synthesis of phenylacetylglutamine by human tissue

SYNTHESIS OF PHENYLACETYLGLUTAMINE BY HUMAN TISSUE*

BY KIVIE MOLDAVE AND ALTON MEISTER

(From the Department of Biochemistry, Tufts University School of Medicine, Boston, Maseachueetts)

(Received for publication, June 25, 1957)

The formation of phenylacetylglutamine is catalyzed by human tissues, but apparently not by those of a large number of other mammals, including the dog, rat, monkey, rabbit, horse, sheep, and cat (2-7). Oral adminis- tration of phenylacetate and benzoate to mammals characteristically results in the excretion of phenylacetylglycine and benzoylglycine, respectively. Man and possibly also the chimpanzee (8) are unique in that orally administered phenylacetate is excreted as phenylacetylglutamine. It has also been found that phenylacetylglutamine is a normal constituent of human urine (9). The phenylacetyl moiety of phenylacetylglutamine apparently arises from phenylalanine; thus, increased excretion of phenylacetylglutamine is observed in patients with phenylpyruvic oligophrenia (9-11). These observations suggest that the metabolic pathways of phenylalanine and glutamine in man (and perhaps the higher primates) are unique in this respect as compared with other mammals.

The present investigation is concerned with an enzyme system present in human liver and kidney that catalyzes the synthesis of phenylacetylglutamine. The experimental evidence indicates that at least two enzymatic entities are involved in phenylacetylglutamine formation. One of these, referred to here as the acylating enzyme, catalyzes the formation of phenylacetylglutamine from phenylacetyl CoA’ and L-glutamine. The other enzyme activity catalyzes the activation of phenylacetic acid by ATP and CoA-SH; such activation appears to involve the intermediate formation of phenylacetyl AMP, and the subsequent formation of phenylacetyl CoA from phenylacetyl AMP and CoA-SH. Related studies on the formation of benzoylglycine from benzoic acid and glycine have also been carried out.

* This investigation was supported in part by research grants (Nos. A-1397 and H-2319) from the National Institutes of Health, United States Public Health Service, and from the National Science Foundation. A preliminary report of this research has appeared (1).

1 The following abbreviations are used in this paper: coenzyme A, CoA or CoA- SH; adenosine triphosphate, ATP; adenosine 5’-phosphate, AMP; phenylacetylglutamine, PAG; acyl-adenylic acid anhydride, acyl-AMP.

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464 PHENYLACETYLGLUTAMINE SYNTHESIS

EXPERIMENTAL

Synthesis of Radioactive L-Glutamine-Randomly labeled CY-L-glutamine was prepared from CY4-L-glutamic acid by a modification (12)2 of the procedure of Levintow and Meister (13). CY4-L-Glutamate (6 mg. ; 300 MC.) was incubated at 37” for 4 hours with ATP, magnesium chloride, ammonium chloride, mercaptoethanol, imidazole buffer, and a purified glutamine synthesis enzyme preparation obtained from peas (14). Unlabeled L-glutamine (54 mg.) was added and the radioactive glutamine was purified on columns of Amberlite XE-64 and IR4B ion exchange resins (12). The yield of pure C4-L-glutamine was 45 mg.; the specific activity was 1.1.5 )( 1Os c.p.m. per mg. (counted with a thin mica window tube).

Synthesis of Acyl CoA and Acyl-AMP Derivatives-Phenylacetyl CoA, benzoyl CoA, isobutyryl CoA, isovaleryl CoA, indoleacetyl CoA, and p- hydroxyphenylacetyl CoA were prepared by treatment of CoA-SK with the corresponding acid anhydrides (15, 16). The acid anhydrides were prepared by refluxing an ethereal solution of the acid chloride with an equimolar quantity of the corresponding silver salt for several hours; after removal of silver chloride by filtration, the anhydride was obtained by evaporation of the ether. Phenylacetic, p-hydroxyphenylacetic, benzoic, and indoleacetic anhydrides were crystallized from ether-petroleum ether; isobutyric and isovaleric anhydrides were obtained as oils.

The acyl CoA derivatives were prepared as follows: CoA-SH (100 pmoles), potassium bicarbonate (150 Lcmoles), and 300 pmoles of the appropriate anhydride were placed in a glass-stoppered test tube containing 2 ml. of water. The tube was flushed with nitrogen and subsequently shaken vigorously at 37” for 5 hours. The mixture was adjusted to pH 2 with dilute hydrochloric acid and extracted four to six times with 3 ml. portions of ether; it was demonstrated that the final ether extract did not contain anhydride as judged by the hydroxamic acid reaction (17). The residual aqueous solution contained between 60 and 80 pmoles of acyl CoA derivative. The solution was neutralized by the addition of solid sodium bicarbonate.

