanine and Tyrosine by ... · THE JOURNAL OF BIOLOGICAL CHEMISTRV Vol....

Page 1: The Hydroxylation of Phenylalanine and Tyrosine by ... · THE JOURNAL OF BIOLOGICAL CHEMISTRV Vol. 266, No. 24, Issue of August 25, pp. 16207-16211,1991 Printed in U.S.A. The Hydroxylation

THE JOURNAL OF BIOLOGICAL CHEMISTRV Vol. 266, No. 24, Issue of August 25, pp. 16207-16211,1991 Printed in U.S.A.

The Hydroxylation of Phenylalanine and Tyrosine by Tyrosine Hydroxylase from Cultured Pheochromocytoma Cells*

(Received for publication, February 22, 1991)

Paula RibeiroS, Dominique Pigeong, and Seymour Kaufman From the Laboratory of Neurochemistry, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892

Pheochromocytoma tyrosine hydroxylase was re- ported to have unusual catalytic properties, which might be unique to the tumor enzyme (Dix, T. A., Kuhn, D. M., and Benkovic, S . J. (1987) Biochemistry 24, 3354-3361). Two such properties, namely the appar- ent inability to hydroxylate phenylalanine and an un- precedented reactivity with hydrogen peroxide were investigated further in the present study. Tyrosine hydroxylase was purified to apparent homogeneity from cultured pheochromocytoma PC12 cells. The pu- rified tumor enzyme was entirely dependent on tetra- hydrobiopterin (BH,) for the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine and hydrogen peroxide could not substitute for the natural cofactor. Indeed, in the presence of BH4, increasing concentrations of hy- drogen peroxide completely inhibited enzyme activity. The PC12 hydroxylase exhibited typical kinetics of tyrosine hydroxylation, both as a function of tyrosine (so.8 Tyr = 15 MM) and BH4 (apparent K, BH, = 210 PM). In addition, the enzyme catalyzed the hydroxyl- ation of substantial amounts of phenylalanine to tyro- sine and 3,4-dihydroxyphenylalanine (apparent K,,, Phe = 100 PM). Phenylalanine did not inhibit the en- zyme in the concentrations tested, whereas tyrosine showed typical substrate inhibition at concentrations 250 MM. At higher substrate concentrations, the rate of phenylalanine hydroxylation was equal to or ex- ceeded that of tyrosine. Essentially identical results were obtained with purified tyrosine hydroxylase from pheochromocytoma PC18 cells. The data suggest that the tumor enzyme has the same substrate specificity and sensitivity to hydrogen peroxide as tyrosine hy- droxylase from other tissues.

Tyrosine hydroxylase, the rate-limiting enzyme in the bio- synthesis of catecholamines, is known to catalyze the hydrox- ylation of both phenylalanine and tyrosine. Ikeda and co- workers (1) reported that crude preparations of rat striatal or adrenal tyrosine hydroxylase catalyzed the hydroxylation of small amounts of phenylalanine. These authors further dem- onstrated that the reaction was not mediated by a liver type of phenylalanine hydroxylase but was catalyzed by a tyrosine hydroxylase which converted phenylalanine to Dopa’ (2).

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “aduertisemerzt” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should he addressed. § Present address: Laboratoire de Recherches Dermatologiques,

Facult6 de Medecine, Avenue de Valombrose, 06034 Nice, France. ’ The abbreviations used are: Dopa, ~-3,4-&hydroxyphenylalanine;

BH,, (6R,6S)-5,6,7,8-tetrahydrobiopterin; 6-MPH,, 6-methyltetra- hydropterin; SDS, sodium dodecyl sulfate.

Subsequently, using highly purified tyrosine hydroxylase from bovine adrenal medulla, Shiman et al. (3) showed that the activity of the enzyme towards phenylalanine was very much increased in the presence of the natural cofactor BH,. When a synthetic cofactor analogue was used, the rate of phenylal- anine hydroxylation was less than 10% that of tyrosine, whereas in the presence of BH, the rates of hydroxylation of the two amino acids were approximately the same (3). Similar results were obtained by Katz et al. (4) with partially purified tyrosine hydroxylase from rat striatum and by Fukami et al. (5) with pure tyrosine hydroxylase from bovine adrenal me- dulla. Other authors reported that phenylalanine is converted to tyrosine and Dopa in rat brain in uiuo (6, 7), as well as in suspensions of intact striatal synaptosomes (8) and in cultures of adrenal chromaffin cells (5).

