[25] Aromatic-l -amino acid decarboxylase from pig kidney

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ACID DECARBOXYLASE

179

[25] A r o m a t i c - L - A m i n o Acid D e c a r b o x y l a s e f r o m P i g K i d n e y

By CARLABORRI VOLTATTORNI, ANNAGIARTOSIO, and CARLO TURANO

Aromatic-L-amino acid decarboxylase (3,4-dihydroxyphenylalanine carboxylase; EC 4.1.1.28) catalyzes the conversion of L-DOPA to dopamine and CO2: NH;

HO

L-DOPA

HO

Dopamine

In addition to its action on DOPA, the enzyme is capable of effecting the decarboxylation of a wide range of naturally occurring (5-hydroxytryptophan, tyrosine, tryptophan, phenylalanine) and synthetic (m-tyrosine, o-tyrosine) amino acids. For this reason, the enzyme is best described as an L-aromatic amino acid decarboxylase. The enzyme has been detected biochemically and, in some cases, cytochemically in the cytosol of peripheral organs including kidney, '-4 liver, 2-4 endocrine glands (adrenal medulla, 3,4pancreatic islets 5) and in the central nervous systemf1,3 A number of purification methods have been reported in recent years yielding homogeneous preparations of DOPA decarboxylase from either p i g 1'6'7 o r guinea pig kidney,8 this tissue being an exceptionally rich source of enzyme. This chapter describes a purification method of pig kidney DOPA decarboxylase that yields large amounts of pure enzyme. I j. G. Christenson, W. Dairman, and S. Udenfriend, Arch. Biochem. Biophys. 141, 356 (1970). 2 j. G. Christenson, W. Dairman, and S. Udenfriend, Proc. Natl. Acad. Sci. U.S.A. 69, 343 (1972). 3 M. K. Rahman, T. Nagatsu, and T. Kato, Biochem. Pharmacol. 30, 645 (1981). 4 T. L. Sourkes, Pharmacol. Rev. 18, 53 (1966). 5 G. Teitelman, T. H. Joh, and D. J. Reis, Proc. Natl. Acad. Sci. U.S.A. 78, 5225 (1981). 6 G. A. Lancaster and T. L. Sourkes, Can. J. Biochem. 50, 791 (1972). 7 C. Borri Voltattorni, A. Minelli, P. Vecchini, and C. Turano, Eur. J. Biochem. 93, 181 (1979). 8 K. Srinivasan and S. Awapara, Biochim. Biophys. Acta 526, 597 (1978).

METHODS IN ENZYMOLOGY, VOL. 142

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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A s s a y Methods

M a n y methods have bccn dcviscd to assay D O P A dccarboxylasc activity and a comprehensive rcvicw has appeared recently.9 In the isolation of D O P A dccarboxylasc, two methods arc of particular interest: the radioisotopic method, based on 14CO2 trapping technique, 1,1°,~and the spcctrophotomctric determination of the amine produccd.~2-14 Alternatively, the enzyme activity can be determined either by following minute changes in absorbancc 15 or by a pH-stat procedure. Morc sensitive methods for assaying D O P A dccarboxylasc activity, not requiring the use of radioisotopcs, have also been described. Thcsc assay proccdurcs, involving the separation by H P L C of amines gcncratcd in the dccarboxylation and thcir fluorimctric or clcctrochcmical dctcction, arc particularly suitable for measuring D O P A dccarboxylasc activity in tissues with a low activity such as serum ~6,~vor brain.18A m o n g these methods, two types of assay arc currently in use in our laboratory. Thc first involves spcctrophotometric amine measurements according to Shcrald et al., 13 as modified by Charteris and John 14 (Method A). Thc second method, developed by our group, is based on the use of a pH-stat for measuring the amount of CO2 liberated; this assay is particularly useful for a rapid asscssmcnt of activity during the purification procedure, although it may be used only at acidic pH, far from the p H optimum of the enzyme (Mcthod B). Method A Principle. T h e f o r m a t i o n o f d o p a m i n e is m e a s u r e d b y its r e a c t i o n w i t h 2 , 4 , 6 - t r i n i t r o b e n z e n e - l - s u l f o n i c a c i d to f o r m t r i n i t r o p h e n y l d o p a m i n e , w h i c h is t h e n e x t r a c t e d i n t o b e n z e n e a n d q u a n t i t a t e d b y m e a s u r i n g its a b s o r b a n c e at 340 n m . 12-14 A l t h o u g h D O P A a l s o f o r m s a d e r i v a t i v e u n d e r t h e s e c o n d i t i o n s , n o p r i o r s e p a r a t i o n o f D O P A f r o m d o p a m i n e is n e c e s s a r y , s i n c e t r i n i t r o p h e n y l - D O P A is n o t e x t r a c t e d into t h e b e n z e n e l a y e r .