Phenylacetyl AMP and benzoyl AMP were prepared by the general procedure of Avison (18). AMP (1 mmole) was dissolved in 10 ml. of 50 per cent aqueous pyridine. Phenylacetic (or benzoic) anhydride (6 mmoles) was added, and the mixture was shaken at 0” for 30 minutes. The mixture was then extracted six times with 10 ml. of cold ether and 0.5 ml. of 2 M potassium bicarbonate was added to the aqueous layer followed by 150 ml. of ice-cold acetone. The mixture was placed at -20” for 18 hours.

* The authors thank Dr. Leon Levintow for advice concerning the modified procedure for the isolation of L-glutamine.


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K. MOLDAVE AND A. MEISTER 465

The product was obtained by centrifugation, and after being washed three times with cold acetone by centrifugation, the precipitate was dried in vacua over phosphorus pentoxide. The product was obtained in 60 to 80 per cent yield and was 90 to 95 per cent pure, based on the hydroxamic acid reaction.

Other Compounds-Disodium ATP was obtained from the Sigma Chem- ical Company and AMP was purchased from Schwarz Laboratories, Inc. CoA-SH was obtained from the Sigma Chemical Company. Phenylacetyl- L-glutamine and the N-acyl derivatives of the other amino acids employed were prepared by allowing the amino acids to react with the appropriate acid chloride (2).

Analytical Procedures-Phenylacetylglutamine formation was determined as follows: The enzymatic reaction mixtures were brought to 70 per cent with respect to ethanol by the addition of absolute ethanol, and the precipitated protein was removed by centrifugation. An aliquot of the supernatant solution was chromatographed on paper strips (Whatman No. 3 MM). A solvent consisting of n-butanol-water-acetic acid (4: 1: 1) was employed for most of the chromatographic determinations. This solvent system separated PAG from pyrrolidonecarboxylic acid, a-ketoglutaric acid, glutamic acid, and glutamine. The Rr values for PAG and the other compounds were, respectively, 0.83, 0.56, 0.44, 0.25, and 0.25 (19). In tert-butanol-formic acid-water (70: 15 : 15)) the respective Rr values were 0.85, 0.76, 0.71, 0.45, and 0.34. Other solvents which gave a satisfactory separation of PAG were 77 per cent ethanol, methanol-pyridine-water (80 : 4 : 20)) and n-propanol-pyridine (80: 20). Glutamine and glutamic acid, which were not separated from each other in the n-butanol- water-acetic acid system, were resolved in other solvents such as tert-

butanol-formic acid. Following chromatography, 1 cm. sections of the paper strips were cut out and counted with a thin mica window tube.

Experiments in which human liver homogenates were incubated with Cl’-L-glutamine and phenylacetyl CoA led to the formation of a radioactive compound which was chromatographically indistinguishable from an authentic sample of PAG in the five paper chromatographic systems. Within experimental error, the amount of PAG recovered in each system was the same. Thus, in the five solvent systems described above, the conversion of glutamine to PAG by human liver homogenate system was 5.9, 5.0, 4.8, 5.4, and 5.5 per cent, respectively. A sample of radioactive PAG (700 c.p.m.), obtained by elution from paper chromatograms, was autoclaved in a sealed tube at 18 pounds pressure for 1 hour in 6 N hydrochloric acid. The solution was evaporated to dryness and the residue was dissolved in water and transferred to a paper chromatographic strip which was developed with the n-butanol-acetic acid-water solvent; virtually all


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(676 c.p.m.) of the radioactivity was recovered in the glutamic acid area. Similar experiments were carried out with Cl*-glutamine and phenylacetate and also with Cl*-phenylacetate and L-glutamine. In experiments in which CL*-phenylacetate was employed, acid hydrolysis of the Cl*-PAG formed gave a single radioactive product which exhibited chromatographic behavior identical with that of phenylacetic acid.

The other acyl amino acids were located on paper chromatograms by a modification of the procedure of Rydon and Smith (20, 21). These were readily separated from glutamine, glutamic acid, cr-ketoglutaric acid, and pyrrolidonecarboxylic acid, and exhibited Rp values which were greater than that of PAG in the n-butanol-water-acetic acid and the tert-butanol- formic acid-water solvents.

Determinations of free sulfhydryl groups were performed by the procedure of Mahler et al. (22).