These observations reinforced the generalized notion that tyrosine hydroxylase shares many of the properties of the more extensively studied phenylalanine hydroxylase of liver. In addition to recognizing the same substrate, the two en- zymes have substantial sequence homology (9, lo), and share mechanistic elements of oxygen activation and substrate hy- droxylation (11). However, according to a report by Dix et al. (12), the tyrosine hydroxylase purified from cultured pheo- chromocytoma PC12 cells has unusual properties which have not been found either with phenylalanine hydroxylase or tyrosine hydroxylase from other tissues. One such unique property of the PC12 enzyme was its apparent lack of phen- ylalanine hydroxylating activity. In contrast to tyrosine, which showed typical kinetic properties (13, 14), phenylala- nine was reported to be inactive as a substrate for the PC12 enzyme, even in the presence of BH, (12). An even more unexpected finding was that tyrosine hydroxylase from PC12 cells could use hydrogen peroxide in place of the pterin cofactor in the hydroxylation of tyrosine (12). Such reactivity with hydrogen peroxide is unprecedented among the aromatic. amino acid hydroxylases, which have a strict specificity for tetrahydrobiopterin or a structurally related pyrimidine (15), and are markedly inhibited by hydrogen peroxide (3, 11).

The findings of Dix and co-workers (12) raise the interest- ing possibility that tyrosine hydroxylase from tumor cells has unique characteristics of substrate specificity and cofactor utilization, presumably reflecting a change in the structure of the enzyme. We have therefore decided to re-examine these properties of pheochromocytoma tyrosine hydroxylase puri- fied from PC12 cells. Our present results do not conform to the earlier findings of Dix et al. (12), but rather support the conclusion that the PC12 hydroxylase has the same kinetic properties as tyrosine hydroxylase from other tissues.

EXPERIMENTAL PROCEDURES

Materials-Phenylalanine, L-tyrosine, ~-3,4-dihydroxyphenyl- alanine, NADH, and dihydropteridine reductase were obtained from

16207

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16208 Phenylalanine Hydroxylation by Tyrosine Hydroxylase

Sigma. Catalase and leupeptin were purchased from Boehringer Mannheim and Fluka, respectively. (6R,6S)-5,6,7,8-Tetrahydrobiop- terin (BH4) and 6-methyltetrahydropterin (6-MPH4) were provided by Dr. B. Schirks Laboratories, Jona, Switzerland. Labeled amino acids ~-[3,5-~H]tyrosine (54 Ci/mmol) and ~-[U-'~C]phenylalanine (493 Ci/mol) were purchased from Amersham and Research Products International, respectively. Thin-layer cellulose plates (20 X 20 cm) were from Eastman Kodak. Activated charcoal Darco-G6O and Dowex 50W-X8 were obtained from J. T. Baker Inc. and Fluka, respectively. Media and solutions for cell culture were from NIH Media Unit. Horse serum was provided by Advanced Biotechnologies Inc. and fetal calf serum was from Gibco. All other chemicals were of reagent grade.

Cells and Culture Conditions-Rat pheochromocytoma PC12 cells (American Type Culture Collections) were grown in 75-cm2 flasks in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum, 5% fetal calf serum, 50 pg/ml streptomycin, and 20 units/ml penicillin. The cells were incubated at 37 "C in a humidified atmos- phere of 95% air and 5% COZ. The medium was changed every 2 days. PC12 cells were grown to confluency and harvested with 0.1% trypsin, 0.02% EDTA in Ca2+ and MP-free phosphate-buffered saline. Cells were washed into 0.01 M sodium phosphate buffer, pH 6.4, containing 0.2 M sucrose (14), and quickly frozen at -70 "C until use. Each flask of PC12 cells generated approximately 2 X 10' cells, and for one purification procedure 100 flasks were used. Rat pheochromocytoma PC18 cells, a subclone of PC12 (16), were a generous gift from Dr. A. W. Tank, University of Rochester, New York. PC18 cells were grown to confluency, harvested, and subsequently frozen, in the same man- ner as that described above for PC12 cells. Approximately 2-4 X IO9 PC18 cells were used for one purification procedure.

