A NEW COFACTOR REQUIRED FOR THE ENZYMATIC CONVERSION OF PHENYLALANINE TO TYROSINE*

Transcription

1 A NEW COFACTOR REQUIRED FOR THE ENZYMATIC CONVERSION OF PHENYLALANINE TO TYROSINE* BY SEYMOUR KAUFMAN (From the Laboratory of Cellular Pharmacology, National Institute of Mental Health, United States Department of Health, Education, and Welfare, United States Public Health Service, National Institutes of Health, Bethesda, Maryland) (Received for publication, September 23, 1957) The enzymatic conversion of phenylalanine to tyrosine, which is described by Equation 1, is a complex reaction requiring at least two enzymes, TPNH and oxygen (3). (1) TPNH + H+ + O2 + phenylalanine -+ TPN+ + Hz0 + tyrosine The partial purification of these two enzymes,2 one extracted from rat liver and the other from sheep liver, has recently been described (3). In the course of further purification of the enzyme from rat liver, it was discovered that another cofactor, in addition to TPNH, was involved in Equation 1. The results of preliminary studies suggest an enzyme-catalyzed interaction between TPNH and the cofactor. The identity of the new cofactor has not yet been established, but it appears to be different from any of the known vitamins and coenzymes; none of those tested can replace the cofactor in the enzyme system under consideration. The cofactor has been purified approximately looo-fold from rat liver, and this communication is primarily concerned with the purification procedure and some of the properties of the new cofactor. EXPERIMENTAL Assay of Cofactor-The first indication that an unsuspected cofactor might be involved in this reaction came from attempts to purify the rat enzyme beyond that point which has already been described (3). It was found that a variety of fractionation procedures led to a very poor recovery of the enzyme units. Almost all of the lost activity of these fractions * Paper II of a series on biological hydroxylation reactions. Preliminary reports on some of this material have been presented (1, 2). 1 The following abbreviations are used: TPNf and TPNH, triphosphopyridine nucleotide and reduced triphosphopyridine nucleotide; DPNH, reduced diphosphopyridine nucleotide; CoA, coenzyme A. 2 Another enzyme system which catalyzes the conversion of phenylalanine to tyrosine, but which requires DPNH instead of TPNH, has been described (4). 931

2 932 CONVERSION OF PHENYLALANINE TO TYROSINE could be restored by the addition of a boiled extract prepared from rat liver. Rat liver enzyme fractions which showed a good dependence upon added cofactor were conveniently prepared by adsorption of the enzyme on calcium phosphate gel and elution with 0.1 N potassium phosphate buffer, ph 7.4. For the assay of the factor, therefore, rat liver enzyme which had been carried through this step was used. The conditions were the same as those of the standard assay (3), except that the time of incuba-.36 I I I I I I I I I I * I I I I I I I I I I I2.I ML. OF FACTOR FIG. 1. Stimulation of tyrosine formation by the cofactor. The assay conditions used were those described in the text. The cofactor preparation used was the concentrated aqueous fluid after precipitation with ethanol and ether. tion was increased to 60 minutes. An excess of the sheep liver enzyme was used, together with an amount of rat enzyme which could lead to the formation of approximately 0.3 pmole of tyrosine in the presence of saturating amounts of the cofactor. The relative instability of the rat enzyme necessitated a weekly redetermination of the amount of this enzyme to be used in the assay. The stimulation of the rate of tyrosine formation by various concentrations of factor3 is shown in Fig. 1. Aliquots of the fractions to be tested for factor activity were chosen so that the measurements were confined to the linear portion of the curve. Zero time controls, where trichloroacetic acid was added before any enzymes, were also included, since it was found that the crude liver fractions contained materials which 3 If rat enzyme which has not been carried through the calcium phosphate gel step is used, a maximal stimulation of 2- to 3-fold can be obtained on the addition of the cofactor.

