Characteristics of the inhibition of aromatic l -amino acid decarboxylase by α-methylamino acids

ARCHIVES

OF

BIOCHEMISTRY

AND

Characteristics

BIOPHYSICS

of the Inhibition

Decarboxylase WALTER

103, g-14 (1963)

LOVENBERG,

of Aromatic

by cY-Methylamino

JACK BARCHAS, HERBERT SIDNEY UDENFRIEND

From the Experimental Therapeutics Branch and Laboratory Heart Institute, Xational Institutes of Health, Received

L-Amino

Acid

Acids WEISSBACH

of Clinical Biochemistry, Bethesda, Maryland

AND

National

June 24, 1963

The characteristics of the inhibition of aromatic L-amino acid decarboxylase from guinea-pig kidney by cy-methyl-DOPA and cr-methyl6hydroxytryptophan have been investigated. Noncompetitive inhibition is obtained when the inhibitor is preincubated with enzyme in the absence of pyridoxal phosphate before the addition of substrate. When the coenzyme is present during the preincubation or when the inhibitor is added at the same time as the substrate the kinetics of inhibition are those of a competitive inhibitor. The inhibition is specific for the L-isomer of the a-methylamino acids and other pyridoxal phosphate requiring enzymes are not affected. The data suggest that specific interaction of the inhibitor, enzyme, and enzyme-bound coenzyme takes place. INTRODUCTION In mammalian tissues a single enzyme, aromatic L-amino acid decarboxylase, catalyzes the decarboxylation of all the naturally occurring aromatic amino acids (1). The also decarboxylates the same enzyme a-methyl analogues of the aromatic amino acids (2), further attesting to the nonspecificity of the enzyme. The decarboxylation of the a-methylamino acids was of special interest since these compounds are potent inhibitors of the enzyme when the natural amino acids are employed as substrates. Sourkes (3), who reported the first studies with cY-methyl-DOPA’ as an inhibitor of the decarboxylation of L-DOPA, stated that the ol-methylamino acids inhibited by interaction with the enzyme rather than with the coenzyme and that the

inhibition

was competitive

1 Abbreviations phenylalanine; ethylamine;

recently, Smith (4) showed that the degree of inhibition was markedly influenced by added pyridoxal phosphate and concluded that the cr-methylamino acids inhibit by combining

in nature. More

used: DOPA, 3,4-dihydroxydopamine, 3,4-dihydroxyphen-

SHTP,

5-hydroxytryptophan;

with

the

coenzyme

in a non-

competitive fashion. We have reinvestigated this problem and have found that the observations of both Sourkes (3) and Smith (4) can be obtained depending on the experimental conditions. Inhibition is competitive in nature when the cr-methylamino acid is preincubated with the enzyme in the presence of pyridoxal phosphate, or when substrate and inhibitor are added simultaneously in the absence of pyridoxal phosphate. However, when the inhibitor is preincubated with enzyme in the absence of coenzyme or substrate noncompetitive inhibition is obtained. Pyridoxal phosphate also markedly influences the degree of inhibition obtained with the a-methylamino acids.

LY-

methyl-DOPA,

3,4-dihydroxy-a-methylphenyl3,4-dihydroxy-aalanine ; ol-methyl-dopamine, methylphenethylamine; a-methyl-5HTP, 5-hydroxy-a-methyl-n, L-tryptophan.

MATERIALS

AND

METHODS

The amino acids and amines were obtained from the California Corporation for Biochemical Research and pyridoxal phosphate from Sigma 9