Enzyme Purification"PC12 cells were thawed in 0.01 M sodium phosphate buffer, pH 6.4, containing 0.2 M sucrose, and leupeptin was added to a concentration of 20 pg/ml. The cell membranes were disrupted by sonication on ice with a Kontes Cell Sonicator a t full power. Generally, five 1-min on/off pulses were delivered to ensure complete disruption of cell membranes. Cell integrity was monitored by light microscopy. After sonication, the enzyme was extracted and purified by the procedure of Kuhn and Billingsley (14). The final enzyme preparation was judged to be pure on the basis of SDS- polyacrylamide gel electrophoresis and densitometry. The specific activity of the pure enzyme was 270 nmol/min/mg when assayed at pH 6.0 and at 37 "C for 13 min, using 1 mM 6-MPH4 and 0.1 mM L- tyrosine.

Tyrosine hydroxylase was purified from PC18 cells by the same method used for PC12 cells (14). The purity of the PC18 enzyme was estimated as greater than 95% by densitometry of SDS-polyacryl- amide gels. The specific activity of this enzyme was 103 nmol/min/ mg when assayed for 15 min at 37 "C and at pH 6.0, with 1 mM 6- MPH, and 0.1 mM L-tyrosine.

Enzyme Assays-The hydroxylation of tyrosine was measured by the tritiated H20 release assay (17) with some of the modifications described by Reinhard et al. (18). The standard assay was carried out in a total volume of 100 p1 and contained the following components: 200 mM sodium acetate buffer, pH 6.0, 2,000 units/ml catalase, 50 milliunits/ml of dihydropteridine reductase, 0.1 mM NADH, 200,000 cpm of [3H]tyrosine with enough nonradioactive tyrosine to produce the desired concentration and pure tyrosine hydroxylase (0.15-0.5 pg). The reaction was initiated with the addition of 100 p M BH4 unless indicated otherwise and the samples were incubated for 15 min at 37 "C. To terminate the reaction, 1 ml of activated charcoal (7.5%, w/v, in 1 M HCl) was added to each sample and, after

containing [3H]OH were radioassayed in 10 ml of 3a70B scintillation centrifugation at 500 X g for 10 min, aliquots of the supernatant

mixture (Research Products International Corp.). In experiments to determine the effect of hydrogen peroxide on

the tyrosine hydroxylating activity of the pure enzyme, the concen- tration of the hydroxylase was increased to 3 pglsample (0.5 pM, final concentration) and the assay was performed with 20 pM [3H]tyrosine (1.0 pCi/pmol) in 20 p M Tris-HC1, pH 8.2, as described by Dix et al.

system of dihydropteridine reductase and NADH were omitted from (12). Catalase, the pterin cofactor, and the cofactor-regenerating

peroxide or BH, were added to final concentrations of 1, 10, and 100 the reaction mixture (12). In some experiments either hydrogen

p ~ , and the samples were incubated for 10 min at 25 "C (12). The blanks contained boiled enzyme with the appropriate amount of either hydrogen peroxide or BH,. After incubation, the reaction was termi- nated with activated charcoal as above, and the [3H]OH produced was radioassayed. In other experiments, the same amount of enzyme

was incubated with 10 ~ L M hydrogen peroxide for up to 20 min, under the conditions described above. Aliquots were assayed at 30-5 inter- vals up to 2 min, and subsequently at 5, 10, 15, and 20 min. Finally, to test for inhibition of BH4-dependent activity by hydrogen peroxide, approximately 3 pg of tyrosine hydroxylase was preincubated with increasing concentration of hydrogen peroxide (100 p~ to 10 mM) at 25 "C. After 10 min, 20 pM [3H]tyrosine (200,000 cpm) was added in the standard assay mixture of catalase, NADH, and dihydropteridine reductase in 200 mM sodium acetate buffer, pH 6.0. The reaction was initiated by the addition of 1 mM 6-MPH4 and the enzyme was assayed for 15 min at 37 "C, as described previously. After incubation, the amount of tyrosine hydroxylated was determined by measuring the release of tritium into H20.