Preparation of Human Acylating Enzyme-Samples of human liver and kidney obtained at autopsy were used as sources of the enzyme activity that catalyzes transfer of the phenylacetyl moiety of phenylacetyl CoA to L-glutamine (19). The tissues were obtained from patients who did not exhibit signs of hepatic or renal disease prior to death or at post-mortem examination.3 The tissues were obtained 5 to 15 hours after death and were homogenized with 3 volumes of cold calcium-free Krebs-Ringer phosphate buffer solution (pH 7.4) (23) in a Potter-Elvehjem glass homogenizer. In general, the activity of such liver preparations was approximately 50 to 60 per cent of that exhibited by similar preparations obtained from liver samples removed surgically.3 The homogenates were centrifuged at 144,000 X g at 2O in a Spinco model L preparative ultracentrifuge for 1 hour. The clear supernatant solution (Stage 1, see Table I) contained almost all of the acylating activity and was fractionated with solid ammonium sulfate as follows: 20 gm. of solid ammonium sulfate were added per 100 ml. of supernatant solution and allowed to stand for 30 minutes at 4’. The resulting precipitate was removed by centrifugation and dis- carded, and additional ammonium sulfate (20 gm. per 100 ml.) was added to the supernatant solution. The precipitate, which contained most of the activity, was dissolved in the minimal quantity of cold distilled water and dialyzed overnight against running tap water at 4’ (Stage 2). A 2 per cent solution of protamine sulfate was added dropwise to the dialyzed solution until no further turbidity occurred upon the addition of protamine. The precipitate, which was inactive, was removed by centrifugation. The supernatant solution was brought to pH 8 with dilute sodium hydroxide and centrifuged to remove a small inactive precipitate (Stage 3). The

3 The authors are indebted to Dr. Charles G. Child for providing the biopsy samples, and to Dr G. J. Gherardi for assistance in obtaining the autopsy material.


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enzyme solution was heated at 50” in a wat,er bath for 5 minutes, cooled rapidly, and the heat-denatured protein was removed by centrifugation (Stage 4). The supernatant solution was adjusted to pH 5 with 0.2 N

acetic acid, centrifuged to remove an inactive precipitate, and then adjusted to pH 8 with 0.1 M potassium hydroxide (Stage 5). Active enzyme was precipitated from this solution by dropwise addition of a 2 per cent solution of ribose nucleic acid previously adjusted to pH 5.5 with sodium hydroxide. The precipitate was isolated by centrifugation and was then extracted by vigorous stirring with 2 volumes of 0.1 M sodium phosphate buffer at pH 7.4 ftir 1 hour. The extract was clarified by centrifugation (Stage 6).

TABLE I

Purification of Acylating Enzyme Activities

Preparation* Cl’-PAG formed per hr. per mg. of proteint

Liver I Kidney

Homogenate. Stage l......

(( 2 ...... d‘ 3 ...... dL 4 ...... 6‘ 5 ...... “ 6 ......

. 118 148

13 71 98

198 212 265 642

* Experimental details are given in the text. t Enzyme activity was determined in a system consisting of 1.26 pmoles of W-L-

glutamine, 2.25 pmoles of phenylacetyl CoA, 150 amoles of sodium phosphate (pH 8.2), and 0.7 ml. of the enzyme fraction in a final volume of 1 ml.; the mixtures were incubated for 1 hour at 37.5”.

Although considerable activity was lost by precipitation with nucleic acid, a significant purification was achieved. The enzyme was stable for several months when stored frozen at -15’; on the other hand, more than 50 per cent of the activity was lost on lyophilization. Results of assays on the various fractions obtained in the course of purification are summarized in Table I. The enzyme activity was purified approximately 90-fold from liver and about 50-fold from kidney; however, the kidney preparation exhibited a higher specific activity than did that obtained from liver.

Preparation of Activating Enzyme System-Acetone powders of human and beef liver mitochondria were prepared as described by Mahler et al. (22) and Schachter and Taggart (16). The acetone powders were extracted for 30 minutes at 4” with 10 volumes of 0.02 M potassium phosphate at pH


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468 PHENYLACETYLG’LUTAMINE SYNTHESIS

7.5. The extract obtained from human liver mitochondria was used in this form. The extract prepared from beef liver mitochondria was fractionated with ammonium sulfate and dialyzed as described (16).

Results

Studies on Acyluting Enzyme System-It was observed early in these studies that incubation of human liver homogenates with C”-n-glutamine and phenylacetate or C4-L-glutamine, phenylalanine, and cu-ketoglutarate led to the synthesis of a radioactive product which was identified as PAG. It was also found that PAG synthesis was increased ‘in the homogenate systems by addition of CoA-SH and ATP, suggesting the participation of phenylacetyl CoA as an intermediate in PAG synthesis. The observation that synthetic phenylacetyl CoA served in place of CoA-SH, ATP, and phenylacetate in the PAG-forming system (19) was consistent with this hypothesis and made possible the purification of the acylating enzyme activity from human liver and kidney as described above (see Table I).

