Enzymatic decarboxylation of isomers and derivatives of dihydroxyphenylalanine

Enzymatic Decarboxylation of Isomers and Derivatives of Dihydroqphenylalaninne’ Theodore Sourkes, Peter Heneage and Yolanda Trano Prom the Merck

Institute

for

Therapeutic

Received

April

Research,

Rahway,

New Jersey

7, 1952

INTRODUCTION

The two n-amino acid decarboxylases, DOPA decarboxylase of mammalian tissues (1) and bacterial tyrosine decarboxylase (2), are analogous in having overlapping substrate specifications (3, 4) as well as in possessingpyridoxal phosphate as coenzyme (5, 6). In a series of publications [summarized in (4)], Blaschko has adduced rules of substrate specificity for these two enzymes. For example, DOPA decarboxylase acts on ortho- or meta-hydroxyphenylalanine; tyrosine decarboxylase attacks only the meta- and para-isomers. On the basis of these generalizations one would expect isomers of DOPA to be decarboxylated by the respective enzymes if the compounds have at least one phenolic hydroxyl group in the appropriate position. To test this hypothesis we have studied the enzymatic decarboxylation of four DOPA isomers. In addition, a number of derivatives of 3,4- and 2,4-DOPA were examined as potential substrates, in order to determine some of the factors affecting the rate of decarboxylation of compounds possessing the basic hydroxyphenylalanine structure. EXPERIMENTAL Enzymes and Reaction Mixtures 1. Lyophilized pig and guinea to Schales and Schales (7). Briefly,

pig kidney preparations were made according the whole kidney, or only the cortex, is blended

1 Presented in part before the American Society of Biological York City, April 15, 1952. 2 The following abbreviations have been used: DOPA = dihydroxyphenylalanine; PA = phenylalanine; DOPS = dihydroxyphenylserine. 185

Chemists,

New

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in the cold with water, centrifuged, and the supernatant is lyophilized. These dried powders were somewhat active without the addition of pyridoxal phosphate, but addition of increasing concentrations of coenzyme gave progressively higher rates, reaching the maximum, under our conditions, at about 5 pg./flask. From the rates with and without added coenzyme we estimate that the apoenzyme was only 22, 32, and 47% saturated with coenzyme in three different pig kidney powders tested. Reaction mixtures were uniformly 2.5 ml. in volume with final concentrations of the constituents as follows: m-substrate, 0.005 M; phosphate buffer, pH 6.8,0.053 M; pyridoxal phosphate, 2rg./ml. Twenty to 50 mg. of lyophilized powder was used in each flask, the amount depending on the activity of the parTABLE Centrifugation

I

of Guinea Pig Kidney Blends

Guinea pig kidneys were blended with 4 vol. water for 2-3 min. Portions were centrifuged in celluloid cups for 25 min. at 2,000 X g and 20,000 X g, respectively. Blending and centrifuging were performed at 2°C. Fractions were tested for DOPA decarboxylase activity manometrically. The Warburg flasks contained in a total volume of 2.5 ml.: DL-~,~-DOPA, 0.005 M; phosphate buffer, pH 6.8, 0.053 M; pyridoxal phosphate, 2 pg./ml.; 1.0 ml. of enzyme solution (or suspension, in the case of residues). Reaction time, 10 min.; incubated at 37”. Fraction

Experiments 4 and 5 (average) Per cent of total /A COz/lO min./ml. activity ml.

QCOp

Total

100

1

-

Experiment 2 3

-

5

Blend

75.6

84

2,000 x g: supernatant residue

58.7 18.8

86 40

79.4 11.9

26.3 21.4 -

16.3 25.4 22.0 3.5 8.0

20,000 x g: supernatant residue

61.8 15.8

86 30

84.0 7.5

34.3 32.1 0 -

16.8 25.6 27.7 2.3 3.1

a &oa = Microliters

18.0

number 4

15.8

16.1

CO2 released/mg. dry matter/hr.