The assay for the hydroxylation of phenylalanine was carried out in 200 mM sodium acetate buffer, pH 6.0, containing 2,000 units/ml of catalase, 50 milliunits/ml of dihydropteridine reductase, 0.1 mM NADH, 300,000 cpm of ['4C]phenylalanine, and varying concentra- tions of unlabeled phenylalanine as indicated in the text, 0.15-0.25 pg of tyrosine hydroxylase, 100 PM BH, or as indicated in the text and HZ0 to a final volume of 50 pl. Samples were incubated at 37 "C for 15 min and the reaction was terminated by the addition of enough trichloroacetic acid to make the final mixture 5% with respect to trichloroacetic acid. After centrifugation for 5 min at 10,000 X g, 10- p1 aliquots of the supernatant were spotted on cellulose TLC plates and the labeled products were separated from the precursor by TLC with I-propanokammonium hydroxide:H20 (81:1, v/v) (4). This sol- vent system routinely generated RF values of 0.65, 0.40, and 0.13 for phenylalanine, tyrosine, and Dopa, respectively. After chromatogra- phy, the separated amino acids were visualized by spraying with ninhydrin, the appropriate areas were cut out from the plates, and the amount of radioactivity in each area was quantitated in 10 ml of scintillation mixture.

Data Analyses-Kinetic parameters were obtained from measure- ments of tyrosine and Dopa production over a concentration range of 10-200 p M phenylalanine or 4-50 p M tyrosine. The latter range excluded concentrations of tyrosine that were markedly inhibitory. For analyses of substrate hydroxylation as a function of the cofactor, BH, was varied between 30 and 250 p~ with either 20 p~ phenylal- anine or 20 p~ tyrosine. All kinetic parameters were determined by direct fit to the Michaelis-Menten equation with the EZ-FIT curve- fitting program (F. W. Perrella, Du Pont). Each K,,, described here is an approximate value, and is therefore expressed either as apparent K,,, or so.5. The latter term was used when inhibition by high levels of tyrosine prevented the determination of a reliable K,,,. Each apparent K,,, for phenylalanine and for tyrosine is the mean of 4-5 inde- pendent determinations, whereas apparent K,,, values for BH4 with either substrate are the average of 2-4 experiments. The standard error for a single kinetic parameter was generally less than 15%.

Additional Procedures-SDS-polyacrylamide gel electrophoresis was performed according to the method of Laemmli (191, and the gels were stained with Coomassie or silver. Densitometry of individual lanes was performed with the "IMAGE" software package (20) using an Apple Macintosh IICX computer and a peripheral COHU solid state video camera. Protein was estimated by the method of Bradford (21) using the Bio-Rad protein assay kit with bovine serum albumin as a standard.

RESULTS

The Kinetics of Tyrosine and Phenylalanine Hydroxylation by Purified PC12 Tyrosine Hydroxylase-Tyrosine hydroxyl- ase was purified to apparent homogeneity from cultured PC12 cells. On the basis of activity measurements, the enzyme was purified nearly 190-fold to a final specific activity of 270 nmol/min/mg. The estimated recovery of activity was 7%. These results agree well with those of Kuhn and Billingsley (14) for their purification of the PC12 hydroxylase. Fig. 1 shows a typical SDS-polyacrylamide gel of the purified en- zyme and establishes the purity of the preparation. Tyrosine hydroxylase appears as the only prominent band on the gel, with a corresponding single peak on the densitometric profile (Fig. 1). The molecular weight ( M J of the enzyme subunit is 60,000, as determined by the relative electrophoretic mobili- ties of protein standards.

The purified tyrosine hydroxylase was shown to catalyze

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Phenylalanine Hydroxylation by Tyrosine Hydroxylase 16209

60 K

FIG. 1. Purified pheochromocytoma tyrosine hydroxylase from PC12 analyzed by SDS-polyacrylamide gel electropho- resis and densitometry. The hydroxylase subunit appears as the only prominent hand, with an estimated molecular weight of 60,000, as determined by the relative motility of suitable protein standards. Densitometric analysis of individual lanes was performed with the IMAGE software package (20) using an Apple Macintosh IICX com- puter and a peripheral video camera, as described under “Experimen- tal Procedures.”

