The Metabolism of Pantothenic Acid*

The Metabolism of Pantothenic Acid*

GENE M. BROWN

From the Division of Biochemistry, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts

(Received for publication, September 15, 1958)

Until now, the biosynthesis of CoA was thought to proceed by way of the following pathway:

pantothenic acid + pantothenylcysteine + pantetheine + 4’-P-pantetheine --) dephospho CoA ---f CoA

The formulation of this pathway was based on: (a) the finding that cysteine supplies the sulfur-containing moiety of CoA (3,4); (b) the activities of pantetheine and pantothenylcysteine as growth factors for Acetobacter suboxydans (4-6) and Lactobacillus helveticus (7); (c) the presence of enzymes in these two organisms and also in Lactobacillus bulgur&s which decarboxylate pantothenylcysteine to pantetheine (4, 7); (d) the report by Hoagland and Novelli (8) that rat liver extracts are able to convert pantothenylcysteine to CoA; and (e) the discovery, separation, and partial purification of the enzymes which convert pantetheine to CoA (8, 9).

It has been reported that 4’-P-pantothenic acid may be synthesized from pantothenic acid and ATP by enzymes present in Lactobacillus arabinosus (10) and Proteus morganii (11). These reports led Ward et a2. (11) to postulate the existence of an alternate route of CoA synthesis which might operate in certain microorganisms and which would include P-pantothenic acid, P-pantothenylcysteine, and P-pantetheine as intermediates. The results of the present investigations establish the existence of such a pathway in both mammalian and microbial systems, and, moreover, indicate that in rat liver and some microorganisms it appears to be the only route by which CoA is synthesized.

EXPERIMENTAL

Pantothenic acid was determined by assaying with Saccharo- myces carlsbergensis strain 4228 (12), for which CoA (13), pantetheine (13)) pantothenylcysteine (4, 14)) and P-pantothenic acid (15) are inactive. Pantetheine was determined with Lactobacillus helveticus strain 80 (13). Pantethine (the disulfide) has half the molar activity of pantetheine in this assay.

Purified bovine intestinal phosphatase was obtained from Pentex, Inc. Pantethine was kindly supplied by Dr. 0. D. Bird of Parke, Davis and Company. The sodium salt of d- pantothenylcystine was a gift from Dr. E. E. Snell.

Microbial Systems

Preparation of Extracts of Proteus morganii-Proteus morganii (ATCC No. 8019) was used as a source of enzymes for the major portion of this work. The handling of the culture, growth, and harvesting of cells in large quantities was carried out as

* These investigations were supported by National Science Foundation Grants G1300 and G4580. Portions of this work have appeared elsewhere as short communications (1, 2).

described in a previous publication (11). After they had been harvested and washed, the moist cells were frozen by immersion in liquid nitrogen and ruptured by putting them through a Hughes press (Shandon Scientific Co., Ltd., London). The material obtained from the press was suspended in approximately an equal volume of 0.02 M phosphate buffer, pH 7.0. The suspension was homogenized briefly in a Potter-Elvehjem homogenizer and centrifuged at 105,000 x g for 1 hour. The clear extract thus obtained was then dialyzed overnight against 200 times its volume of 0.02 M phosphate buffer at pH 7.0.

Metabolism of Paniothenic Acid ,by Extracts of P. morganii- The ability of the dialyzed extract to metabolize pantothenic acid is shown in Table I. In the presence of ATP and in the absence of cysteine, pantothenic acid was quantitatively converted into a product which could not be measured by assay with S. carlsbergensis. However, all of the pantothenic acid was recovered by subsequent treatment of the product with intestinal phosphatase. This product of pantothenic acid metabolism previously has been reported to be 4’-P-pantothenic acid’ (ll), and the enzyme responsible for the reaction has been named pantothenic acid kinase (11). When cysteine was included with ATP in the reaction mixture, again all of the pantothenic acid was converted to a product which could not substitute for panthothenic acid in the X. carlsbergensis assay. However, only about one-half of this product was reconverted to pantothenic acid by treatment with phosphatase. This observation indicated that at least two enzymes were present, pantothenic acid kinase and an enzyme which catalyzed a cysteine-dependent reaction of pantothenic acid. The latter enzyme will be referred to as the “coupling enzyme,” since evidence presented below indicates that it catalyzes the synthesis of P-pantothenylcysteine from P-pantothenic acid and cysteine. Other data in Table I establish that the amount of pantothenic acid which reacted in the presence of cysteine and was not recovered by phosphatase treatment approximately equalled the amount of phosphorylated pantetheine which was produced. Thus, the crude extract contained all of the enzymes necessary to convert pantothenic acid, ATP, and cysteine to one or more forms of phosphorylated pantetheine (i.e., P-pantetheine, dephospho-CoA, and CoA).

The relative amounts of the kinase and the coupling enzyme present in the dialyzed extract are indicated in Fig. 1. A comparison of curves 1 and 2 suggests that P-pantothenic acid was being formed as an intermediate which was then used

1 The abbreviation used is : Tris, tris (hydroxymethyl)amino- methane; hereafter the use of the terms P-pantothenic acid, P-pantothenylcysteine, and P-pantetheine will denote that the phosphate group is attached to position 4’ of the pantothenic acid moiety.