9 A. J. Culvenor and W. Lovenberg, in "Methods in Biogenic Amine Research" (S. Parvez, T. Nagatsu, I. Nagatsu, and H. Parvez, eds.), p. 375. Elsevier, Amsterdam, 1983. to K. Lloyd and O. Hornykiewicz, Brain Res. 22, 426 (1970). 11 F. Lamprecht and J. T. Coyle, Brain Res. 41, 503 (1972). 12C. Streffer, Biochim. Biophys. Acta 139, 193 (1967). 13A. F. Sherald, J. C. Sparrow, and T. R. Wright, Anal. Biochem. 56, 300 (1973). 14A. Charteris and R. John, Anal. Biochem. 66, 365 (1975). t5 M. H. O'Leary and R. L. Baughn, J. Biol. Chem. 252, 7168 (1977). 16M. K. Rahman, T. Nagatsu, and T. Kato, Biochem. Pharmacol. 30, 645 (1981). 17M. K. Rahman, T. Nagatsu, and T. Kato, Life Sci. 28, 485 (1981). 18T. Nagatsu, T. Yamamoto, and T. Kato, Anal. Biochem. 100, 160 (1979).

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AROMATIC-L-AMINO ACID DECARBOXYLASE

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Continuous extraction of trinitrophenyl-dopamine into benzene is essential for quantitative recovery of the compound.14

Reagents Potassium phosphate buffer, 0.1 M, pH 6.8 L-DOPA, 5 m M 2,4,6-Trinitrobenzene-l-sulfonic acid (4.3 mM in potassium phosphate buffer, 0.1 M, pH 7.5) Benzene Uvasol (Merck) Enzyme solution to be assayed Procedure. The standard reaction mixture contains 100/xl of 0.1 M potassium phosphate buffer, pH 6.8, 25 tzl of 5 mM L-DOPA, enzyme (usually 5-10 ~1), and water to a final volume of 250/~1. After 5 min of incubation at 25 ° the reaction is stopped by heating at I00° for 1 min. Benzene (1.5 ml) and 2,4,6-trinitrobenzene-l-sulfonic acid (1 ml of a 4.3 m M solut;.~n in 0.1 M potassium phosphate buffer, pH 7.5) are added and the reaction producing trinitrophenyl-1-dopamine is carried out at 42 ° for 1 hr with continuous shaking. The concentration of trinitrophenyl-ldopamine in the benzene layer is measured in a spectrophotometer using 8 3 4 0 n m = 12,400 M -1 cm -~. Definition of Unit and Specific Activity. One unit of enzyme activity is defined as the amount of protein which catalyzes the production of 1 nmol of amine in 1 min under the conditions specified for the spectrophotometric assay. Specific activity is expressed as units per milligram of protein; the latter is measured by the biuret method with bovine serum albumin as a standard.

Method B Principle. The enzyme activity is determined by measuring the number of acid equivalents which must be added to the reaction mixture to keep the pH constant during the decarboxylation of DOPA. Reagents L-DOPA, 22 m M Pyridoxal phosphate, 10 ~ M HC1, 2.5 m M Enzyme solution to be assayed Procedure. The determination of the enzyme activity is carried out in a pH-stat apparatus (Radiometer-Copenhagen) using a 5 ml thermostatted (37 °) reaction vessel, separate electrodes, and a 2.5-ml injecting

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CATABOLISM OF THE AROMATIC AMINO ACIDS