>

substrate, pM

FIG. 2. The activity of pure tyrosine hydroxylase from PC12 cells as a function of either phenylalanine or tyrosine concentration in the presence of BH,. Each incubation contained, in addition to the components listed under “Experimental Proce- dures,’’ 0.15-0.50 pg of pure tyrosine hydroxylase, either 300,000 cpm of [’‘Clphenylalanine or 200,000 cpm of [“Hltyrosine, 100 PM BH,, and sufficient unlabeled phenylalanine or tyrosine to generate the desired final concentrations. In each assay, the incubation time was 15 min. Dopa formation from added tyrosine ( a ) was measured by the tritium release assay. Phenylalanine hydroxylation to tyrosine ( b ) and to Dopa (c) was determined by thin layer chromatography. Total hydroxylation of phenylalanine is indicated as the sum of tyrosine and Dopa formation ( d ) . V is expressed as nanomole of substrate hydroxylated per rnin/mg of enzyme. The data are typical of four independent experiments.

the hydroxylation of phenylalanine to tyrosine and Dopa. The results in Fig. 2 illustrate the kinetics of tyrosine and Dopa formation with increasing concentrations of phenylalanine or tyrosine, each in a concentration range of 4-200 pM and in the presence of 100 p~ BH,. At low substrate concentrations ( 4 0 0 p ~ ) , the rate of phenylalanine hydroxylation to tyro- sine and Dopa (Fig. 2d) was generally slower than that of tyrosine to Dopa (Fig. 2a). This resulted in substantially different apparent K,,, and so.5 values of 100 and 15 pM for phenylalanine and tyrosine, respectively, indicating a greater affinity of the enzyme for tyrosine. In contrast, a t higher substrate levels (2100 p ~ ) the rate of phenylalanine hydrox- ylation generally exceeded that of tyrosine hydroxylation. Furthermore, unlike tyrosine, phenylalanine did not appear to be inhibitory at any of the concentrations used. The con-

version of phenylalanine to tyrosine and Dopa displayed traditional Michaelis-Menten kinetics with no evidence of substrate inhibition by phenylalanine (Fig. 2d). Conversely, the formation of Dopa from added tyrosine was inhibited at substrate concentrations higher than 50 p~ (Fig. 2a). Inhibi- tion by tyrosine in the presence of BH, has been reported for tyrosine hydroxylase from a variety of different tissues (3, 4, 11).

The effects of BH, on the hydroxylation of tyrosine and phenylalanine were investigated with each substrate a t a constant concentration of 20 p ~ , while the cofactor was varied between 30 and 250 p ~ . The results are illustrated in Fig. 3. The formation of tyrosine and Dopa from phenylalanine obeyed Michaelis-Menten kinetics and was characterized by an apparent K, of 84 p~ BH,. Conversely, the hydroxylation of tyrosine to Dopa did not saturate even a t 250 p~ BH4, and the apparent K,,, for the cofactor was 210 p ~ . There was no visible inhibition of tyrosine hydroxylase activity by BH, in the concentration range tested with either tyrosine or phen- ylalanine substrates.

The Effects of Hydrogen Peroxide on the Hydroxylation of Tyrosine by Purified Tyrosine Hydroxylase from PC12 Cells- I t is generally well established that high levels of hydrogen peroxide (>250 p ~ ) can significantly inhibit the activity of adrenal tyrosine hydroxylase (3). To ascertain if the pheo- chromocytoma enzyme has a similar sensitivity to hydrogen peroxide, aliquots of the purified PC12 hydroxylase were preincubated with increasing amounts of hydrogen peroxide for 10 min at 37 “C, prior to assaying for tyrosine hydroxyl- ation. The results are illustrated in Fig. 4. Pre-exposure of the enzyme to hydrogen peroxide drastically inhibited its subsequent ability to catalyze the hydroxylation of tyrosine to Dopa. At 500 p~ hydrogen peroxide the reaction rate was inhibited by more than 40%, and by 5 mM the enzyme was essentially inactive. These results are consistent with earlier studies of adrenal tyrosine hydroxylase (3).