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TABLE I

Metabolism of pantothenic acid and synthesis of phosphorylated pantetheine by crude extract of P. morganii

Complete system: pantothenic acid, 0.083 pmole; cysteine, 20 rmoles; ATP, 20 pmoles; MgC12, 10 rmoles; and 0.2 ml. of extract in a volume of 2 ml. of 0.05 M Tris buffer, pH 7.4. Incubation was for 3 hours at 37’. The reaction mixtures were heated for 5

minutes in a 100’ water bath and centrifuged to remove denatured protein. Aliquots were removed for treatment with intestinal phosphatase. To each aliquot was added 0.1 ml. of a suspension

of intestinal phosphatase containing 40 mg. per ml., 0.1 ml. of 1 M sodium bicarbonate buffer, pH 8.5, and enough water to make a total volume of 1 ml. These reaction mixtures were incubated for 2 hours at 37’ and assayed for either pantothenic acid or pantetheine. Each reaction mixture was also assayed for pantothenic acid and pantetheine before treatment with intestinal

phosphatase.

Pantothenic acid present

Reaction mixture After

reaction

Complete...... 0 Omit cysteine. 0

Omit ATP..... 830 Omit panto-

thenic acid. 0

After reaction and intestinal phosphatase

treatment

jwldes x 104

400 830

830

0

F ‘antothenic acid ot recovered by

intestinal phosphatase

treatment

pm&s x 104

430

0 0

0

Phos&oryl-

pant&h&x formed

/m7les x IO’

460

80 90

60

as substrate in the reaction catalyzed by the coupling enzyme. The linearity of the curves at lower concentrations of extract indicates the feasibility of assaying for either pantothenic acid kinase or the coupling enzyme.

Separation of Coupling Enzyme from Pantothenic Acid Kinase-

In order to decide whether or not the coupling enzyme would work equally well with phosphorylated and nonphosphorylated pantothenic acid, it was necessary to separate this enzyme from pantothenic acid kinase. In the fractionation work described below one unit of coupling enzyme is defined as the amount which catalyzes the cysteine-dependent inactivation of 1 pg. of pantothenic acid in 3 hours at 37”. Protein was determined by measuring absorbancy at 280 rnp (16). All of the operations were

carried out at 4” with solutions cooled to the same temperature. The dialyzed crude extract was separated into 4 fractions by

treatment with a saturated solution of ammonium sulfate adjusted to pH 7.0 with ammonium hydroxide. Each ammonium sulfate precipitate was dissolved in 0.02 M phosphate buffer, pH 7.0, and dialyzed overnight against the same buffer before being tested for enzyme activity. It was found that the protein fraction precipitating between 0 and 38 per cent saturation (ammonium sulfate fraction I) contained large amounts of pantothenic acid kinase and no coupling enzyme (Table II). At the other extreme, the fraction which precipitated between 50 and 56 per cent saturation contained a small amount of coupling

enzyme and was free of the kinase. Both enzymes were present in the two intermediate fractions, although ammonium sulfate fraction III contained the bulk of the coupling enzyme and a relatively modest amount of kinase. The specific activity of the coupling enzyme present in this fraction was 4.2 units per mg. of

protein, a purification of about 6.5-fold over the crude extract.

0 0.05 0.10 0.15 0.20 0.25 EXTRACT OF I? MORGANII, ML.

FIG. 1. The relative amounts of pantothenic acid kinase and coupling enzyme present in dialyzed crude extracts of P. morgunii. Reaction mixtures and incubation conditions were as described in Table I. Curves 1 and I show the amounts of P-pantothenic acid present at the end of the incubation period in the presence and absence of cysteine. Curve 3 shows the amount of product formed in the presence of cysteine, which did not yield pantothenate by treatment with intestinal phosphatase. Thus, Curve S represents the amount of product formed by the coupling enzyme.

TABLE II

Enzynzatic activities of anz?nonium sulfate fractions

Reaction mixtures and incubation conditions were as described in Table I. Coupling enzyme was determined according to directions in the text.

I Coupling enzyme Ammonium sulfate Pantothenic acid

fraction* kinase Total units Specific activityt

(units/mg. protein)

I ++++ 0 0

II ++++ 1380 2.5

III ++ 2650 4.2

IV 0 720 2.0

* Ammonium sulfate fractions I, II, III, and IV were those protein fractions which were precipitated between 0 to 38, 38 to

44,44 to 50, and 50 to 56 per cent saturation, respectively. t The crude extract which was used as the starting material

contained 0.64 unit per mg. of protein.

The coupling enzyme was obtained free of the last traces of the kinase by treatment of ammonium sulfate fraction III with calcium phosphate gel. This treatment was carried out at a ratio of 3 parts of gel to 4 parts of protein. The gel was added to 20 ml. of ammonium sulfate fraction III containing 1600 units of enzyme and 390 mg. of protein. dfter stirring for 10 minutes to allow adsorption to occur, the suspension was centrifuged and the gel washed with 20 ml. of distilled water. Elution was effected with 10 ml. of M ammonium sulfate, pH 7.0. The eluate (hereafter referred to as the gel eluate) was dialyzed overnight against 0.02 M phosphate buffer, pH 7.0, to remove the ammonium sulfate. It was found that none of the pantothenic acid kinase had adsorbed to the gel. However, about 80 per cent of the coupling enzyme was adsorbed and was quantitatively


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372 Metabolism of Pantothenic Acid Vol. 234, No. 2

TABLE III

Metabolism of pantothenic acid by eluate from calcium phosphate gel

The complete reaction mixture consisted of: pantothenic acid, 0.083 pmole; ATP, 20 pmoles; cysteine, 20 pmoles; MgClg, 10 pmoles; gel eluate, 0.1 ml., ammonium sulfate I, 0.1 ml. in a total volume of 2 ml. of 0.04 M Tris buffer, pH 7.4. Incubation conditions and treatment with intestinal phosphatase were as described in Table I. The amount of pantothenic acid inactivated by the coupling enzyme was determined according to directions in the text.