The formation of PAG from phenylacetyl CoA and r,-glutamine was directly proportional to enzyme concentration over the range investigated (1 to 12 mg. of total protein per ml.). PAG was not formed in the absence of human enzyme, in experiments with boiled enzyme preparations, or with preparations obtained from beef or rat liver. Several other studies carried out with the purified human kidney acylating enzyme preparation are described in Fig. 1. The formation of PAG was studied over the pH range 5.5 to 10; very little activity was observed below pH 7.4, while the reaction proceeded at optimal rate at relatively alkaline values of pH. It was also found that the enzyme preparation catalyzed the synthesis of phenylacetylglycine at a slow rate (Fig. 1, A). The curve describing the time-course of the formation of PAG at pH 8.2 (Fig. 1, B) levels off after 4 hoursof incubation, at a point corresponding to approximately 50 per cent conversion of the added glutamine to PAG. That the reaction did not go to completion may probably be ascribed to partial destruction of phenylacetyl CoA and also to inactivation of enzyme; furthermore, some conversion of glutamine to pyrrolidonecarboxylic acid, cr-ketoglutarate, and glutamic acid occurred during the long incubation period. The effect of L-

glutamine concentration on PAG formation is described in Fig. 1, C; under the conditions employed, maximal activity was observed with about 50 pmoles of L-glutamine. Studies in which phenylacetyl CoA concentration was varied indicated that saturation of the system was achieved with somewhat lower levels of the CoA derivative (Fig. 1, 0).

The finding that the enzyme preparation also catalyzed the formation of phenylacetylglycine is of interest. It was observed that glycine at fairly high concentrations (0.138 M) decreased formation of PAG from CY4-gluta-


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mine and phenylacetyl CoA by approximately 35 per cent and that, in the presence of 0.055 M glutamine, the synthesis of phenylacetylglycine was

5 6 7 8 910 PH HOURS

c 2 1000 0 a. 800

*’

-* 600

x 400

E' 200 i

0 y21i L- %h&

FIG. 1. Studies with the purified kidney acylating enzyme preparation. A, pH- dependence of the formation of phenylacetylglutamine, Curve 1, and phenylacetylglycine, Curve 2. The reaction mixtures consisted of 2.25 pmoles of phenylacetyl CoA, 1.26 rmoles of CWL-glutamine or 0.96 pmole of CWglycine, 1 mg. of enzyme (Stage 3, Table I), and 150 Mmoles of buffer in a final volume of 1.0 ml.; incubated at 37.5” for 1 hour. Sodium acetate and sodium phosphate buffers were used below pH 8.2 and tris(hydroxymethyl)aminomethane and sodium borate buffers were employed at values of pH greater than 8.2. B, time-course of the formation of phenylacetylglutamine from phenylacetyl CoA and glutamine. The reaction mixtures consisted of 1.26 pmoles of CP-L-glutamine, 2.25 cmoles of phenylacetyl CoA, 150 amoles of sodium phosphate (pH as indicated), and 1 mg. of enzyme in final volume of 1.0 ml.; incubated at 37.5”. C, effect of glutamine concentration on phenylacetylglutamine formation. The reaction mixtures consisted of 2.25 rmoles of phenylacetyl CoA, 150 pmoles of sodium phosphate (pH 8.2)) 1 mg. of enzyme, 1.26 pmoles of W-L-

glutamine, and Cl*-L-glutamine to give the indicated concentrations in a final volume of 1.0 ml.; incubated for 1 hour at 37.5”. D, effect of phenylacetyl CoA concentration on phenylacetylglutamine formation. The reaction mixtures consisted of 1.26 pmoles of Cia-L-glutamine, 150 amoles of sodium phosphate (pH 8.2), 1 mg. of enzyme, and phenylacetyl CoA as indicated in a final volume of 1.0 ml.; incubated for 1 hour at 37.5”.


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decreased about 15 per cent; however, these findings do not permit definite conclusions. The question as to whether the synthesis of PAG and that of phenylacetylglycine are catalyzed by the same enzyme remains for future study. It is possible that the human enzyme preparations contain several related enzymatic activities; as described below, these preparations also catalyzed the formation of benzoylglycine from benzoyl CoA and glycine.

The synthesis of PAG from phenylacetyl CoA was accompanied, within experimental error, by equimolar formation of free sulfhydryl groups. Similar studies were carried out with benzoyl CoA and glycine; here also, a close relationship was found betlveen formation of the acylamino acid and of free sulfhydryl groups (Table II).