titular preparation. Substrate was tipped from a side arm of the Warburg flask at zero time. When it was desired to stop the reaction, 0.5 ml. of 2 N H#O, was added from a second side arm. By running control flasks in which the acid was tipped in at zero time, appropriate corrections were made for “bound COz” in all experiments. The gas phase was nitrogen. In some experiments, guinea pig kidney was blended with 4 vol. water, centrifuged in the cold, and the supernatant used without further treatment, such as lyophilizing. Since DOPA decarboxylase is in the aqueous portion of the blend, this treatment results in some degree of purification, with minimal losses of enzyme, as shown in Table I. The first three columns demonstrate the separation of the enzyme into supernatant and residual fractions, the data being averaged from

DIHYDROXYPHENYLALANfNE

187

two experiments. The &co2 values presented in the remaining columns indicate that in three of the five experiments substantial removal of inactive ballast material was achieved by centrifugation at 20,000 X 8. Attempts to make a dried kidney preparation by acetone precipitation always resulted in total loss of enzyme activity. 2. Tyrosine decarboxylase was obtained as acetonized preparations of StreptoCOCCUSfuecalis R cells grown on yeast extract-peptone-glucose medium. These enzyme preparations were compared with the live, resting cell suspensions from which they were made, and it was found that relative activities with the substrates tested ran closely parallel. The Warburg flasks contained 1 mg. of bacterial N when live cells were used or 10 mg. of the acetonized cells in 3.0 ml. final volume; substrate, 0.004 M; acetate buffer, pH 5.5, 0.067 M (M/15) (final concentrations). When the acetonized enzyme was used, 5 or 10 pg. of pyridoxal phosphate was added per flask, depending on the activity of the unfortified preparation. A number of “blank” flasks were usually run in which buffer alone replaced the buffersubstrate solutions in the side arm. These blanks registered a small release of gas which did not increase for more than 5-10 min. after tipping in the side arm contents. CO2 volumes recorded from experimental flasks were routinely corrected by subtracting this blank volume.

Substrates All substrates except 3,4-DOPA were synthesized in the Research and Development Laboratories of Merck & Co., Inc., by Dr. Gustav A. Stein and Mr. Herman A. Bronner. We are indebted to them and to Dr. Karl Pfister for making the compounds available to us. DL-3,4DOPA was purchased from Eastman Kodak Company. The other compounds studied (m-mixtures) were 2,5-DOPA; 3,5-DOPA; 2,4-PAPA; 3-methoxy-4-hydroxyphenylalanine; 3,4-dimethoxyphenylalanine; 2,4dimethoxyphenylalanine; N-methyl-S, 4-DOPA; N-(2-cyanoethyl)-3,4DOPA; and 3,4-dihydroxyphenylserine (3,4-DOPS). The last-named compound was an optically inactive material prepared by the method of Dalgliesh and Mann (8). These authors believe the product to be one of the two possible racemic modifications. Blaschko and coworkers (9) have determined the composition of this mixture and have found that one of the two stereoisomers present yields, on decarboxylation, artereno1 having the natural configuration. Measurement of Rates studies indicated that the rate of decarboxylation

Preliminary was linear with respect to time for up to E-20 min. with the mammalian and acetoniaed bacterial enzymes, and considerably longer with the resting cell suspension. For purposes

of this study we chose to measure the evolution of gas during the IO-min. period immediately after tipping in the substrate. Occasionally, as described below, longer incubation periods were used. All determinations were made at 37”.

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SOURKES,HENEAQE AND TRANO RESULTS 1. Decarboxylation of DOPA Isomers

Table II shows that all the isomers are decarboxylated, but at widely different rates. The figures represent averages of ten determinations in practically all of which the order of rates was the same. While these figures are not to be considered as “biological constants” they at least show the relative susceptibility of the compounds to enzyme action. When both phenolic hydroxyl groups were in positions satisfying the structural requirements for decarboxylation, there did not seem to be any resulting increases in rate. For example, 3,5-DOPA, which con-