Reaction mixture Pantothenic acid product

formed by coupling HlZYllltZ

jmoles x 104

Complete................................ Omit ammonium sulfate I.. Omit gel eluate. Omit cysteine..

500 0 0 0

removed by the eluting agent. The gel eluate contained 1360 units of coupling enzyme with a specific activity of 39 units per mg. of protein. This value represented an over-all purification of Bl-fold over the starting material.

Data obtained from experiments performed with the gel eluate are given in Table III. It is evident that the reaction catalyzed by the coupling enzyme was completely dependent on the presence of ammonium sulfate fraction I, a fraction which was rich in pantothenic acid kinase. The requirements for cysteine and the gel eluate are also clearly demonstrated by the results in Table III.

Requirement of CTP for Synthesis of P-pantothenylcysteine- The results presented above suggest that P-pantothenic acid is the obligate substrate for the coupling enzyme. It should be possible, then, to replace the requirements for pantothenic acid and pantothenic acid kinase with P-pantothenic acid. This corollary was found to be true from the results of the experiments described below. In the course of these experiments a unique function for CTP was discovered.

P-pantothenic acid was formed by incubating 0.08 Mmole of pantothenic acid, 20 pmoles of ATP, 10 Imoles of MgClz and 0.1 ml. of ammonium sulfate fraction I in a total volume of 1

TABLE IV

Requirement of CTP for synthesis of P-pantothenylcysteine

The reaction mixtures contained: P-pantothenic acid, 0.083

pmole; ATP, 5 rmoles; cysteine, 20 pmoles; MgC12, 10 rmoles; 0.1 ml. of gel eluate in a total volume of 2 ml. of 0.04 M Tris buffer, pH 7.4. Incubation conditions were as described in Table I.

Addition to reaction mixture Phosphopantothenic acid disappearance

pmoles x 102

None.................................... Boiled crude extract. Ammonium sulfate I, 0.2 ml..

CTP,5pmoles.................... .._... ATP,5pmoles.......................... GTP, 5 pmoles. ITP,5pmoles............................

UTP, 5pmoles ,......: .._ ..__....

CDP, 5 pmoles..

CMP, 5 pmoles..

1.3 1.9 4.4 6.9

1.3 1.7 1.8 1.7

2.9 1.5

ml. of 0.08 M Tris buffer, pH 7.4, for 3 hours at 37”. Assays showed that, under these conditions, all of the pantothenic acid was converted to P-pantothenic acid. The reaction mixture was heated (5 minutes in a 100” water bath) and centrifuged to remove denatured protein. To the solution, which contained P-pantothenic acid, was added 20 pmoles of cysteine and 0.1 ml. of the gel eluate and the total volume adjusted to 2 ml. with water. The reaction mixture was reincubated at 37” for 3 hours and the cysteine-dependent disappearance of P-pantothenic acid was measured by determining the amount of pantothenic acid which could be liberated by treatment with intestinal phosphatase. Unexpectedly, it was found that only a small amount of P-pantothenate disappeared. Much more disappeared when either 0.2 ml. (5 mg. of protein) of ammonium sulfate fraction I or CTP was added to the reaction mixture. The activating effects of these substances, as well as the relative inactivity of other nucleotides and boiled extract, are shown in Table IV. Additional experiments have revealed that, under the conditions given in Table IV, maximal activation can be achieved with as little as 0.2 pmole of CTP per 2 ml. Since the activating factor in ammonium sulfate fraction I was heat labile and nondialyzable, it was considered likely that it was nucleoside diphosphate kinase which would be required to resynthesize CTP as it was being used in the coupling reaction. This likelihood was supported by the finding that the crude extract could be completely inactivated by treatment with Dowex 1 (chloride form) and reactivated by the addition of either substrate amounts of CTP or catalytic amounts of CTP together with substrate amounts of ATP. It was established by paper chromatographic methods that incubation of CTP and cysteine with the gel eluate resulted in the formation of no compound which conta.ined both cytosine and sulfur. Thus it seems likely that CTP functions in some way in the activation of P-pantothenic acid. The coupling enzyme has not yet been purified enough to decide whether, during the reaction, orthophosphate or pyrophosphate is cleaved from CTP to yield CDP or CMP.

The results presented in Tables III and IV indicate con-, elusively that P-pantothenic acid can be used as substrate for the coupling enzyme and that pantothenic acid cannot be used.

The product formed from P-pantothenic acid and cysteine by the purified coupling enzyme was present in such small quantities that isolation of enough of it for a chemical analysis was not feasible. However, sufficient evidence was obtained by paper chromatographic methods to identify the dephosphorylated product as pantothenylcysteine. For this purpose, reaction mixtures were treated with intestinal phosphatase and chromato- graphed on paper strips together with an authentic sample of pantothenylcystine. The developing solvent consisted of 95 parts butanol, 5 parts acetic acid, and enough water to saturate. Zones of migration were determined by the bioautograph tech- nique by using as the assay organism L. helveticus strain 80-PC (a mutant strain which will use pantothenylcystine as a growth factor (7)). The RF value (0.57) of the material from the reaction mixture corresponded with the RF value (0.59) of authentic pantothenylcystine. An additional active compound of RF

0.18 was also found and identified as the mixed disulfide of pantothenylcysteine and cysteine.