TABLE II

Release of Free Sulfhydryl Groups and Synthesis of Acylamino Acids from Acyl CoA Derivatives*

Acyl CoA derivative Amino acid I -SH W-AC

r lamino acid

ormed

Phenylacetyl CoA 42 “ “ CWGlutamine 250 230

Bensoyl CoA 23 “ “ Cl’-Glycine 162 110

* The reaction mixtures consisted of 0.42 rmole of L-glutamine-R-CL’ (1.15 X 10’ c.p.m. per mg.) or 0.15 pmole of glycine-l-C?* (4.8 X 10” c.p.m. per mg.), 50 rmoles of sodium phosphate (pH 8.2), 4 mg. of human kidney enzyme from Stage 3, Table I, and 0.75 rmole of phenylacetyl CoA or 0.72 pmole of benzoyl Co.4 in a final volume of 0.6 ml.; incubated for 1 hour at 37.5”.

Spec@city of Human Acyluting Enzyme System-The observation that the purified human kidney enzyme preparation catalyzed formation of phenylacetylglycine and benzoylglycine as well as PAG synthesis led us to test other acyl CoA derivatives and amino acids in this system. In par- ticular, it seemed desirable to examine acyl CoA derivatives that might arise in the course of metabolism of certain amino acids, such as tyrosine, valine, and leucine. A recent report (24) describing the presence of indole- acetylglutamine in human urine prompted us to test indolylacetyl CoA. However, as summarized in Table III, none of these acyl CoA derivatives was active in acylating glutamine, and only phenylacetyl CoA and benzoyl CoA were active in the acylation of glycine. Similar results were obtained with the purified human tissue preparations.

Experiments were also carried out with glutamic acid, phenylalanine, and leucine. None of these amino atids was acylated in the presence of phenylacetyl CoA. A rat liver preparation catalyzed the formation of


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Ii. MOLDAVE AND A. MEISTER 471

TABLE III

Specificity of Acylating Enzyme System with Respect to Acyl CoA Derivatives’

Acyl CoA derivative

Phenylacetyl CoA. Isobutyryl CoA. Isovaleryl CoA. Benzoyl CoA. Indolylacetyl CoA. p-Hydroxyphenylaeetyl CoA

Cl‘-A&amino acid formed

C~QJ3utamine

Liver Kidney --

9tI#molcr mpmolcs

40.0 102.0 0 0

0 0

0 0

0 0

0 0

0.Glycine

Liver Kidney

m~malcs qmder

4.8 15.4 0 0

0 0

58.0 86.0

0 0

0 0

* The reaction mixtures consisted of 0.42 rmole of CWL-glutamine or 0.32 amole of Cl’-glycine, acyl CoA derivative, and human kidney or liver enzyme from Stage 1, Table I (8 mg. of protein), in a final volume of 0.33 ml.; incubated for 1 hour at 37.5”. The concentrations of acyl CoA derivatives were as follows: phenylacetyl CoA, 0.75 pmole; isobutyryl CoA, 0.9 amole; isovaleryl CoA, 0.63 pmole; benzoyl CoA, 0.72 #mole; indolylacetyl CoA, 0.44 pmole; and p-hydroxyphenylacetyl CoA. 0.75 pmole .

TABLE IV

Specificity of Acylating Enzyme System with Respect to Amino Acid*

Source of enzyme

Human kidney L-Glutamine-R-0’ ‘I “ Glycine-I-0 “ ‘I L-Glutamic acid-R-C*’ “ “ nn-Rhenylalanine-3-W “ “ nn-Leucine-2-W

Rat liver Glycine-1-U’ ‘I “ n-Glutamine-R-C**

Labeled substrate Cl’-Acyhmino acid formed

WQ&tnOkS

2.36

34 0 0 0

114 0

* The reaction mixtures consisted of 2.25 pmoles of phenylacetyl CoA, 150 amoles of sodium phosphate (pH 8.2), radioactive amino acid, and either 1 mg. of human kidney enzyme or 4 mg. of rat liver enzyme from Stage 3, Table I, in final volume of 1 ml.; incubated for 1 hour at 37.5”. The concentrations of radioactive amino acids were as follows: glutamine (1.15 X 10’ c.p.m. per mg.), 1.26 pmoles; glycine (4.8 X 10d c.p.m. per mg.), 0.96 pmole; glutamic acid (2.5 X 10’ c.p.m. per mg.), 4.2 #moles; phenylalanine (5.0 X lOa c.p.m. per mg.), 4.5 pmoles; and leucine (2.0 X 10’ c.p.m. per mg.), 3.9 bmoles.

phenylacetylglycine but did not catalyze PAG formation from glutamine and phenylacetyl CoA (Table IV).