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TABLE I PURIFICATION OF PIG KIDNEY DOPA DECARBOXYLASE

Fraction 1. Crude extract 2. Ammonium sulfate fractionation 3. DEAE-Cellulose chromatography 4. QAE-Sephadex chromatography 5. Sephadex G-100

Volume (ml)

Protein (mg)

Total activity (U)

Specific activity (U/mg)

1,090 375

76,300 26,250

913,800 689,400

11.9 26.3

-75

140

1,260

474,500

376.6

52

44

308

365,600

1,187

40

42

130

292,900

2,253

32

Recovery (%)

syringe. The solution under examination is mechanically stirred. A solution containing 300/xl of 10/xM pyridoxal phosphate, 2.375 ml of water, and enzyme (usually 25/zl) is placed in the reaction vessel of the pH-stat apparatus and the pH is adjusted to 5.8 with 2.5 mM HCI. The reaction is primed by the addition of 300/~1 of 22 mM DOPA, previously adjusted to pH 5.8. The total volume of the reaction mixture is 3 ml. The titration is performed with 2.5 mM HC1. Purification Procedure. All operations are performed at 4°. A summary of the purification is given in Table I. Step 1. Extraction. Fresh pig kidneys (about 800 g) are minced in a meat grinder and homogenized with a half volume of 0.1 M potassium phosphate buffer, pH 6.8 in a Waring blender. The homogenate is centrifuged for 30 min at 10,000 g in a Sorvall RC-2B centrifuge. The precipitate is washed once with a half volume of the homogenizing medium, centrifuged, and the wash is added to the original supernatant. The supernatant fluid is considered a crude extract and referred to as fraction 1 in Table I. Step 2. Ammonium Sulfate Fractionation. Crystalline ammonium sulfate is added gradually to the crude extract to 25% saturation. After stirring for 30 min, the precipitate is removed by centrifugation for 30 min at 10,000 g and discarded. Additional solid ammonium sulfate is added until 55% saturation is reached. During the precipitation procedure the pH is maintained at 6.8 with 1 M dipotassium phosphate. The precipitate is collected by centrifugation for 30 min at 10,000 g and dissolved in 0.01 M potassium phosphate buffer, pH 7.8, containing 0.01 mM pyridoxal phosphate, 0.1 m M EDTA, and 1 mM 2-mercaptoethanol (buffer A). This fraction is dialyzed for 24 hr against several changes of buffer A. The

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AROMATIC-L-AMINO ACID DECARBOXYLASE

183

dialyzed solution ( - 3 0 0 ml) is stirred with 5 g cellulose for 15 min and then centrifuged for 4 hr at 48,000 g in a Sorvall RC-2B centrifuge. The supernatant fluid is collected (fraction 2 in Table I). Step 3. DEAE-Cellulose Column Chromatography. Fraction 2 is applied to a DEAE-Cellulose column (4 x 20 cm) which has been equilibrated with buffer A minus pyridoxal phosphate and EDTA. The column is washed with 0.02 M potassium phosphate buffer, pH 7.8, containing 1 m M 2-mercaptoethanol, until the absorbance at 280 nm of the effluent is 0.1 or less, and is then eluted with 0.05 M potassium phosphate buffer, pH 7.8, containing 1 m M 2-mercaptoethanol. Fractions (15 ml) are collected and those with the highest enzyme activity are pooled (fraction 3 in Table I). Step 4. QAE-Sephadex Column Chromatography. Solid ammonium sulfate is added to fraction 3 to 55% saturation and the resulting precipitate is collected by centrifugation, dissolved in 0.1 M potassium phosphate buffer, pH 7.2, containing 0.01 mM pyridoxal phosphate, 0.1 mM EDTA, and 1 m M 2-mercaptoethanol (buffer B), and dialyzed for 24 hr against several changes of buffer B. The dialyzed material ( - 4 0 ml), after centrifugation, is applied to a QAE-Sephadex column (1.5 x 27 cm) equilibrated with buffer B minus pyridoxal phosphate and EDTA (Buffer C). Elution is carried out by a linear gradient: the mixing chamber contained 500 ml of buffer C and the reservoir chamber the same volume of 0.08 M NaC1 dissolved in buffer C. The enzyme was eluted at a flow rate of 5 ml/ hr between the 79th ml and the 123th ml (fraction 4 in Table I). Step 5. Sephadex G-IO0Superfine Fractionation. Fraction 4 is brought to 55% saturation with (NH4)2SO4,the precipitate collected by centrifugation, and dissolved in about 2 ml of 0.1 M potassium phosphate buffer, pH 6.8, containing 0.1 mM dithiothreitol. The solution is applied to a Sephadex G-100 superfine column (3.2 x 92 cm) equilibrated with the same buffer. Fractions of 1.5 ml are collected and the enzyme is eluted as a symmetrical peak (fraction 5 in Table I). The fractions with the highest activity are pooled, concentrated to 1.5 ml by vacuum dialysis in collodion bags, and stored at - 2 0 °. The final specific activity ranges from 2000 to 2800 U/mg (Method A). Properties