Sulfur-containing Compounds Serving as Substrate for Coupling

Enzyme-Of a large number of sulfur-containing compounds which were tested, only those listed in Table V were used as substrate by the coupling enzyme. Compounds which were


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February 1959 G. M. Brown 373

ineffective included homocysteine, thioglycollic acid, thiomalic acid, glutathione, mercaptoethanol, cysteic acid, and taurine as well as such nonsulfur compounds as serine and ethanolamine. It can be observed that, on the basis of their equal sulfur content, cystine, &mercaptoethylamine, the disulfide of the latter compound, and cr-methylcysteine were about 0.05, 0.25, 0.1, and 0.1 as effective, respectively, as cysteine. As discussed above, the product formed when cysteine was used as substrate was P-pantothenylcysteine. The small amount of phosphorylated pantetheine which was produced (Table V) resulted from the presence of a small amount of P-pantothenylcysteine decarboxylase in the gel eluate. With /3-mercaptoethylamine as substrate, P-pantetheine should be produced directly. The data in Table V show this to be the case. The excellent agreement between the amount of P-pantothenate which disappeared and the amount of P-pantetheine which was formed leaves no doubt about the stoichiometry of the reaction. The product formed from (Y- methyl cysteine presumably was N-(P-pantothenyl)-a-methyl cysteine. Treatment of this product with P-pantothenylcysteine decarboxylase and phosphatase did not yield a product active in the pantetheine assay. It can not be said whether or not the product was decarboxylated by this treatment.

Formation of P-pantetheine from P-pantothenylcysteine-The results in Table I established that crude extracts were able to synthesize phosphorylated pantetheine, but, as mentioned above, the purified coupling enzyme (gel eluate) formed P-pantothenylcysteine as its product. When other protein fractions were tested, it was discovered that nearly all of the enzyme which decarboxylates P-pantothenylcysteine to P-pantetheine was present in the ammonium sulfate fraction II (see Table II for a description of this preparation). The product was identified as P-pantetheine by demonstrating that pantetheine could be formed by treating the reaction mixtures with either intestinal phosphatase or the phosphomonoesterase of prostate phosphatase.2

The results of tests for the presence of P-pantothenylcysteine decarboxylase in various other microorganisms are given in Table VI. The P-pantothenylcysteine used in these studies was synthesized with the purified coupling enzyme. In addition to ammonium sulfate fraction II (prepared from extracts of P. morganii), extracts of Escherichia coli also appeared to represent a good source of the enzyme. Baker’s yeast and extracts of Neurospora crassa also possessed some activity, but none could be detected in preparations of L. helveticus and L. arabinosis.

All of these extracts were prepared from cells ruptured in a Hughes press. Significantly, neither these enzyme preparations, nor the crude extracts of P. morganii, were able to decarboxylate pantothenylcystine in the presence or the absence of cysteine (added as reducing agent) and/or ATP (Table VI).

Distribution of Pantothenic Acid Kinase and Coupling Enzyme among Microorganisms-Pantothenic acid kinase has been reported to occur in L. arabinosus (8). We have confirmed this report and, in addition, have found this enzyme in extracts of E. coli and baker’s yeast as well as in extracts of P. morganii,

Extracts of E. coli were also found to be rich in the coupling enzyme. The E. coli enzyme resembled the P. morganii en-

zyme in that CTP was demonstrated to be specifically required when Dowex l-treated extracts3 were used as a source of enzyme.

2 Kindly supplied by Dr. Gerhard Schmidt. 3 Dowex treatment was carried out according to the directions

given by Novelli and Schmeta (17).

TABLE V

Sulfur compounds used as substrates by coupling enzyme Reaction mixtures and incubation conditions were as described

in Table IV. The gel eluate was used as a source of the coupling

enzyme .

Sulfur compound added f \mount added -pantothenate disappearance

None .._ Cysteine.

Cysteine Cysteine........ Cysteine.

&Mercaptoethylamine. B-Mercaptoethylamine. P-Mercaptoethylamine.

P-Mercaptoethylamine. @-Mercaptoethylamine Cystine Disulfide of @-mercapto-

p?Loles pm&s x 10

0 0 2.5 290

5.0 540 7.5 730

10.0 830

5.0 140 10.0 26s 20.0 590

30.0 730 40.0 800 10.0 120

ethylamine. 20.0 440 or-Methylcysteine. 40.0 420

L

Source of Enzyme

Ammonium sulfate II..

Extract of E. coli.. Extract of baker’s yeast. Extract of Neurospora crassa

Extract of L. helveticus. Extract of L. arabinosus

Jmoles x 104

0 0

0 180 280

140 240 594

750 800

TABLE VI

Decarbozylation of P-pantothenylcysteine Reaction mixtures contained: either P-pantothenylcysteine,

0.015pmole or pantothenylcystine, 0.029 pmole; 0.2 ml. of the indi-

cated extract in 1 ml. of 0.04 h% Tris buffer, pH 7.4. When ATP and/or cysteine were added to reaction mixtures containing pantothenylcystine, 10 *moles of each were used.