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Properties of Activating Enzyme System-Preparations of human liver mitochondria catalyzed the formation of PAG from glutamine, phenylace-

TABLE V Activation of Phenylacetate and Benzoate*

Additions

Phenylacetate + CoA-SH + ATP “ + “ + “ “ + ATP “ + CoA-SH

CoA-SK + ATP Phenylacetate + CoA-SH + ATP

“ + “ + “ “ + “ + “

Phenylacetyl AMP + CoA-SH “ “ + CoA-SH

+ “ Benzoaie + CoA-SH + ATP#

“ + “ + I1 ‘I + “ + “ r, + “ + I‘

Benzoyl AMP + CoA-SHg “ “ + “ “ “ 0 ‘, “ + CoA-SHQ

CoA-SH$

Enzyme preparationst

Human liver nitochondrial

enzyme -~

+

+

+

+

Z*

+I

+

z

+

:r

+t +

+

+

+

Human kidney mzyme wage

3, Table I)

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

T Acylamino acid

formed

m~mo1c.v

34.0 36.0

1.7 3.3 0

15.0 0 0

25.0 3.0

22.0 5.3

29.0 28.0 43.0 60.0 18.0 25.5

1.0 2.1 0

* The reaction mixtures consisted of MgCll (0.77 pmole), sodium phosphate, pH 8.2 (50 rmoles), glutathione (4.9 rmoles), Cl’-L-glutamine (0.42 amole), and, as indicated, CoA-SH (0.13 pmole), ATP (2.5 rmoles), phenylacetate (5 pmoles), benzoate (10 #moles), phenylacetyl AMP (0.46 rmole), benzoyl AMP (0.35 pmole), human liver mitochondrial preparation (6 mg.), and human kidney acylating enzyme preparation from Stage 3, Table I (7.7 mg.), in a final volume of 0.7 ml.; incubated for 2 hours at 37.5”.

t Enzyme additions are denoted by plus signs. $ Beef liver mitochondrial preparation, 8 mg. 0 Glycine-1-C” (0.32 pmole) was substituted for glutamine.

tate, ATP, and CoA-SH; these preparations also catalyzed PAG synthesis from phenylacetyl CoA and glutamine. In contrast, the purified human acylating enzyme preparations were active in catalyzing only the latter reaction. As indicated by the data given in Table V, omission of ATP or of CoA-SH resulted in a very marked reduction in the formation of PAG


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from phenylacetate and glutamine by the human liver mitochondrial preparation. Furthermore, addition of the acylating enzyme preparation did not increase PAG formation, indicating that the mitochondrial preparation possessed a considerable level of acylating activity. A preparation obtained from beef liver mitochondria catalyzed PAG formation from phenylacetate, CoA-SH, and ATP in the presence, but not in the absence, of human acylating enzyme. These results indicate that, although beef liver mitochondrial preparations are capable of activating phenylacetate (cf. Schachter and Taggart (25)), such preparations are incapable of catalyzing the transfer of the phenylacetyl group to glutamine.

Human liver mitochondrial preparations also catalyzed the formation of benzoylglycine from benzoate, glycine, ATP, and CoA-SH (Table V). Evidence for the participation of benzoyl CoA as an intermediate in this reaction has been reported (16).

The participation of phenylacetyl AMP in PAG synthesis is described in Table V. It was found that phenylacetyl AMP could serve in place of phenylacetate and ATP in the human liver mitochondrial system. How- ever, phenylacetyl AMP was active only in the presence of CoA-SH. When CoA-SH was omitted, relatively little PAG was formed, a finding consistent with the belief that phenylacetyl AMP reacts with CoA-SH to yield phenylacetyl CoA. Analogous results were obtained in the benzoylglycine-forming system. Thus, benzoyl AMP replaced benzoate and ATP in the formation of benzoylglycine; in these experiments also, addition of CoA-SH was required for synthesis of the acylamino acid. It is of interest that benzoyl AMP and phenylacetyl AMP were active in relatively low concentrations (0.35 to 0.46 pmole per 0.7 ml.); in experiments carried out with benzoate and phenylacetate at these concentration levels (in the presence of CoA-SH and ATP) significant acylamino acid formation did not occur.