10

LOVENBERG

Chemical Company. a-Methyl-DOPA, or-methyldopamine, ol-methyl5HTP, a-methylserotonin, and cY-methyltryptamine were obtained through the courtesy of Merck Sharpe and Dohme and the Upjohn Company. Aromatic L-amino acid decarboxylase was partially purified from guinea-pig kidney2 according to the procedure described by Clark et al. (F). The enzyme preparation used in most experiments, about lo-fold purified, was prepared from a high-speed supernatant fraction of a guinea-pig kidney homogenate by ammonium sulfate precipitation. Where indicated the enzyme preparation was further purified by adsorption onto alumina Cy and elution (G). The decarboxylase from Streptococcus fuecalis was prepared from cells which had been grown as described by Epps (7). The cells were harvested by centrifugation, suspended in 2 volumes of water and sonicated for 30 min. in a Raytheon 10 kc sonic oscillator. The resulting suspension was then centrifuged for 1 hr. at 100,OOOy. The supernatant fraction obt.ained was applied to a Sephadex G-25 column and the protein fraction which emerged from the column was used in the experiments reported here. Enzyme Assay. Incubations with tryptophan (6.7 X lop3 &f) or 5HTP (1.5 X lo+ M) were carried out in air at 37”, in a metabolic shaker, and contained enzyme, 10m3&f iproniazid and either 0.08 M phosphate buffer or Tris buffer pH 8.4 in a final volume of 3 ml. When DOPA was used incubation conditions were modified as shown in the text. The enzyme was generally preincubated for 3 min. (with or without inhibitor) before the addition of substrate. Pyridoxal phosphate, when used, was added during the preincubation period and, unless otherwise stated, the final concentration was 7 X 10m5M. At the end of the incubation period the samples were placed in a boiling water bath, diluted with water, and centrifuged to remove protein. The amount of amine formed was determined in the supernatant solution by assay procedures which were essentially those described in a previous report (1). Serotonin and tryptamine were separated from the corresponding amino acids by the use of Permutit columns and dopamine was separated from DOPA by means of an IRC-50 column. Decarboxylation of the 01methyl-amino acids was assayed fluorometrically as described for the desmethyl analogues (1). The assay of tryptophan decarboxylation was modified when serotonin and a-methyl-serotonin were used as inhibitors since these amines inter2 Contrary to the report of Awapara et al. (5) a similar aromatic L-amino acid decarboxylase, but with lower activity, was obtained from rat liver.

ET AL. fered in the normal assay. In these experiments a 0.5 ml. aliquot of the incubation mixture was placed in a 60-ml. shaking bottle containing 5 ml. of 1 N NaOH. The tryptamine was then extracted into 15 ml. of benzene. After washing the organic phase with a second portion of 1 N NaOH t,o remove residual phenolic indoleamines, 10 ml. of the benzene phase were transferred to a shaking tube containing 1.0 ml. of 0.1 N HCI. The tube was shaken for 5 min. and 0.5 ml. of the aqueous acid phase was transferred to a cuvette containing 1.0 ml. borate buffer, pH 10.0. The tryptamine fluorescence was measured in the spectrophotofluorometer as previously described (8). The reaction was quantified by carrying internal standards through the entire procedure. RESULTS

AND

DISCUSSION

Efect of Pyridoxal Phosphate on the Decarboxylation of Aromatic Amino Acids. It is known that pyridoxal phosphate is the coenzyme for mammalian aromatic L-amino acid decarboxylase. However, in guinea-pig kidney the coenzyme is firmly bound to the enzyme and with most substrates less than a 20 % stimulation is observed when pyridoxal phosphate is added to enzyme preparations purified 50- to Wfold. Among the naturally occurring substrates L-DOPA shows the largest stimulation (doubling of activity) upon addition of exogenous pyridoxal phosphate to the incubation. However, the a-methylamino acids tested exhibited an

1’

Eo 0

” 10-G

MOLARITY

/

1’ 10-s

IO.4

OF PYRIDOXAL

II

10-3 PHOSPHATE

FIG. 1. The effect of pyridoxal phosphate on the decarboxylation of aromatic amino acids. The incubations contained 0.08 M phosphate buffer pH 8.4 when 5HTP and a-methyl-5HTP were substrates and 0.08 M phosphate buffer pH 7.0 with DOPA and cY-methyl-DOPA. The substrate concentrations used were 5HTP, 1.5 X 10M3 M; a-methyl-5HTP, 3 X 10d3 M; DOPA, 1 X 10-Z M; a-methyl-DOPA, 1 X 10-Z M.