Recently, Dix et al. (12) reported that the PC12 hydroxylase has a unique reactivity with hydrogen peroxide, such that low micromolar amounts of hydrogen peroxide could substitute for the pterin cofactor in the hydroxylation of tyrosine to Dopa (12). At 10 p~ hydrogen peroxide and in the absence of the pterin cofactor, tyrosine hydroxylation was reported to be

>

100 -

80 -

4:E 20 0 50 100 150 200 250

BHq. )In FIG. 3. The effect of tetrahydrobiopterin (BH,) on the hy-

droxylation of tyrosine to Dopa (a) and the hydroxylation of phenylalanine to tyrosine (b) , and to Dopa (c) by pure tyrosine hydroxylase from cultured PC12 cells. Total phenylalanine hydroxylation to tyrosine and Dopa is shown in d. The condi- tions employed in this experiment were identical to those described in the legend to Fig. 2, except that the concentration of each substrate was held constant a t 20 p ~ , and the cofactor was varied between 30 and 250 p ~ . V is the rate expressed in nanomole of substrate hydrox- ylated per min/mg of enzyme. Each curve is typical of 2-4 separate determinations.

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16210 Phenylalanine Hydroxylation by Tyrosine Hydroxylase

100 $ 1

0 6 8 1 0

H 2 0 2 , mP1

FIG. 4. The effect of preincubation with hydrogen peroxide on the activity of PC12 tyrosine hydroxylase. The enzyme was preincubated for 10 min at 25 “C with varying concentrations of hydrogen peroxide. After this period all the components of the stand- ard [3H]tyrosine hydroxylase assay were added, as described under “Experimental Procedures,” and the reaction was initiated by the addition of 1 mM 6-MPH4. Tyrosine hydroxylation was measured by the tritium-release method after a 15-min assay at 37 “C. The data are expressed as the percentage of control samples which were prein- cubated in the absence of hydrogen peroxide. The rate of tyrosine hydroxylation in control samples was 257 nmol/min/mg. Each data point is the average of duplicate determinations.

TABLE I The effect of hydrogen peroxide on the activity of pure tyrosine

hydroxylase from cultured PC12 cells Pure tyrosine hydroxylase (TH) was incubated in a total volume

of 100 p1 of 20 mM Tris-HC1, pH 8.2, containing 20 p~ [3H]tyrosine (1.0 pCi/Nmol) and varying concentrations of either hydrogen per- oxide (H,O,) or tetrahydrobiopterin (BH,), as under “Experimental Procedures.” Tyrosine hydroxylation was measured after a 10-min reaction at 25 “C. Blanks contained boiled enzyme and the appropri- ate amount of either hydrogen peroxide or BH,. The data are ex- pressed as the average of three independent experiments.

[THI [J%I [H,Ozl [Hydroxylation] * M IrM P M f l M

0.5 1 0 0.2

100 0.5

0 1.9 0.5 0 1 ND“

0 10 ND 0 100 ND

10 0

a ND, no detectable tyrosine hydroxylation.

stoichiometric with respect to the amount of enzyme, whereas higher concentrations of peroxide severely inhibited the re- action (12). These apparently conflicting effects of hydrogen peroxide were tested in the present study, but the results, summarized in Table I, were inconsistent with those of Dix et al. (12). The purified PC12 enzyme was assayed for tyrosine hydroxylating activity at 25 “C and in 20 mM Tris-HC1, pH 8.2, such as described by Dix et al. (12). Hydrogen peroxide or BH, were added to final concentrations of 1-100 p ~ , and the reaction was allowed to continue for up to 20 min. After 10 min, there was no measurable enzyme activity with any of the concentrations of hydrogen peroxide tested (Table I). Similarly, no activity could be detected after assaying 0.5 p~ enzyme with 10 pM hydrogen peroxide for various time pe- riods, ranging from a 30-s incubation up to 20 min (data not shown). Substantial enzyme activity was detected, however, when BH4 was substituted for hydrogen peroxide (Table I). Approximately 0.2 ptM tyrosine was hydroxylated following a 10-min incubation of 0.5 pM enzyme with 1 p~ BH, even at basic pH and in the absence of a regenerating system for BH4. At 10 p~ BH4 the amount of substrate hydroxylated became stoichiometric with respect to the amount of enzyme, and at

100 pM the hydroxylation of tyrosine was increased nearly 4- fold to 1.9 FM. Collectively, the results rule out the possibility that hydrogen peroxide, under present conditions, can substi- tute for the pterin cofactor in the enzymatic hydroxylation of tyrosine.