Utilization of phenylacetyl AMP would be expected to be associated with a stoichiometric disappearance of CoA-SH in the absence of glutamine. As reported elsewhere (26) incubation of phenylacetyl AMP and CoA-SH with the human liver mitochondrial preparation is associated with the disappearance of free sulfhydryl groups. These experiments were carried out with an experimental system similar to that employed in the studies described in Table V, except that glutamine and glutathione were omitted from the reaction mixture. Disappearance of free sulfhydryl groups was also observed in the course of the reaction between CoA-SH and benzoyl AMP. In the experiments with phenylacetyl AMP and benzoyl AMP, the decrease in the concentration of free sulfhydryl groups was of the same order of magnitude as that of acylamino acid formation observed under the conditions described in Table V (26).


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DISCUSSION

The experimental findings demonstrate that CoA-SH and ATP are required for PAG synthesis, and strongly indicate that phenylacetyl AMP and phenylacetyl CoA are intermediates in this process, which may be represented by the following reactions:

(1) Phenylacetate + ATP + phenylacetyl AMP + pyrophosphate (2) Phenylacetyl AMP + CoA-SH -+ phenylacetyl CoA + AMP (3) Phenylacetyl CoA + L-glutamine + phenylacetylglutamine + CoA-SH

According to this interpretation, activation involves a two step mechanism analogous to that proposed for the activation of fatty acids (27, 28). The formation of acyl-AMP derivatives was originally suggested by Berg for the acetate-activating system (29). The present results indicate that phenylacetyl AMP is active in the presence of CoA-SH in the PAG-forming system, and that benzoyl AMP exhibits similar behavior in the benzoylglycine-forming system. The previous demonstration (16) and present confirmation of the activity of benzoyl CoA in the benzoylglycine-forming system suggest that benzoylglycine and PAG are formed by similar mecha- nisms.

Although the human liver mitochondrial system catalyzed all three of the reactions given above, an enzyme activity capable of catalyzing only Reaction 3 was isolated and purified. The question as to whether individ- ual enzymes are responsible for Reactions 1 and 2 remains for future study. It is of interest that mitochondrial preparations obtained from beef liver catalyzed the activation of phenylacetate. However, the reaction between phenylacetyl CoA and glutamine is apparently specifically catalyzed by human tissues. The difference in specificity between the acylating systems of human tissues and those of other mammals may be due to a qualitative difference in the respective enzymes. On the other hand, if separate acylating enzymes are involved in benzoylglycine, phenylacetylglycine, and PAG synthesis, the present results may be interpreted to mean that human liver and kidney possess an acylating enzyme which is not present in other species.

The benzoyl moiety of benzoylglycine appears to arise predominantly from ingested benzoate, although there is evidence for the conversion of relatively small amounts of phenylalanine to benzoylglycine (9, 30, 31). On the other hand, it appears probable that the phenylacetyl moiety of PAG is derived from phenylalanine. The abnormally high excretion of PAG by patients with phenylpyruvic oligophrenia is compatible with this interpretation. Furthermore, we have observed formation of PAG in experiments with human liver homogenates in which phenylalanine, cy- ketoglutarate, ATP, CoA-SH, were incubated with glutamine. It appears


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probable, therefore, that PAG represents a product of the metabolism of phenylalanine and that phenylacetic acid arises by oxidative decarboxylation of phenylpyruvate formed from phenyalanine by transamination. De- carboxylation of phenylpyruvate might take place by reactions analogous to those previously considered for pyruvate (32) and a-ketoglutarate (33).

The authors wish to acknowledge the assistance of Miss Rita Indresano.

SUMMARY

1. The tnzymatic synthesis of phenylacetylglutamine, which appears to be catalyzed only by human tissues (and perhaps those of higher primates), has been studied with use of preparations of human liver and kidney. Homogenates of human liver and kidney and preparations of human liver mitochondria catalyzed the synthesis of phenylacetylglutamine from L-glutamine and phenylacetate; adenosine triphosphate (ATP) and coenzyme A (CoA-SH) are requird for synthesis.

2. Homogenates of human liver and kidney, human liver mitochondrial preparations, and the supernatant fractions of human liver and kidney homogenates obtained by high speed centrifugation catalyzed the formation of phenylacetylglutamine from phenylacetyl CoA and glutamine. Similar preparations from rat and beef liver were not active. The enzyme that catalyzes this reaction has been purified from human liver and kidney. This enzyme preparation also catalyzed benzoylglycine formation from benzoyl CoA and glycine, and, at a relatively low rate, the synthesis of phenylacetylglycine from phenylacetyl CoA and glycine. Indolylacetyl CoA, isobutyryl CoA, isovaleryl CoA, and p-hydroxyphenylacetyl CoA were inactive in the acylation of glycine and glutamine. Benzoyl CoA was not active in the acylation of glutamine, nor was phenylacetyl CoA active in acylating glutamic acid, phenylalanine, or leucine. The formation of phenylacetylglutamine from phenylacetyl CoA and glutamine (and of benzoylglycine from benzoyl CoA and glycine) was accompanied by stoichiometric liberation of free sulfhydryl groups.