Relative

Rates of Decarboxylation

TABLE II of DOPA Isomers and Methoxyphenylalanine Derivatives

In experiments with S. faecalis R the Warburg flasks contained in a total volume of 3.0 ml.: 1 mg. of bacterial nitrogen (live cells) or 10 mg. of acetoneprecipitated cells; nL-substrate, 0.004 M; acetate buffer, pH 5.5, 0.067 M (M/15); pyridoxal phosphate (added to acetonized cells only), 5-10 pg./flask. For the kidney enzymes, flask contents were as in Table I; the kidney fraction used was the supernatant obtained by centrifuging 20% water blends at 2,000 X Q for 25 min. at 2”. Figures represent relative rates of decarboxylation (IO-min. incubation) and are averages of ten replications. Substrate

3,4-DOPA 2,5-DOPA 3,5-DOPA 2,4-DOPA 3-Methoxy-4-hydroxy-PA 3,4-Dimethoxy-PA 2,4-Dimethoxy-PA

S. faecalis R

Pig

100 4 18 113 9 0 0

100 88 24 71 -

Kidney

Guinea pig

100 112 23 42 0 0 0

tains two meta-groups, was acted upon more slowly by all preparations than was 2,4-DOPA which possesses only one hydroxyl in a position required by the kidney and bacterial enzymes, respectively. This is similar to Epps’s observation (3) that tyrosine is decarboxylated more rapidly than 3,4-DOPA by the bacterial enzyme. Our finding that 2,5-DOPA is decarboxylated by S. juecalis enzyme, although admittedly at a very slow rate, is at some variance with the report of Stanley (10) that this compound is decarboxylated only very slowly by acetone-dried cells and not at all by intact cells. Indeed, we would not consider the low rate which we observed significant but for the fact that when live cells are used, gas continues to be evolved long

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DIHYDROXYPHENYLALANINE

after the initial lo-min. period. Its decarboxylation by the bacterial tyrosine decarboxylase would, of course, be expected on the basis of the rules Blaschko has adduced. Another isomer, 2,3-DOPA, which we have not tested, was found to be decarboxylated by both mammahag kidney and bacterial enzymes in Blaschko’s laboratory (11). Comparison of results for pig and guinea pig enzymes shows that there is a species difference in the rate at which 2,5- and 2,QDOPA are decarboxylated. 2. Effect of Methylation

of the Phenolic Hydroxyl

Groups

Gale (12) has pointed out that the amino acids attacked by bacterial decarboxylases possess, in addition to the a-amino and a-carboxyl groups, another polar group, which probably aids in the attachment of substrate to the apoenzyme. Phenylalanine is an exception, inasmuch as McGilvery and Cohen observed that it is acted upon by S. faecalis preparations (13). However, in the case of tyrosine, if the polarity of its phenolic hydroxyl is diminished by ether or ester formation, the bacterial decarboxylase is inactive (12). We have found that methylation of both hydroxyls in 2,4- and 3,4-DOPA results in compounds not attacked by either the mammalian or bacterial enzymes (Table II). When only one hydroxyl is methylated, as in 3-methoxy-4-hydroxyPA,2 the substrate structural requirements of the bacterial enzyme are sufficiently met, and decarboxylation occurs. The kidney enzyme does not act on this compound, which is in agreement with its recognized inability to act on tyrosine. 3. Substitution on. the Amino Group N-Methyl-S, 4-DOPA and N-methyl-a, 4-DOPS have been tested by Blaschko (4, 14) who found they are not decarboxylated by kidney (or bacterial) preparations. We have confirmed this with the first-named compound and have found that N-(2-cyanoethyl)-3,4-DOPA is not attacked either. Thus it seems that both hydrogens must be available on the nitrogen atom to permit enzymatic attack. 4. Substitution on the /T-Carbon Atom (3,4-DOPS) Using our standard system we were not able to demonstrate unequivocally the decarboxylation of 3,4-DOPS by the kidney enzyme. Blaschko, after obtaining similar negative results (9), later showed that if one incubates large amounts of kidney extract with 3,4-DOPS and for periods up to 5 hr., one can observe a release of CO2 and formation