P-pantetheine* produced from

P-pantothenyl- cyst&e Pantothenylcystinet

w?zoles x 10” /moles x 104

145 0 149 0

27 0 7 0 0 0 0 0

* P-pantetheine was measured indirectly by treating the reaction mixtures with intestinal phosphatase and assaying for pan-

tetheine. t The addition of either cysteine or ATP or both to reaction

mixtures containing pantothenylcystine did not result in the

formation of any pantetheine or phosphorylated pantetheine.

No coupling enzyme activity could be detected in extracts of baker’s yeast, L. arabinosus, and L. helveticus.

Jdammalian Systems

The evidence presented above which establishes roles for P- pantothenic acid and P-pantothenylcysteine as intermediates in CoA biosynthesis by microorganisms led the author to reinvesti- gate CoA biosynthesis in mammalian systems.

Preparation of Extracts of Rat Liver and Rat Kidney-Six adult white rats were decapitated and their livers and kidneys immediately removed and chilled. An equal weight of 0.02 ELI phosphate buffer, pH 7.0, was added to the pooled organs and homogenates were prepared by treatment in a Waring Blendor.


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374 Metabolism of Pantothenic Acid Vol. 234, n-o. 2

Soluble extracts were obtained by centrifuging the homogenates at 105,000 x g for 1 hour. The extracts were dialyzed overnight against 200 times their volumes of 0.02 M phosphate buffer at pH 7.0.

Metabolism of Pantothenic Acid by Mammalian Extracts- The abilities of the liver and kidney extracts to metabolize pantothenic acid (see Table VII) suggest that they contained enzymes similar to those found in P. morganii, namely, pantothenic acid kinase and a coupling enzyme which catalyzes a cysteine-dependent reaction of pantothenate. For the sake of convenience, the mammalian enzyme will also be referred to as the coupling enzyme, although evidence presented la.ter indicates that this enzyme is different in some respects from the bacterial

T.4BLE 1'11

Reactions of pantothenic acid catalyzed by dialyzed extracts of rat liver and rut kidney

The complete reaction mixture contained: pantothenic acid, 0.083 pmole; ATP, 20 pmoles; cysteine, 20 pmoles; MgC12, 10 wmoles; and 0.2 ml. of extract of either rat liver or rat kidney in a t,otal volume of 2 ml. of 0.04 nf Tris buffer, pH 7.4. Incubation

conditions and treatment with intestinal phosphatase were as described in Table I.

Reaction mixture

-

A

Pantothenic acid present

fter reaction

A. Rat liver Complete.

Omit cysteine. Omit ATP

B. Rat kidney

Complete. Omit cysteine. Omit ATP

I

.I

‘moles x 104 pm&s x 101

0

0 833

0

800

833

0 300 0 683

833 833

ifter reaction and treatment with

intestinal phosphatase

Metabolized pantothenic acid not

recovered by treatment with

ltestinal phosphatase

jmoles x 104

833

33

533 150

TABLE VIII

Relative amounts of pantothenic acid kinase and coupling enzyme present in ammonium sulfate fractions of rut liver extracts

Reaction mixtures were as described in Table VII except that

0.4 ml. of the fraction to be tested was used per reaction mixture. In those systems in which coupling enzyme activity was to be assessed, an excess of pantothenic acid kinase, prepared from

Proteus morganii, was used in order to make the assay specific for the coupling enzyme. The added kinase was entirely free of the coupling enzyme. Incubation conditions and treatment

with intestinal phosphatase were as described in Table I.

Ammonium sulfate fraction*

Pantothenic acid metabolized

Kinase activity Coupling activity

pmoles x 104 /.moles x IO’

A (O-31)..... B (31-40). .

C (40-46). D (46-54). E (54-60).

F (60-66).

0 450 183 780

433 820 400 830

20 830 0 167

* The figures in parenthesis represent the per cent saturation with ammonium sulfate.

TABLE IX

Inability of mammalian coupling enzyme to utilize pantothenic acid as substrate

The complete reaction mixture was the same as that described in Table VII, except that a source of pantothenic acid kinase (purified from P. morganii) m-as added and ammonium sulfate

fraction A (see Table VIII) was used in place of the crude extract. Incubation conditions and treatment with intestinal phosphatase were as described in Table I.

Reaction mixture Pantothenic acid metabolized and

not recovered by intestinal phosphatase treatment

Complete ..........................

Omit pantothenic acid kinase ...... .I Omit cysteine ..................... .’

pm&s x 104

475 0 0

coupling enzyme. The relative amounts of the kinase and the coupling enzyme present in the two extracts are evident from the data given in Table VII.

Separation of Mammalian Coupling Enzyme from Pantothenic Acid K&use-Preparations of the coupling enzyme were obtained free of pantothenic acid kinase by fractionation of the dialyzed rat liver extract with a saturated solution of ammonium sulfate adjusted to pH 7.0. All of the operations were carried out at 4”. A 40-ml. portion of extract was used as starting material. Each fraction was dissolved in 20 ml. of 0.02 M

phosphate buffer, pH 7.0, and dialyzed overnight against the same buffer before being tested for enzyme activity. Table VIII contains a description of the six fractions which were obtained and a summary of their abilities to metabolize pantothenic acid. Fractions A and E contained little or no pantothenic acid kinase; however, the two preparations were rich in the coupling enzyme. Both enzymes were present in fractions B, C, and D. The requirement for a source of pantothenic acid kinase in order for fraction A to carry out the cysteine-dependent reaction is shown in Table IX. Thus the mammalian coupling enzyme resembles the bacterial enzyme in that neither can use pantothenic acid as substrate.