DECARBOXYLASE I

I

INHIBITION

BY a-METHYLAMIXO TABLE

I

I

11

ACIDS I

EFFECT OF PREINCUBATI~N AND PYRIDOXAL PHOSPHATE ON INHIBITION OF 5HTP L)ECARBOXYLATION BY a-METHYL-BHTP Per cent inhibition Concentration m-methyl-SHTP

Preincubation (3 min.) Pyridoxal phosphate

5 1 5 1

0

5 IO PREINCUBATION

20 PERIOD

30

x x x x

10-7 IO-6 IO-” 10-b

Standard ployed.

5 18 56 75 incubation

iYo preincubation

h-0 h-0 Pyridoxal pyridoxal pyr’doxa’ phosphate phosphate phosphate

24 55 94 100 conditions

12 19 4G A7

13 15 Gl 79 were

em-

(MINUTES)

FIG. 2. Effect of preincubation on aromaticL-amino acid decarboxylase activity. Enzyme was preincubated at 37” at pH 8.4, with or without inhibitor, cu-methyl-5HTP (5 X lo-’ M) and pyridoxal phosphate (7 X 10e5 iv), for the indicated times. The addition of substrate (and pyridoxal phosphate to those tubes that did not contain any during the preincubation) initiated the enzymatic reaction. A, Enzyme + pyridoxal phosphate; B, enzyme + pyridoxal phosphate + inhibitor; C, enzyme; 11, enzyme + inhibitor.

almost absolute requirement for pyridoxal phosphate for their decarboxylation. These effects are shown in Fig. 1, where the coenzyme requirements for decarboxylation of 5HTP, DOPA, and their a-methyl analogues are compared. It is also seen in Fig. 1 that high concentrations of coenzyme inhibit decarboxylation of all the amino acids. This very likely is an indication of nonenzymatic interaction between the coenzyme and these substrates. Such a reaction has already been demonstrated with L-DOPA (9). Preincubation Studies. Preincubation of the enzyme at 37” in the absence of coenzyme, substrate, or inhibit,or resulted in a decrease in enzyme activity which was nearly linear with time (Fig. 2, curve C). When pyridoxal phosphate was present during the preincubation, an initial rise in enzyme activity was observed, followed by a slow inactivation (curve A). In the presence of an a-methylamino acid the rate of inactivation observed during the preincubation was more rapid (curve D) but could be partially pre-

vented by having pyridoxal phosphate present during preincubation (curve B). However, the inactivation produced by preincubating with inhibitor could not be reversed by adding coenzyme at the end of the preincubation. It should be noted that the 3-min. preincubation period employed in most of the studies presented below resulted in only slight inactivation of the enzyme even in the absence of pyridoxal phosphate. Similar effects were produced when preincubations were carried out at pH 7.4 or 9.0. However, no inactivation was observed if the preincubation was done at 4”, even in the absence of coenzyme. The Inhibition of Amino Acid Decarboxylation by a-Methylamino Acids. When studied as inhibitors of amino acid decarboxylation the a-methylamino acids exhibit quite different properties depending upon the presence or absence of added pyridoxal phosphate and whether or not they had been preincubated with the enzyme before the addition of substrate. As can be seen in Table I a given concentration of cY-methylamino acid produced much less inhibition when pyridoxal phosphate was present during the preincubation. If no preincubation was employed, pyridoxal phosphate had little, if any, effect on the inhibition obtained with the a-methylamino acids. The nature of the inhibition obtained with the cr-methylamino acids, under varying conditions, can be seen in a series of double

12

LOVENBERG

ET AL.

reciprocal plots shown in Fig. 3. The inhibition by cy-methyl-5HTP was found to be competitive with substrate, with or without added coenzyme, when no preincubation was employed (Figs. 3A and 3B). When as short a preincubation as 3 min. was used, however, the inhibition became noncompetitive with substrate in the absence of pyridoxal phosphate (Fig. 3D) but was still competitive in the presence of added pyridoxal phosphate (Fig. 3C). Comparable results were obtained with a-methyl-DOPA as an inhibitor. The above findings suggest that the FIG. 3C

‘O60-

.-METHYL

5HTP i

‘1: p

.5

I

2

3 I/s

4

5

x 104

OOI

I

2I

d

FIN. 311

l/s FIG.

oo+-*-2++

--+

b

I& FIG.