3 S. KAUFMAN 933 contribute to the final color in the tyrosine assay. A unit of factor activity is defined as the amount which leads to the formation of an additional 1.0 pmole of tyrosine under the conditions of the standard assay. To rule out the possibility that the observed stimulation of tyrosine formation was an artifact restricted to this particular chemical determination of tyrosine, the stimulation was also measured (in one experiment) by following, with the aid of paper chromatography, the conversion of phenylalanine-cl4 to tyrosine-c14, as previously described (3). The results (Table I) indicate that the activity of the cofactor is the same when measured by these two different procedures. TABLE I Stimulation of Tyrosine Formation by Factor As Determined by Two Different Methods The system contained the following components (in micromoles): potassium phosphate buffer, ph 6.8, 100; TPNH, 1.0; phenylalanine-c14, 1.5; rat enzyme, 2.2 mg. of protein; sheep enzyme, 4.3 mg. of protein. The cofactor, added as indicated, was 0.4 ml. of the crude boiled rat liver extract. The final volume was made up with water to 1.25 ml. and incubated for 60 minutes at 25. For the radioactive determination, the tyrosine and phenylalanine were separated by paper chromatography as previously described (3). Tyrosine formed in absence of factor.. I presence of factor.. Method of tyrosine determination I I Calorimetric I Radioactive pvz& jmmole The purification of the factor was followed by determining organic matter by the dichromate oxidation procedure as described by Johnson (5). This value agreed well with a dry weight determination on the crude boiled liver extract before the introduction of any inorganic salts. In some of the purification steps, the optical density at 250 rnp was also followed. Puri&ation of Factor-A brief survey of various tissues indicated that this factor was apparently not widely distributed. Extracts4 prepared from the following tissues were found to be inactive: beef spleen, heart, brain, thyroid, kidney, pancreas, rabbit muscle, bakers yeast, brewers yeast,, Escherichia coli (strain 4157), rice bran, wheat, bran, and cabbage. Extracts from beef adrenal glands showed some activity. On the other hand, all liver extracts so far tested (rat, sheep, rabbit, beef) were active, * In each case the boiled extract was prepared in the same manner as will be described in detail for the rat liver preparation.

4 934 CONVERSION OF PHENYLALANINE TO TYROSINE but none of them was as active as the rat liver extract which contains to 0.4 units per ml. of boiled extract. It was therefore decided to purify the factor from boiled extracts of rat liver. All of the steps were carried out at 24 unless specified otherwise. All additions were made with mechanical stirring. Water distilled from glass was used throughout the procedure. The livers were removed and homogenized in a Waring blendor with 1.5 volumes of water, and the homogenate was slowly added to another 1.5 volumes of boiling water. After the addition was complete, the mixture was boiled for another minute and cooled as quickly as possible. The cooled homogenate was centrifuged for 15 minutes at 18,000 X g, and the residue was discarded. The opalescent, yellow supernatant fluid was adjusted to ph 1.6 to 1.7 with 1 N HzS04 and stirred for 20 minutes. The precipitate was centrifuged at 3000 X g for 20 minutes and the supernatant fluid brought to ph 6.15 with 2 N KOH added dropwise. 3 volumes of absolute ethanol and 7 volumes of ether were added slowly with stirring for 20 minutes. The mixture was centrifuged at 3000 X g for 15 minutes, and the residue was discarded. The supernatant fluid was concentrated almost to dryness under reduced pressure. The resulting concentrate was taken up in enough water so that the volume at this stage was one-tenth that of the initial extract. This fraction, as well as the boiled extract, is stable for months when stored at ml. of this fraction were adjusted to ph 6.5 to 7.0 with 1 N KOH and put on a 2.4 X 4.2 cm. column of Dowex 1 (Cl-) (10 per cent cross-linked, 100 to 200 mesh) at room temperature. The effluent fluid was saved, and the column was washed with 50 ml. of water. The washings and the effluent were combined and adjusted to ph 3.0 with 2 M acetic acid. The acidified solution was cooled to 4 and applied to a 2.4 X 7.0 cm. column of Dowex 50-X2 in the sodium form (200 to 400 mesh) in the cold. After all of the solution had been added, the column was eluted with 0.05 M potassium phosphate buffer, ph ml. fractions of the eluate were collected. Usually, the activity begins to come off after 70 ml. of the eluting fluid have been added to the column. The above fractions were combined and stored at 4. The over-all procedure usually leads to an 800 to looo-fold purification with a 20 to 30 per cent yield of units of activity. Properties of Cofactor-The factor is completely destroyed by ashing and is exceedingly labile to alkali. The activity is completely lost when it is treated at 50 for 10 minutes in 0.1 N KOH. The loss on alkali treatment is not reversed by a subsequent incubation with glutathione or ascorbic acid, nor is it prevented by carrying out the incubation in an evacuated Thunberg tube. On the other hand, the factor is relatively stable to acid, surviving 10 minutes at 50 in N HCl. However, at loo, the same con-