Requirement of Nucleoside Triphosphate for P-pantothenylcysteine Synthesis-The mammalian coupling enzyme was

different from the bacterial enzyme in that CTP was not specifically required for the synthesis of P-pantothenylcysteine. The data of Table X indicate that all of the nucleoside triphosphates were approximately equally active in the crude mammalian system. The disappearance of P-pantothenate in the absence of added nucleoside triphosphate was probably caused by residual ATP which was present in the preparation of P- pantothenic acid. Only ATP was added at a high enough concentration to result in maximal disappearance of P-panto-

thenate. The relatively large amounts of nucleoside triphosphate required, when compared to the amount of CTP required in the bacterial system, might be a result of the presence in the enzyme preparation of large amounts of nucleotidases. Alterna- tively, the mammalian enzyme might be inherently different

from the bacterial enzyme in the amount of nucleoside triphosphate it needs to function.

Sulfur-containing Compounds Active in Mammalian Xystem- Of several compounds which were tested, only those listed in Table XI were used as substrate by the mammalian coupling enzyme. With the esception of glutathione, these compounds


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TABLE X TABLE XI NucleosiJe triphosphate requirement for P-pantothenylcysteine Sulfur compounds used as substrate by mammalian coupling enzyme

synthesis by mammalian coupling enzyme Reaction mixtures were as described in Table VII. Conditions Reaction mixtures contained: P-pantothenic acid, 0.075 rmole; of incubation and treatment with intestinal phosphatase are de-

scribed in Table I. cysteine, 10 pmoles; MgCls, 10 rmoles; 0.2 ml. of Dowex-treated ammonium sulfate fraction A (see Table VIII) ; and additions as shown in a total volume of 2 ml. of 0.04 M Tris buffer, pH 7.4. The amount of P-pantothenic acid which disappeared was determined by measuring the amount of pantothenic acid which could be generated by treatment with intestinal phosphatase. Incu- bation conditions were as described in Table I.

Addition to reaction mixture P-pantothenic acid* disappearance

None CTP, 1 pmole.. ATP, 1 pmole. ATP, lOpmoles..... GTP, 1 /*mole.. UTP, 1 pmole..

pm&s x 104

325

467 490 750 510

517

* P-pantothenic acid was synthesized enzymatically with a Dowex-treated preparation of ammonium sulfate I (see Table II).

corresponded with those which the bacterial enzyme could use (see Table V); however, the two enzymes differed markedly in the amounts of substrate required. The concentration of cysteine required in the mammalian system was only 0.1 of that required in the bacterial system. Based on an equal sulfur content, fl-mercaptoethylamine, its disulfide, and cystine were about 0.1, 0.05, and 0.5 as effective as cysteine, respectively in the mammalian system. For comparative purposes, these same compounds, given in the same sequence, were 0.25,0.1, and 0.05 as effective as cysteine in the bacterial system. The activity of glutathione, noted in Table XI, undoubtedly resulted from its enzymatic hydrolysis to cysteine.

The difference in the amounts of pantothenic acid which disappeared and the phosphorylated pantetheine which was produced in reaction mixtures containing cysteine and a crude extract of rat liver (Table XI) suggests that P-pantothenylcysteine decarboxylase was present in relatively small quantities in the extract. With @-mercaptoethylamine as substrate, the amount of P-pantetheine produced was about one half the amount of pantothenate metabolized (Table XI). This result was somewhat surprising since the product of this reaction should be P-pantetheine. It should be noted, however, that these experiments were done with crude enzyme preparations and, therefore, the possibility cannot be discounted that some of the

Substrate

None . . . . Cysteine............ Cysteine............ Cysteine............ Cysteine............ Cystine............. Cystine............. Cystine............. Cystine............. P-Mercaptoethyl-

amine p-Mercaptoethyl-

amine. Disulfide of &mer-

captoethylamine. Disulfide of B-mer-

captoethylamine or-Methyl cysteine.. Glutathione (re-

duced) ,

Amount substrate

added

pm&s

0.1 0.2 0.3 0.5

0.1 0.2 0.3

0.5

2.0

4.0

2.0

4.0

5.0

10.0

Pantothenic acid metabolized and not recovered by intestinal phos-

phatase treatment

pm&s x IO"

0 165

391 591 600

190 450 558

650

383

650

358

683 600

320

antetheine present after intestinal

phosphatase treatment*

/.tmoles x 10’

0 63 59 60

58

30 39

40

189

308

163

363

* These values have been corrected for an enzyme blank of 30 X 10m4 fimoles per 2 ml. of reaction mixture.

TABLE XII Abilities of rat liver and rat kidney extra& to decarboxylale

P-pantothenylcysleine* and their inabilities to decarbozylate pantothenylcystine

Reaction mixtures contained: P-pantothenylcysteine, 0.0165 pmole, or pantothenylcystine, 0.029 pmole; cysteine, 10 pmoles (added as reducing agent) ; and 0.2 ml. of extract in a volume of 1 ml. of 0.04 M Tris buffer, pH 7.4. Incubation conditions and treatment with intestinal phosphatase, where desired, were as described in Table I.