Comparison of the Substrate Specificities and Cofactor Re- quirements of Tyrosine Hydroxylase Purified from PC12 and PC18 Cells-Tyrosine hydroxylase was purified from cultured PC18 cells by the same method used for PC12 (14). The specific activity of tyrosine hydroxylase in crude extracts of PC18 cells was 0.6 nmol/min/mg of protein, and the enzyme was purified 167-fold with a yield of 2-3%. The final specific activity of the enzyme (103 nmol/min/mg of protein) was thus considerably lower than that obtained with the PC12 hydroxylase (270 nmol/min/mg of protein). Nonetheless, the PC18 enzyme was judged to be at least 95% pure, based on densitometric analyses of SDS-polyacrylamide gels both after Coomassie and silver staining. The relatively low fold purifi- cation and low specific activity of this enzyme probably reflect some loss and/or inactivation of the enzyme during the puri- fication procedure.

The hydroxylation of tyrosine by the purified PC18 hy- droxylase displayed the same low SO.& for tyrosine and the same marked substrate inhibition with concentrations of ty- rosine above 50 p ~ . In the presence of 100 p~ BH, the so.& for tyrosine was 5.8 p ~ . The latter was increased to 113 p ~ , with no indication of substrate inhibition, when the natural cofactor was substituted by the synthetic analogue 6-MPH4. The apparent K, for BH, in the presence of 20 p~ tyrosine was 230 p ~ . The PC18 hydroxylase was shown to catalyze the hydroxylation of substantial amounts of phenylalanine to tyrosine and to Dopa, in a manner analogous to the PC12 enzyme. The apparent K, for phenylalanine with 100 PM BH4 was 71 p~ and the apparent K, for BH4 with 20 p~ phenyl- alanine was 130 p ~ . Finally, the PC18 hydroxylase displayed the same strict dependence on the pterin cofactor as the enzyme from PC12. PC18 tyrosine hydroxylase activity could not be detected when 0.5 p~ enzyme was assayed with either 10 or 100 p~ hydrogen peroxide in the absence of BH, (12).

DISCUSSION

Pheochromocytoma tyrosine hydroxylase was recently de- scribed as having unique characteristics of substrate specific- ity and cofactor utilization. These findings raised the inter- esting possibility that the catalytic properties of the tumor enzyme, and perhaps its structure, might be different from those of the enzyme from normal tissues. The present results, however, do not support these earlier observations. Rather, the data suggest that the pheochromocytoma enzyme is essen- tially indistinguishable from tyrosine hydroxylase in other tissues.

The hydroxylation of tyrosine displayed typical biphasic kinetics with low for the substrate (520 pM) and pro- nounced inhibition at high substrate concentrations. The inhibition was evident at physiological concentrations of ty- rosine (11), suggesting that substrate inhibition is of signifi- cance for the regulation of the enzyme in uiuo. This same pattern of inhibition has been reported in studies of pure and partially purified tyrosine hydroxylase from various tissues (3,4, 11), and also in rat brain after in vivo administration of large doses of tyrosine (22).

When the hydroxylation of tyrosine was measured as a function of the cofactor, the apparent K, for BH4 was in the same high micromolar range (>200 p M ) as that for unacti- vated tyrosine hydroxylase either from brain or adrenal me- dulla (23). In other systems, there is ample evidence that the

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Phenylalanine Hydroxylation by Tyrosine Hydroxylase 16211

K,,, for the pterin cofactor decreases as much as 20-fold following activation of the enzyme, for example, by phos- phorylation (23). This is believed to represent a sensitive and crucial mechanism for the regulation of tyrosine hydroxylase in vivo because the physiological concentration of BH4 in most tissues (10-20 PM) is but a small fraction of the K , for the unactivated enzyme (11, 23). Therefore, a decrease in K , following activation would increase tyrosine hydroxylation manyfold. Consistent with this notion, recent experiments have shown that the K , for the pterin cofactor was decreased significantly when the pure PC12 enzyme was assayed with tyrosine under phosphorylating conditions.2 This suggests that a similar interaction between kinetic parameters and state of phosphorylation may influence the activity of this enzyme in vivo.