5

x 10-4

3B

3. Double reciprocal plots showing the effects of pyridoxal phosphate and preincubation on the nature of the inhibition. The concentration of or-methyl-5HTP used was 5 X 10e6 M in A and B and 10e6 M in C and D. A, No preincubation; with pyridoxal phosphate. B, No preincubation; without pyridoxal phosphate. C, Preincubation; with pyridoxal phosphate. D, Preincubation; without pyridoxal phosphate. FIG.

34

41

J 5

x 10-4

30

cY-methylamino acids react with enzymebound coenzyme. However, the interaction does not appear to be a nonspecific combination with the coenzyme alone. That the a-methylamino acids combine directly with the enzyme is certain since they are themselves substrates of this enzyme, with a K, in the range of 5HTP and DOPA (about 5 X 10p5) and a V,,, comparable to that of the weaker substrates (about 0.1 pmole decarboxylated per hr.) (2). The decarboxylated products of the or-methylamino acids (corresponding amines), as well as the amines formed from the natural aromatic amino acids, also inhibit amino acid decarboxylation, although to a lesser degree than do the cY-methylamino acids. This is particularly apparent when a substrate with a high K, value, such as tryptophan, is used (Table II). Here, too, pyridoxal phosphate partially reverses the inhibition. Another factor which demonstrates that

DECARBOXYLASE TABLE

INHIBITION

II

INHIBITION OF TRYPTOPHAN DECARBOXYLATION BY SEROTONIN AND a-METHYLSEROTONIN, WITH AND WITHOUT ADDED PYRIDOXAL PHOSPHATE Per cent inhibition Concentration of amine (4,)

10-S 10-h IO--”

a.Methylserotonin + Pyridoxal phosphate

9G 81 35

Serotonin

+ pyrpoPoxa, Pyridoxal phosphosphate phate

100 9G 83

65 26 0

83 57 28

communication.

EFFECTS

L ISOMERSOF~~-METHYL-DOPA

5HTP

No pyridoxal phosphate

there is interaction between the a-methylamino acids and the enzyme is the stereospecificity of the inhibition. It was found (Table III) that only the L form of a-methylDOPA was an effective inhibitor whereas both isomers would be expected to react nonenzymatically with free pyridoxal phosphate . Favoring the postulate that the cr-methylamino acids inhibit mammalian aromatic-namino acid decarboxylase by a unique interaction with both enzyme and bound coenzyme, rather than by nonspecific interaction with the coenzyme alone, are the findings that other pyridoxal phosphate requiring enzymes are not affected by these inhibitors. It has been observed that tyrosine transaminase is not inhibited by ol-methyl-DOPA.3 Furthermore, Sourkes (3) has reported that the L-amino acid decarboxylase from Streptococcus faecalis is not inhibited by the a-methylamino acids. Since the mammalian and bacterial enzymes have different properties with respect to pH optima and substrate specificity the latter studies were repeated using L-DOPA as substrate for both enzymes, cr-methylDOPA as inhibitor, and incubating under identical conditions of pH, temperature, etc. Exogenous pyridoxal phosphate was removed from the crude bacterial enzyme by Sephadex treatment and from the mammalian enzyme by the usual purification B., personal

TABLE III OF THE INHIBITORY

COMPARISON D AND

13

ACIDS

ON

Per cent inhibition by a-Methyl-DOPA

10-z 10-h

L-isomer

D-isomer

87 32

0 0

The standard incubation (1.5 X 1OW M) was employed. TABLE

containing

5HTP

IV

EFFECT OF ~-METHYL-DOPA AND BACTERIAL AMINO ACID Mammalian enzyme

Control Control + 10-S M aMethyl-

OF THE

DECARBOXYLATION

Concentration of inhibitor (U)

The incubations contained substrate at a concentration of 6.7 X 10s3 fi1. Pyridoxal phosphate, where added, was employed at a concentration of 1 x lo* M.