Stability. The purified enzyme is relatively stable in 0. I M potassium phosphate buffer containing 0.1 m M dithiothreitol. No loss of activity is observed on storage at - 2 0 ° for at least 2 months. The activity gradually

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CATABOLISM OF THE AROMATIC AMINO ACIDS

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decreases, however, on repeated freezing and thawing. Even when incubated at 25°, the enzyme activity remains unchanged for at least 4 hr. Furthermore, no activity is lost on lyophilization. Physicochemical Properties. The enzyme is shown to be homogeneous by polyacrylamide disc gel electrophoresis and by ultracentrifugation analysis. The apparent molecular weight of pig kidney DOPA decarboxylase is estimated by ultracentrifugation analysis to be 103,000. 7 The enzyme is composed of two subunits each with a molecular weight of about 50,000 as judged by SDS-polyacrylamide gel electrophoresis. In most preparations a faint band corresponding to an Mr -40,000 is also present. The enzyme shows maximal activity at pH 6.8. The amino acid composition of the enzyme has been determined, t9 HoloDOPA decarboxylase contains 1 mol of pyridoxal phosphate per mol of protein 1,7 bound to the e-amino group of a lysyl residue of the apoenzyme. 2° The enzymatic activity is stimulated (-30%) by added pyridoxal phosphate. The coenzyme can be removed by hydroxylamine treatmerit. 21The resolved enzyme is inactive in the absence of added pyridoxal phosphate; addition of coenzyme restores 40-60% of the original activity. The enzyme is also inactivated by treatment of the holoenzyme with NaBH4. The absorption spectrum is characterized, in addition to the usual absorption maximum at 280 nm, by two absorption maxima, at 420 and 335 nm, respectively. The ratio of the intensity of these two peaks, A335/ A420, is 3.5-3.8 at pH 6.8 and increases with increasing pH. 7 After reduction of the enzyme with NaBH4, only a single maximum at 330 nm is observed. 2~ Once the pyridoxal phosphate removal procedure has been performed, the apoenzyme no longer exhibits the absorption maximum at 420 nm, but shows a shoulder in the range of 335 nm. 21 Addition of substrates (aromatic amino acids in L form) or substrate analogs (aromatic amino acids in D form) causes an increase in absorbance at 420 nm which disappears as the substrates are decomposed but remains unchanged in the presence of substrate analogs. 22,23 19 C. Borri Voltattorni, A. Min¢lli, C. Cirotto, D. Barra, and C. Turano, Arch. Biochem. Biophys. 217, 58 (1982). ~0 F. Bossa, F. Martini, D. Barra, C. Borri Voltattorni, and C. Turano, Biochem. Biophys. Res. Commun. 78, 177 (1977). 21 C. Borri Voltattorni, A. Minelli, and C. Turano, FEBS Lett. 17, 231 (1971). A. Fiori, C. Turano, C. Borri Voltattorni, A. MineUi, and M. Codini, FEBS Lett. 54, 122 (1975). 23 C. Borri Voltattorni, A. MineUi, and P. Dominici, Biochemistry 22, 2249 (1983).