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of arterenol, the latter demonstrated and quantitated by pharmacological means (15). The decarboxylation of DOPS thus occurs, presumably by the action of DOPA decarboxylase, but at extremely slow rates. According to Beyer (16), on the other hand, DOPS is decarboxylated by kidney supernatant at rates comparable with those seen when 3,bDOPA is substrate. The reason for this discrepancy is not clear. We considered the possibilit’y that 3,4-DOPS is, more rapidly decarboxylated in the adrenal medulla, where the product of the reaction, arterenol, is stored. The results of eight experiments are given in Table III. For each experiment a different batch of beef adrenal glands was collect,ed at a nearby abattoir, and carried to the laboratory on cracked TABLE

III

by Beef Adrenal Medulla Homogenates Warburg flasks contained 800 mg. wet weight of homogenized tissue per flask; other components of the reaction mixtures as in Table I; incubated at 37”.

Decarboxylation

of 3,4-DOPA

and S,$-DOPS

Experiment No.

Incubation period milt.

1 2 3 4 5 6 7 8

10 30 30 30 30 30 60 60

3,4-DOPA

15 28 62 31 46 13 30 25

3, I-DOPS

9 33 -4 4 19 2 11 15

ice, to be used at once. The medullary portion was dissected out and homogenized in water. With increased amounts of tissue in the flasks, decarboxylation of 3,4-DOPS was observed in five of the eight experiments. In one of these, the DOPS was decarboxylated at a rate slightly greater than that for 3,4-DOPA, but in the remainder the rate was less. Our data for beef medulla DOPA decarboxylase are of the same order as those recently reported by Langemann (17). Guinea pig adrenals contain little or none of this enzyme (9, 18). 5. Paper Chromatography The Rf values were determined for the compounds used in this work. Two-hundredths ml., containing 10-20 pg. of the compound dissolved

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DIHYDROXYPHENYLALANINE

in 50% acidified ethanol, was spotted on filter paper, and the chromatogram was developed according to the usual techniques. Reagents used were ninhydrin (0.1% in water-saturated n-butanol) or the ferricyanide reagent of James (19). Some variation in Rf values was observed from run to run, but the data in Table IV are quite typical. TABLE IV RI Values of DOPA Isomers and Derivatives Ten to 20 Fg./spot; solvent: phenol, saturated with water; atmosphere: air saturated with HCl. Chromatograms were run for 24 hr. at 20/23”, then air-dried overnight and sprayed. N = ninhydrin reagent; F = ferricyanide reagent. Compound

Rf

3,4-DOPA 2,4-DOPA 3,5-DOPA 2,5-DOPA 3,4-DOPS 3-Methoxy-4-hydroxy-PA 3,4-Dimethoxy-PA 2,4-Dimethoxy-PA N-Methyl-3,4-DOPA N-Cyanoethyl-3,4-DOPA

0.33 0.37 0.29 0.40 0.22 0.75 0.93 0.94 0.67 0.67

(N) (N) (N) (N) (N) (N) (N) (N) (F) (F)

DISCUSSION

This and other studies on the substrate specificity of tyrosine and DOPA decarboxylases make it clear that any alteration in the substituents on the tyrosine and DOPA structures affects the rate of decarboxylation. Indeed some of these substitutions render the compounds resistant to enzymic attack. Decarboxylation of all DOPA isomers (except 2,6-DOPA) by both decarboxylases is to be expected by extrapolation of results with the monohydroxyphenylalanines. Our data with four of the six possible racemic DOPA mixtures are in qualitative agreement with Blaschko’s empirical rules. The differences in rates of decarboxylation of the various isomers, however, are not explicable on the basis of assigning independent contributions from each of the two phenolic hydroxyls in determining the rates. For example, 3,5-DOPA possessestwo meta-groups (only one of which is sufficient to ensure the action of either enzyme), yet it is decarboxylated more slowly than 2,4-DOPA. Likewise a second phenolic group on the tyrosine molecule, to form 3,4-DOPA, results actually in a decreased rate of decarboxylation by the bacterial enzyme