3. It was found that phenylacetyl adenosine 5’-phosphate (AMP) could replace phenylacetate and ATP in the synthesis of phenylacetylglutamine by human liver mitochondria preparations provided that CoA-SH was present. Similarly, benzoyl AMP served in place of benzoate and ATP in the formation of benzoylglycine (hippuric acid).

4. The experimental evidence suggests that phenylacetylglutamine syn- t,hesis involves the activation of phenylacetate by ATP yielding phenylacetyl AMP, which reacts with CoA-SH to form phenylacetyl CoA. Although activation of phenylacetate is catalyzed also by beef mitochondrial preparations, acylation of glutamine by phenylacetyl CoA is catalyzed only by preparations of human liver or kidney.


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BIBLIOGRAPHY

1. Moldave, K., and Meister, A., Federation Proc., 16. 222 (1957). 2. Thierfelder, H., and Sherwin, C. P., Ber. them. Ges., 47.2630 (1914). 3. Sherwin, C. P., J. Biol. Chem., 31,307 (1917). 4. Sherwin, C. P., Wolf, M., and Wolf, W., J. Biol. Chem., 37, 113 (1919). 5. Shiple, G. J., and Sherwin, C. P., J. Am. Chem. Sot., 44, 618 (1922). 6. Shiple, G. J., and Sherwin, C. P., J. Biol. Chem., 52,463 (1922). 7. Ambrose, A. M., Power, F. W., and Sherwin, C. P., J. Biol. Chem., 101,669 (1933). 8. Power, F. W., PTOC. Sot. Exp. Biol. and Med., 22, 598 (1935). 9. Stein, W. H., Paladini, A. C., Him, C. H. W., and Moore, S., J. Am. Chem. Sot.,

76. 2848 (1954). 10. Wolf, L. I., Biochem. J., 49, p. ix (1951). 11. Meister, A., Udenfriend, S., and Bessman, S. I’., J. Clin. Invest., 36, 619 (1956). 12. Levintow, L., Eagle, H., and Piez, K. A., J. Biol. Chem., 337, 929 (1957). 13. Levintow, L., and Meister, A., J. Biol. Chem., 202, 265 (1954). 14. Elliott, W. H., J. Biol. Chem., 201, 661 (1953). 15. Simon, E. J., and Shemin, D., J. Am. Chem. Sot., 76, 2520 (1953). 16. Schachter, D., and Taggart, J. V., J. Biol. Chem., 203,925 (1953). 17. Lipmann, F., and Tuttle, L. C., J. Biol. Chem., 169.21 (1945). 18. Avison, A. W. D., J. Chem. Sot., 732 (1955). 19. Moldave, K., and Meister, A., Biochim. et biophys. ucta, 24, 654 (1957). 20. Rydon, H. N., and Smith, P. W. G., Nature, 169,922 (1952). 21. Meister, A., Levintow, L., Greenfield, R. E., and Abendschein, P. A., J. Biol.

Chem., 216, 441 (1955). 22. Mahler, H. R., Wakil, S. J., and Bock, R. M., J. Biol. Chem., 204,453 (1953). 23. Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Manometric techniques and

tissue metabolism, Minneapolis, 119 (1949). 24. Jepson, F. B., Biochem. J., 64, 14P (1956). 25. Schachter, D., and Taggart, J. V., J. Biol. Chem., 206,263 (1954). 26. Moldave, K., and Meister, A., Biochim. et biophys. acta, 26, 434 (1957). 27. Jencks, W. P., and Lipmann, F., J. Biol. Chem., 226, 207 (1957). 28. Moyed, H. S., and Lipmann, F., J. Back, 72,117 (1957). 29. Berg, P., J. Biol. Chem., 222. 1015 (1956). 30. Schreier, K., Altman, K. I., and Hempelmann, L. H., Proc. Sot. Exp. Biol. and

Med., 67, 61 (1954). 31. Armstrong, M. D., Chao, F.-C., Parker, V. J., and Wall, P. E., Proc. Sot. Ezp.

Biol. and Med., 60, 675 (1955). 32. Lynen, F., and Reichert, E., Angew. Chem., 62, 47 (1951). 33. Sanadi, D, R., and Littlefield, J. W., J. Biol. Chem., 195, 683 (1951).


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Kivie Moldave and Alton MeisterHUMAN TISSUE

PHENYLACETYLGLUTAMINE BY SYNTHESIS OF

1957, 229:463-476.J. Biol. Chem.

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