/ Pantetheine present

P-pantetheinewhich was formed might have been metabolized Enzyme and substrate used

to a product or products which would not yield pantetheine by After reaction and

After reaction treatment with

treatment with intestinal phosphatase. Further purification of intestinal phosphatase

the mammalian coupling enzyme will be necessary to explain j.lmoles x 104 p?mles x JO’

this discrepancy. A. Rat liver Decarboxylation of P-pantothenylcysteine by Liver and Kidney P-pantothenylcysteine 0 100

E&acts-The results given in Table XII establish the abilities Pantothenylcystine 0 of dialyzed extracts of rat liver and rat kidney to decarboxylate None...................... 0 43

P-pantothenylcysteine. The yield of P-pantetheine in this ‘. Rat kidney reaction was about 35 per cent. This finding supports the P-pantothenylcysteine. 0 68

evidence cited above that the liver extracts contained relatively Pantothenylcystine 0

small amounts of P-pantothenylcysteine decarboxylase. It None...................... 0 15

should be noted that these same enzyme preparations were * P-pantothenylcysteine was produced with the purified cou- completely ineffective in producing pantetheine from panto- pling enzyme of P. morganii.


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ROUTE I tH3 w CH,-C-CH-C-NH-CH,-CHrCOOH ROUTE 2

ATP

/

OH ~H~&H

PANTOTHENIC ACID ‘YTE’NE

y43 i? FH3 I: L 0 500”

CH@-CH-C-NH-CH,CH,COOH

b LH bH

CH2-t-YH-C-NH-CH&-NH-CH-CH,

3 SH

L03H2 PANTOTHENYLCYSTEINE

~LPH~SPH~PANTOTHENIC ACI c~ CYSTEINE

1

- co, CTP OR ATP

FH3 FI f I

CH2-F--CH-C-NH-CH,-CH,-C-NH f-- 51

b

C NH CH,-CH,-C-NH-CH,-CH,

CH, H b

bo,H, &H

4:PHOSPHOPANTOTHENYLCYSTEINE PANTETHEINE

1 -co,

tH3 fl if CH,-F-:H-C-NH-CH,-CH,-C-NH-CH,-CH+H$H COENZYME A

b C H,OH

bO,H,

FIG. 2. The reactions concerned in the proposed routes for the biosynthesis of CoA. The reaction indicated by a broken arrow is the only one shown for which no enzyme has been found.

thenylcystine, with or without cysteine added as a reducing agent.

DISCUSSION

The reactions involved in the two proposed routes for CoA biosynthesis are given in Fig. 2. The results of the present investigations demonstrate conclusively that P. morganii, E. coli, and mammalian systems synthesize CoA by way of Route 1 (Fig. 2). It is considered likely that Route 1 also operates in L. arabinosus, yeast and Neurospora crassa since all of these organisms contain at least one of the three enzymes necessary for the operation of Route 1, and since no pantothenylcysteine decarboxylase could be detected in any of them (see Table XIII). Other studies which support this conclusion are those which show that these three organisms, as well as P. morganii and E. coli, synthesize and accumulate P-pantothenic acid when large amounts of pantothenate are included in their growth media (7, 18). Of the enzymes listed in Table XIII only panto-

TABLE XIII Distribution among microorganisms of enzymes concerned

in meta,bolism of pantothenic acid

P. morganii.. E. coli

L. arabinosus. Yeast. Neurospora crassa.

L. helveticus

+ + +

: + - -

+ + -

+ + - + +

- - - - +

thenylcysteine decarboxylase was detected in L. helveticus. Also, whole cells of this organism when supplied with large amounts of pantothenate were not able to accumulate P-pantothenic acid (18). These facts, together with the additional fact that mutant strains of L. helveticus can use pantothenylcysteine a.s a growth factor (7), suggest that this bacterium when forced to use pantothenic acid as a growth factor might synthesize its CoA by way of Route 2. Acetobacter suboxydans, which can also utilize pantothenylcysteine as a growth factor (4, 5), and Lactobacillus bulgaricus, which contains pantothenylcysteine decarboxylase (7), are other microorganisms which could possibly utilize Route 2. However, until the presence of an enzyme which synthesizes pantothenylcysteine from pantothenic acid and cysteine can be demonstrated, the question of whether or not Route 2 operates in any system cannot be answered un- equivocally.

In 1954, Hoagland and Novelli (8) studied CoA biosynthesis in rat liver extracts and reached the conclusion that the probable pathway was by Route 2 (Fig. 2). This conclusion, which is contrary to the one reached in the present publication, was partially based on the ability of their enzyme preparation to form pantetheine from pantothenic acid, cysteine, and ATP. An examination of their analytical methods revealed that P- pantetheine would also be active in their assays for pantetheine. Since their enzyme preparation, being crude, almost certainly contained pantothenic acid kinase, it now seems likely that their system was synthesizing P-pantetheine via Route 1 instead of pantetheine by way of Route 2. The report by these workers that their enzyme preparation would readily decarboxylate pantothenylcystine (8) is somewhat harder to reconcile with the results of the present investigations. The data of Table XIV demonstrate that the combination of pantothenic acid, cysteine,


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and ATP was far more effective as a precursor of phosphorylated pantetheine than was pantothenylcystine of pantetheine, or of phosphorylated pantetheine when ATP was included in the reaction mixture. It should be noted that no pantetheine what- ever was formed until the concentration of pantothenylcystine had been raised to a high level and, even then, the amount of pantetheine produced represented a yield of only 0.5 per cent. It was also found that no pantothenic acid was formed from pantothenylcystine, a finding which eliminates the possibility that the almost negligible yields of pantetheine was a result of its enzymatic degradation to pantothenic acid.