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Fluorescence measurements on the holoenzyme show that an emission maximum occurs at 380 nm when 335 nm light is used for excitation and a very feable emission occurs at 490 nm when the excitation wavelength is 420 nm. 7 Active Site. The amino acid sequence around the coenzyme binding lysine of pig kidney DOPA decarboxylase has been determined. 2° A chymotryptic phosphopyridoxyl peptide has been isolated and sequenced: it shows clear homologies with amino acid sequences of pyridoxyl peptides from coenzyme binding sites of various amino acid decarboxylases. Recently, chemical modification studies have provided information regarding the identification of possible amino acid residues at or near the active site of pig kidney DOPA decarboxylase: an arginine residue 24and a sulfhydryl residue 25 are probably involved in the substrate-binding site of the enzyme; a histidyl residue 26 seems to be involved in the catalysis. Substrate Specificity and Kinetic Constants. The enzyme was found to decarboxylate DOPA, 5-hydroxytryptophan, and m- and o-tyrosine in L form at readily measurable rates, but does not decarboxylate the corresponding aromatic D-amino acids, thus indicating its stereospecificity. However, D isomers of DOPA, 5-hydroxytryptophan, m-tyrosine, and otyrosine exert an inhibitory effect on the decarboxylase activity, The Ki values are 4.6 × 10-4 M for D-DOPA, 1.0 × 10-3 M for D-m-tyrosine, and 2.2 × 10-3 M for o-o-tyrosine 23 (o-m-tyrosine and D-o-tyrosine were prepared from the corresponding racemic forms by the action of L-amino acid oxidase and catalase23). Therefore, correct kinetic measurements can be obtained only in the presence of substrates in L form. A summary of the kinetic parameters for DOPA, 5-hydroxytryptophan, m-tyrosine, and otyrosine in L form is given in Table II. It can be seen that unnatural substrates, i.e., o- and m-tyrosine, have a decarboxylation rate higher than that of natural ones. The enzyme also decarboxylates p-tyrosine, phenylalanine, and tryptophan, but the reaction rate is much slower with those substrates. It must be stressed that kinetic measurements with DOPA decarboxylase are complicated by many side reactions, such as the spontaneous decarboxylation of the substrate DOPA, 1 the nonenzymatic reaction of DOPA with free coenzyme,l and the gradual inactivation of the enzyme following a product deamination reaction. 15,27 24 B. Tancini, P. Dominici, D. Barra, and C. Borri Voltattorni, Arch. Biochem. Biophys. 238, 565 (1985). 25 p. Dominici, B. Tancini, and C. Borri Voltattorni, Bull. Mol. Biol. Med. 9, 1 (1984). 26 p. Dominici, B. Tancini, and C. Borri Voltattorni, J. Biol. Chem. 260, 10583 (1985). 27 M. H. O'Leary and R. L. Baughn, Nature (London) 253, 52 (1975).

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CATABOLISM OF THE AROMATIC AMINO ACIDS

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TABLE II KINETIC PARAMETERSOF PIG KIDNEY DOPA DECARBOXYLASE

Amino acid

gm (M × 10-4)

Vmax (nmol min -I mg -1)

L-DOPA L-5HTP L-m-Tyrosine ° L-o-Tyrosine °

1.8 2.3 5.2 1.2

2390 420 4940 5650

a

m- and o-Tyrosine in L form were prepared from the corresponding racemic forms by the action of D-amino acid oxidase and catalase) 3