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SOURKES,HENEAGE AND TRANO

(3). Thus, (a) position of the phenolic groups and (b) their mutual influence are of importance in effecting enzyme-substrate union and, consequently, the rate of decarboxylation of these compounds. 2,6-DOPA is a special case in possessing two phenolic groups both ortho- to the side chain; hence, it would be expected to be acted upon only by the mammalian enzyme. Comparative tests of the two types of enzyme using 2,6-DOPA as substrate would, therefore, be of interest, at least in checking the prediction. The interaction between the substituents on the benzene ring is further exemplified by results with the methoxylated phenylalanines. The failure of the enzymes to decarboxylate the 2,4- and 3,4-dimethoxy derivatives attests to the role of hydrogen bonding between substrate and enzyme. However, 3-methoxy-4-hydroxy-PA (“3-methoxy-tyrosine”), which possesses the para-OH necessary for the action of the bacterial enzyme, is decarboxylated only very slowly by S. jaecalis preparations; it is possible that the adjacent methoxy substituent hinders formation of the enzyme-substrate complex by exerting an attraction for the proton of the phenolic group (20) and by thus, in a sense, competing with the apoenzyme. In regard to side-chain structure, the necessity for an unsubstituted a-amino group has been confirmed with a new 3,4-DOPA derivative bearing a 2-cyanoethyl group. The presence of an alcoholic group on the p-carbon atom reduces the rate of decarboxylation considerably, but not completely (comparison of 3,4-DOPA and 3,PDOPS) by kidney (15) and adrenal medulla, as well as by the bacterial enzyme (4). SITMMARY 1. The substrate specificities of tyrosine decarboxylase of Streptococcus jueculis R and of dihydroxyphenylalanine (DOPA) decarboxylase of pig and guinea pig kidney have been studied. 2. The quantitative data reveal that interaction between substituents on the benzene ring of the substrates, as well as the position of the groups, affects the rate of decarboxylation. 3. Decarboxylation of 3,4-DOPA and 3,4-DOPS has been detected in the medulla of beef adrenal glands. 4. The RI values of the compounds studied have been determined. REFERENCES 1. HOLTZ, P., HEISE, R., AND LUDTKE, K., Arch. ezptl. Path. Pharmakol. 191, 87 (1938).

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DIHYDROXYPHENYLALANINE

2. GALE, E. F., Biochem. J. 34, 846 (1940). 3. EPPS, H. M. R., Biochem. J. 38, 242 (1944). 4. BLASCHKO, H., Biochim. et Biophys. Acta 4, 130 (1950). 5. UMBREIT, W. W., BELLAMY, W. D., AND GUNSALUS, I. C., Arch.

Biochem.

7,

155 (1945). 6. GREEN, D. E., LELOIR, L. F., AND NOCITO, V., J. Biol. Chem. 161,559 (1945). 7. SCHALES, O., AND SCHALES, S., Arch. Biochem. 24, 83 (1949). 8. DALOLIESH, C. E., AND MANN, F. G., J. Chem. Sot. 1947, 658. 9. BLASCHKO, H., HOLTON, P. AND STANLEY, G. H. S., Brit. J. Pharmacol. 3, 315 (1948). 10. STANLEY, G. H. S., Biochem. J. 44,373 (1949); cf. BLASCHKO, H.,AND STANLEY, G. H. S., Biochem. J. 42, iii (1948). 11. BLASCHKO, H., AND LANGEMANN, H., Biochem. J. 46, vii (1951). 12. GALE, E. F., Advances in Enzymol. 6, 1 (1946). 13. MCGILVERY, R. W., AND COHEN, P. P., J. Biol. Chem. 174, 813 (1948). 14. BLASCHKO, H., J. Physiol. 66, 50P (1939). 15. BLASCHKO, H., BURN, J. H., AND LANOEMANN, H., Brit. J. Pharmacol. 6, 431 (1950). 16. BEYER, K. H., in Chemical Factors in Hypertension. Advances in Chemistry, Series No. 2, American Chemical Society, Washington, D. C., 1950. 17. LANGEMANN, H., Brit. J. Pharmacol. 6, 318 (1951). 18. SCHAPIRA, G., Compt. rend. sot. biol. 140, 173 (1946). 19. JAMES, W. O., Nature 161, 851 (1948). 20. WULF, 0. R., LLIDDEL, U., AND HENDRICKS, S. B., J. Am. Chem. Sot. 66, 2287 (1936).