5 S. KAUFMAN 935 ditions lead to essentially complete loss of activity. Although the crude boiled liver extract, as well as the ethanol-ether supernatant fluid, can be stored for months at - 20, the Dowex 50 eluates lose activity very rapidly under these conditions. They are somewhat more stable at 4. The cofactor is extractable with butanol although the partition coefficient is unfavorable. In contrast to butanol, no detectable activity can be extracted into ethyl acetate or ether from neutral or acid solutions. Compounds Tested for Cofactor Activity-The following compounds have been tested and found to be inactive (the final concentration in the assay tube is given in micromoles or, when specified, in micrograms) ascorbic acid (0.5 to 2.5), glutathione (l.o), thiamine (0.5), vitamin B, (100 r), cysteine (2.0), epinephrine (O.l), pyridoxal phosphate (0.5), Fe& (), 3,4-dihydroxyphenylalanine (0.1)) riboflavin monophosphate ()) thiamine monophosphate (0.5), folic acid (100 +y), acetylcholine (O.l), choline (0.1)) lipoic acid (1.0)) CoA (0.3)) N-methylnicotinamide (1.0)) diphosphopyridine nucleotide (0.06), thyroxine (O.l), triiodothyronine (O.l), DPNH (0.8)) biotin (0.5)) methylthioadenosine (0.1)) adenosine 2-phosphate (0.1)) adenosine 3-phosphate (O.l), adenosine 5-phosphate (O.l), adenosine (O.l), inosine (O.l), uridine (O.l), and cytidine (0.1). In addition to these compounds tested singly, ascorbic acid and FeClz (6) and ascorbic acid and vitamin Blz (7) were tested together and found to be inactive. Enzymatic Studies Dependencies in Presence of Factor--It was found that the dependencies already described (3) for the conversion of phenylalanine to tyrosine were unchanged by the presence of the factor. Both enzymes, TPNH, phenylalanine, and oxygen were still required, and the stimulation was the same whether TPNH was added in stoichiometric amounts or generated from catalytic amounts of TPN+ in the presence of glucose and an excess of glucose dehydrogenase. Lag Period: Studies with Unresolved Enzymes-As previously described (3), the conversion of phenylalanine to tyrosine can be followed by measuring the phenylalanine-dependent disappearance of TPNH spectrophotometrically. It was found that there was a lag of several minutes before the rate of TPNH oxidation in the presence of phenylalanine exceeded that of the control. Because it was felt that the lag period might provide a clue to the primary reaction in the sequence which leads to tyrosine formation, this phenomenon was studied in some detail. In particular, conditions were sought which might shorten or eliminate the lag period. In Table II, the results of experiments carried out with unresolved enzyme fractions are summarized. It has already been reported (3) that the lag period could be shortened by a preliminary incubation of both enzymes

6 936 CONVERSION OF PHENYLALANINE TO TYROSINE aerobically with TPNH. For comparison, the results of a separate experiment performed under these conditions are included in Table II (Ex- TABLE Effect of Preliminary Incubation under Various Conditions on Lag Period The reactions were carried out in 1.0 cm. Beckman cuvettes containing the following components (in micromoles): potassium phosphate buffer, ph 6.8, 100; n-phenylalanine, 2.0; TPNH, 0.13; rat enzyme, 2.4 mg. of protein; sheep enzyme, 1.5 mg. of protein. Final volume, 1.0 ml. Incubations carried out at room temperature. As an example of the manner in which all the anaerobic preincubation experiments were carried out, the details of the procedure used in Experiment 2 will be outlined. Two Thunberg tubes, each containing sheep enzyme in the side bulb and TPNH and the buffer in the main compartment, were evacuated for 2 minutes with a mechanical pump. The tubes were closed, and the contents of the side bulbs were tipped in. After a 15 minute incubation at room temperature, the tubes were opened and the contents were immediately pipetted into two Beckman cuvettes containing rat enzyme and rat enzyme plus phenylalanine, respectively, and the rate measurements were begun. The reported values in each case have been corrected for any TPNH oxidation which occurred in the absence of phenylalanine (Cuvette 1). Experiment NC Conditions of incubation No preincubation Preincubation with sheep enzyme Anaerobic, with TPNH without TPNI Aerobic, with TPNH Anaerobic, with TPNH followed by exposure to air II Ratio, initial rate* final rate Rate (A optical density to 3 min 0.33 to 6 min 4.33 X 1000) per min. to 9 min to 20 min Preincubation with rat enzyme Anaerobic, with TPNH Aerobic, with TPNH Preincubation with both enzymes Aerobically with TPNH * First 6 minutes. periment 8). Subsequently, it was discovered that oxygen is not required during this preincubation. Moreover, if the preincubation is carried out