One reported difference between the PC12 hydroxylase and that from other tissues was the apparent inability of the tumor enzyme to hydroxylate phenylalanine (12, 14). The present results, however, show that phenylalanine is an ex- cellent substrate for the PC12 hydroxylase. Although the Km(so.6) for the substrate was generally lower with tyrosine, the rate of phenylalanine hydroxylation was comparable to that of tyrosine and, in fact, exceeded tyrosine hydroxylation at higher substrate concentrations. Phenylalanine did not inhibit enzyme activity in the concentration range tested, in contrast to tyrosine which caused severe inhibition at high substrate concentrations. Furthermore, with phenylalanine as the substrate, the apparent K , for BH, was nearly 3-fold lower than in the presence of tyrosine as the substrate. These observations are consistent with several other studies of pu- rified striatal and adrenal tyrosine hydroxylase (3-5, l l ) , as well as studies of intact striatal synaptosomes (8) and adrenal chromaffin cells (5). Collectively, the evidence suggests that in these tissues phenylalanine is an important source of tyrosine, and may also serve as a precursor of small amounts of catecholamines in vivo.

Previously, the tumor hydroxylase was reported to have the ability to use hydrogen peroxide in place of BH, in the hydroxylation of tyrosine to Dopa (12). This unusual reactiv- ity with hydrogen peroxide was attributed in part to a unique redox state of the iron in tyrosine hydroxylase, which was believed to permit hydrogen peroxide activation, and the subsequent transfer of the peroxide oxygen to tyrosine during hydroxylation (12). However, in the present study we have been unable to detect any tyrosine hydroxylase activity when hydrogen peroxide was substituted for BH,. Moreover, prein- cubation of the enzyme with increasing concentrations of hydrogen peroxide was shown to inhibit and eventually elim- inate all measurable activity, in a manner analogous to that described for adrenal tyrosine hydroxylase and liver phenyl- alanine hydroxylase (3, 11). Our findings suggest that pheo- chromocytoma enzyme has the same sensitivity to hydrogen peroxide as other aromatic amino acid hydroxylases.

There are many discrepancies between the present results and those of Dix et al. (12), notwithstanding our great care in duplicating their enzyme purification procedure (14) and, where possible, their assay conditions. It should be empha- sized that in the previous study, tyrosine hydroxylase was assayed in the absence of catalase and either at pH 7.2 or 8.2 (12). These conditions would not have permitted optimal

P. Ribeiro and S. Kaufman, unpublished results.

enzyme activity (3, 13, 24), and thus were modified for meas- urements of kinetic parameters both with tyrosine and phen- ylalanine. However, we were still able to detect the hydrox- ylation of small amounts of phenylalanine to tyrosine and Dopa, even under the most extreme assay conditions described by Dix et al. (12), namely in the absence of catalase and at pH 8.2 (data not shown). It is unlikely, therefore, that changes in assay conditions could account for the discrepancy in our findings. On the other hand, the techniques that were used in the previous study to measure phenylalanine hydroxylation (12), which did not include the use of radiolabeled phenylal- anine, may have been too insensitive for the detection of relatively small amounts of product formed. This could ex- plain the earlier failure to detect tyrosine and Dopa formation, even when the amounts formed were readily detected with the radioisotopic methods employed in the present study.

In addition to the PC12 hydroxylase, we have succeeded in purifying and partially characterizing tyrosine hydroxylase from cultured pheochromocytoma PC18 cells, a subclone of PC12 (16). The pure PC18 hydroxylase had essentially the same kinetic properties as the PC12 enzyme, both with respect to tyrosine and phenylalanine substrates. Moreover, the PC18 hydroxylase displayed the same strict dependence on the pterin cofactor as the enzyme from PC12. Hydrogen peroxide was equally ineffective as a cofactor for tyrosine hydroxylase from both PC18 and PC12 cell lines.

In summary, two preparations of pheochromocytoma tyro- sine hydroxylase have been investigated with a view to iden- tifying and characterizing potentially unique properties of catalysis. The results reported here do not support the con- clusion that there is a difference between the pheochromocy- toma enzyme and tyrosine hydroxylase from nontumor tis- sues, but rather suggest that their kinetic properties and presumably their structures are essentially indistinguishable.

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