Inhibitors. DOPA decarboxylase is inhibited by carbonyl reagents such as hydroxylamine, thiosemicarbazide, semicarbazide, amino-oxyacetate, and by sulfhydryl reagents such as mercuribenzoate, iodoacetamide, N-ethylmaleimide, and dithiobisnitrobenzoic acid. L-cz-Methyl-a-hydrazino-3,4-dihydroxyphenylpropionic acid (carbiDOPA or MK 485) and 2,3,4-trihydroxybenzylhydrazine (Ro 4-5127), whose precursor is the trihydroxybenzylhydrazine seryl derivative (Ro 44602), are powerful inhibitors of pig kidney DOPA decarboxylase. 28,29 Both MK 485 and Ro 4-4602 are compounds used clinically in combination with DOPA for Parkinson's disease as they enhance the DOPAinduced increase of catecholamines in brain. This can be attributed to a poor penetration of these drugs through the blood-brain barrier leading to a preferential inhibition of DOPA decarboxylase activity in peripheral tissues. Moreover, an extensive series of compounds, structurally related to substrates and devoid of the substituted hydrazine function, have been examined for their inhibitory effect in vitro and in vivo on DOPA decarboxylase activity. 3°-32In most cases, only a rough indication of the affinity of a given compound for the enzyme (expressed as I~/2) is provided; for some of these compounds a direct spectrophotometric determination of :s C. Borri Voltattorni, A. Minelli, and P. Borri, FEBS Lett. 75, 277 (1977). 29 C. Borri Voltattorni, A. Minelli, and P. Borri, Life Sci. 28, 103 (1981). 30 G. Bartholini and A. Pletscher, Pharmacol. Ther. 1, 407 (1975). 3J Z. Huszti, E. Kasztreiner, G. Szilagyi, J. Kosaty, and J. Borsy, Biochem. Pharmacol. 22, 2267 (1973). 32 L. Ellenbogen, R. G. Kelly, R. J. Taylor, Jr., and C. S. Stubbs, Biochern. Pharmacol. 22, 939 (1973).

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AROMATIC-L-AMINO ACID DECARBOXYLASE

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the inhibitor binding is now available. 33 Inactivation of DOPA decarboxylase as a result of abortive transamination has been also observed: the enzyme catalyzes, in addition to the usual decarboxylation, a decarboxylation-dependent transamination which converts DOPA into 3,4-dihydroxyphenylacetaldehyde and simultaneously enzyme-bound pyridoxal phosphate into pyridoxamine phosphate. 15,27 Similar reactions occur when DOPA decarboxylase acts on a-methylDOPA, 15'21,34 m-tyrosine, 15 serotonin, 34,35 o-5-hydroxytryptophan, and o-tryptophan. 23 Finally, several recently developed a-fluoromethyl derivatives of DOPA, namely OL-a-monofluoromethyl-fl-(3,4-dihydroxyphenyl) alanine, OL-a-difluoromethyl-fl-(3,4-dihydroxyphenyl) alanine and DL-o~monofluoromethyl-fl-(2,3-dihydroxyphenyl) alanine, were shown to behave as highly potent irreversible and selective inhibitors of DOPA decarboxylase. 36-4° They act via an enzyme-activated "suicide mechanism": inactivation occurs because the compounds are decarboxylated by the enzyme generating a highly reactive intermediate which alkylates the enzyme. 41

33 E. Barboni, C. Borri Voltattorni, M. D'Erme, A. Fiori, A. Minelli, and M. A. Rosei, Life Sci. 31, 1519 (1982). 34 E. Barboni, C. Borri Voltattorni, M. D'Erme, A. Fiori, A. Minelli, M. A. Rosei, and C. Turano, Biochern. Biophys. Res. Cornnrnun. 99, 576 (1981). 35 C. Borri Voltattorni and A. Minelli, Bull. Mol. Biol. Med. 5, 52 (1980). 36 p. Bey, in "Enzyme-activated Irreversible Inhibitors" (N. Seiler, M. J. Jung, and J. Kock-Weser, eds.), p. 27. Elsevier/North Holland Biomedical Press, Amsterdam, 1978. 37 j. Kollonitsch, A. A. Patchett, S. Marburg, A. L. Maycock, L. M. Perkins, G. A. Doldouras, D. E. Duggan, and S. D. Aster Nature (London) 274, 906 (1978). 38 B. W. Metcalf, P. Bey, C. Danzin, M. J. Jung, P. Casara, and J. P. Vevert, J. Am. Chem. Soc. 100, 2551 (1978). 39 M. G. Palfreyman, C. Danzin, P. Bey, M. J. Jung, G. Ribereau-Gajon, M. Aubry, J. P. Vevert, and A. Sjoerdsma, J. Neurochern. 31, 927 (1978). 40 M. J. Jung, M. G. Palfreyman, J. Wagner, P. Bey, G. Ribereau-Gajon, M. Zraika, and J. Kock-Weser, Life Sci. 24, 1037 (1979). 41 A. L. Maycock, S. D. Aster, and A. A. Patchett, Biochemistry 19, 709 (1980),