7 S. KAUFMAN 937 under anaerobic conditions, the rat enzyme is no longer needed (Experiment 2) and this (it may be emphasized again) differs from the requirements for aerobic preincubation. Anaerobically, TPNH is still required (Experiment 3). If the anaerobic preincubation is followed by a brief exposure to air, the lag period is fully restored (Experiment 5). These results suggest the possibility that TPNH interacts with the sheep enzyme or a cofactor bound to that enzyme. That this interaction may represent a reduction of the cofactor by TPNH is made probable by the results of Experiment 5, Table II, where it was found that the reaction which takes x I? ( cj without factor IO MINUTES FIG. 2. Stimulation of the phenylalanine-dependent oxidation of TPNH by the cofactor. The system contained the following components (in micromoles) : potassium phosphate buffer, ph 6.8, 100; n-phenylalanine, 2.0; TPNH, 0.13; rat enzyme, 1.2 mg. of protein; sheep enzyme, 0.4 mg. of protein. The cofactor, added as indicated, was 0.1 ml. of an eluate from a Dowex 50 column in the sodium form. Silica cells; light path, 1.0 cm. The final volume was made up to 1.0 ml. with water. In each case, a control in which phenylalanine was omitted was included, and the reported values have been corrected for any oxidation of TPNH which occurred in the absence of phenylalanine. place during the anaerobic preincubation of TPNH with the sheep enzyme can be reversed by a short exposure to air.k Lag Period: Experiments with Resolved Enzymes--Direct evidence in support of the idea that a cofactor is indeed involved in the reaction (re- 5 A consideration of the maximal amounts of cofactor which could be present in the system suggests a rapid turnover of this component in the catalytic cycle which leads to tyrosine formation. The duration of the lag period is far greater than the probable turnover time of the cofactor. One possible interpretation of the data concerning the lag period is that, during the preincubation with TPNH and the sheep enzyme, the cofactor is slowly converted from a catalytically inactive form to an active form, which then participates in the reaction involving TPNH, phenylalanine, and oxygen. Other interpretations of these data, assuming the involvement of inhibitors, are also possible.

8 938 CONVERSION OF PHENYLALANINE TO TYROSINE actions) which leads to a shortening of the lag period has been obtained from experiments which employ the isolated cofactor and rat and sheep enzymes6 which have both been essentially completely resolved with respect to the cofactor. In a system containing both the rat and sheep enzymes, it was found that the addition of the cofactor, although greatly stimulating the rate of the phenylalanine-dependent oxidation of TPNH, did not eliminate the lag period (Fig. 2). Moreover, in contrast to the earlier observation with TABLE Requirement for Factor in Reduction of Lag Period The conditions were the same as those described in Table II, except for the amount of sheep enzyme, which was 0.6 mg. of a fraction which had been resolved with respect to the cofactor. The cofactor, added as indicated, was 0.1 ml. of a Dowex 50 eluate. All of the incubations were carried out with sheep enzyme under anaerobic conditions. Conditions of preincubation (anaerobic in presence of sheep enzyme) No added factor Factor added * First 6 minutes No preincubation Preincubation No preincubation Preincubation Same as Experiment 4 but no TPNH Experiment NO. III Ratio, initialrate final rate I Rate (A optical density X 1000) per min. 0 to 3 min T 3 to 6 min to 9 min to 20 min cruder sheep enzyme, the anaerobic preliminary incubation of the purified sheep enzyme with TPNH was no longer effective in shortening the lag period; the addition of the factor was required. As shown in Experiment 5, Table III, in the presence of added factor, TPNH is still needed. The results of these experiments are consistent with the view that some kind of interaction between TPNH and the factor takes place, and that this reaction is an early and perhaps the primary step in the complex sequence which ultimately leads to tyrosine formation. More direct evidence for a stoichiometric reaction between TPNH and the factor must await the availability of larger amounts of the purified factor. 6 Kaufman S., and Levenberg, B., unpublished procedure.

9 S. KAUFMAN 939 SUMMARY The partial purification and some of the properties of a new cofactor which is involved in the conversion of phenylalanine to tyrosine have been described. The factor does not replace any of the other components of the system; reduced triphosphopyridine nucleotide (TPNH), phenylalanine, oxygen, and two enzyme fractions are still required. Experiments which suggest an enzyme-catalyzed interaction between TPNH and the cofactor have been discussed. The author wishes to thank Mr. Richard Funk for skilful technical assistance during the course of this work. BIBLIOGRAPHY 1. Kaufman, S., Biochim. et biophys. acta, 23, 445 (1957). 2. Kaufman, S., Federation Proc., 16, 203 (1957). 3. Kaufman, S., J. Biol. Chem., 226, 511 (1957). 4. Mitoma, C., Arch. Biochem. and Biophys., 60,476 (1956). 5. Johnson, M. J., J. Biol. Chem., 181, 707 (1949). 6. Udenfriend, S., Clark, C. T., Axelrod, J., and Brodie, B. B., J. Biol. Chem., 208, 731 (1954). 7. Uchida, M., Suzuki, S., and Ichihara, K., J. Biochem., Japan, 41, 41 (1954).

10 A NEW COFACTOR REQUIRED FOR THE ENZYMATIC CONVERSION OF PHENYLALANINE TO TYROSINE Seymour Kaufman J. Biol. Chem. 1958, 230: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-1