3 LaDu,

BY or-METHYLAMINO

ON MA~XMALIAN DECARBOXYL~SE Bacterial enzyme

Specific activitya

Per cent inhibition

Specific activitya

Per cent inhibition

938 787

17

028 686

0

525

44

628

0

DOPA

Control + 10-E M 01. MethylDOPA

u Specific activity is expressed as pg. dopamine formed per hr. per mg. protein. The reaction mixture contained 1.5 X 1OF M phosphate buffer pH 6.0, 10d3 M DOPA, but no added pyridoxal phosphate. A sample containing 0.48 mg. protein of the Al Cy eluate was used for the mammalian enzyme and 0.42 mg. protein of the Sephadex treated bacterial extract was used. Each incubation mixture was assayed for amine formation at 0, 5, and 10 min. after the addition of substrate to start the reaction.

procedure. Thus, the two enzyme preparations contained only the pyridoxal phosphate which was bound to the enzyme. As shown in Table IV, only the mammalian enzyme was inhibited. Obviously this could not be the case if cy-methyl-DOPA interaction were mainly with pyridoxal phosphate. It is evident that cy-methyl-DOPA, and related cr-methylol-methyldHTP, amino acids have a high degree of specificity for inhibiting mammalian aromatic L-amino acid decarboxylase which can only be explained by a specific interaction with this

14

LOVENBERG

enzyme. On the other hand, the inhibition is modified both in its intensity and in its characteristics by the presence of coenzyme and substrate. The studies would indicate that the inhibitor binds to the active site of the enzyme and to the enzyme-bound prosthetic group. If these interactions occur then the reason for both the stereo and enzymatic specificity of this inhibition is apparent. The relief of this inhibition by exogenous pyridoxal phosphate may then be due to the reactivation of the bound pyridoxal phosphate in a manner comparable to that reported by Nishimura el al. (10). They found that exogenous pyridoxal phosphate, or cu-keto acids, activated aspartic acid P-decarboxylase by reactivating enzyme-bound coenzyme which had been internally inactivated. ilwapara et al. (5) have reported findings which suggest a similar effect of added pyridoxal phosphate on the unre-

ET AL.

solved aromatic n-amino acid decarboxylase from rat liver. REFERENCES 1. LOVENBERG, W., WEISSBACH, H., AND UDENFRIEND, S., J. Biol. Chem. 33’7, 89 (1962). 2. WEISSBACH, H., LOVENBERG, W., AND UVENFRIEND, S., Biochem. Biophys. Res. Commun. 3, 225 (1960). 3. SOURKES, T. L., Arch. Biochem. Biophys. 51, 444 (1954). 4. SMITH, S. E., Brit. J. Pharmacol. Chemotherap. 15, 319 (1960). 5. A\VAPARA, J., SANDMAN, R. P., AND HANLY, C., Arch. Biochem. Biophys. 93, 520 (1962). 6. CLARK, C. T., WEISSBACH, H., AND UDENFRIEND, S., J. Biol. Chem. 210, 139 (1954). 7. EPPS, H. M. R., Biochem. J. 39, 42 (1945). 8. SJOERDSMA, A., OATES, S. A., ZALTZMAN, P., AND UDENFRIENV, S., J. Pharmacol. Exptl. Therap. 126, 217 (1959). 9. SCHOTT, H. F., AND CLARK, W. G., J. Biol. Chem. 196, 449 (1952). J. S., MANNING, J. M., AND 10. NISHIMURA, MEISTER, A., Biochemistry 1, 442 (1962).