Thompson and Bird (19) reported in 1954 that pantothenylcystine (or pantothenylcysteine) could not replace pantothenic acid as a nutritional requirement for chicks and rats under the same conditions in which pantethine (or pantetheine) and CoA were fully active. This report is consistent with the results of the present work. It would now appear that pantothenylcysteine is a relatively inert compound when fed to animals. It can neither be phosphorylated to P-pantothenylcysteine nor hydrolyzed to pantothenic acid. Pantothenylcysteine is decarboxylated to pantetheine in such small quantities that this reaction is of questionable significance.

In view of the findings of the present publication, the metabolic importance of pantetheine must be reassessed. With a few possible exceptions (discussed above), pantetheine appears to be off the biosynthetic pathway for CoA formation. Its wide- spread distribution among natural materials (20, 21) would apparently result from the degradation of CoA or P-pantetheine. Both of these compounds are readily degraded to pantetheine by phosphatases. It is significant that pantetheine is found in largest quantities in those materials which have been allowed to autolyze (20). However, the destruction of CoA by dephos- phorylation to pantetheine does not result in a metabolic cul de sac since pantetheine may be converted to P-pantetheine and thence to CoA by the action of certain kinases present in microorganisms (11) and animal tissues (9).

The finding that fi-mercaptoethylamine will react, in place of cysteine, with P-pantothenic acid to yield P-pantetheine directly was somewhat surprising in view of the reports that this compound will replace cysteine for CoA synthesis neither in rat liver (8) nor in resting cell suspensions of L. ara6inosus (3) and P. morganii (22). The inactivity of @-mercaptoethylamine in the bacterial experiments might have been a result of the impermeability of the cells to the compound. On the other hand, since this compound is not as effective a substrate as cysteine, it might not have been added to the bacterial suspensions in large enough amounts. The reported inactivity of fl-mercaptoethylamine in rat liver preparations (8) was almost certainly a result of its not being tested in high enough concentrations.

At present, the function of CTP in the synthesis of P-pantothenylcysteine is not known. It is possible that it functions in the activation of the carboxyl group of P-pantothenic acid in a manner similar to the way ATP functions in the activation of pantoic acid for the synthesis of pantothenic acid (23). An alternate possibility is that CTP reacts with P-pantothenic acid to give cytidine diphosphopantothenic acid which might serve as the true substrate for the addition of cysteine. In order to decide which of these explanations, if either, is correct, the coupling enzyme must be purified free of contaminating nucleotidases.

TABLE XIV Formation of pantetheine and phosphorylated pantetheine

by rat liver extracts Reaction mixtures contained: cysteine, 20 pmoles; MgC12, 10

pmoles; 0.2 ml. of dialyzed rat liver extract; and substrates as shown in a total volume of 2 ml. of 0.04 M Tris buffer, pH 7.4. When pantothenic acid was used as substrate 0.083 rmole was added. Incubation conditions and treatment with intestinal phosphatase were as described in Table I.

/ Pantetheine present

Substrate ATP added After

reaction

None . . Pantothenic acid. Pantothenylcystine, 0.165 pmole. Pantothenylcystine, 0.165 pmole. Pantothenylcystine, 0.80 pmole. Pantothenylcystine, 0.80 pmole.

~moles jmoles x lo’ jmoles x 104

0 0 32 20 0 130 0 0 40

20 0 38 0 42 96

20 36 92

After reaction and intestinal phosphatase treatment

SUMMARY

Extracts of Proteus morganii, Escherichia co&, rat liver, and rat kidney contained the following three enzymes involved in the biosynthesis of coenzyme A: (a) pantothenic acid kinase, (6) the enzyme which converts 4’-phosphopantothenic acid and cysteine to 4’-phosphopantothenylcysteine (referred to as the coupling enzyme), and (c) phosphopantothenylcysteine decarboxylase which catalyzes the formation of 4’-phosphopantetheine. Fractionation of extracts of P. morganii and rat liver yielded preparations of pantothenic acid kinase and the coupling enzyme which were free one from the other. 4’-Phosphopantothenic acid, but not pantothenic acid, was used as substrate by both the bacterial and mammalian coupling enzymes. Phospho- pantothenylcysteine, but not pantothenylcysteine, was decarboxylated by extracts of P. morganii, E. coli, rat kidney, and rat liver. These results led to the conclusion that the major pathway of coenzyme A biosynthesis is as follows: pantothenic acid + 4’-phosphopantothenic acid + 4’-phosphopantothenylcysteine -+ 4’-phosphopantetheine + coenzyme A.

Sulfur-containing compounds which were used as substrate for the bacterial and the mammalian coupling enzymes were: cysteine, @-mercaptoethylamine, the disulfide of the latter compound, a-methyl cysteine, and cystine. Of these cysteine was much more effective than any of the others. The significance of the activities of the other compounds was discussed.

Cytidine triphosphate (CTP) was specifically required for the reaction catalyzed by the bacterial coupling enzyme. The possibility that the function of CTP in this reaction is in the activation of the carboxyl group of 4’-phosphopantothenic acid was discussed. The mammalian coupling enzyme differed from the bacterial enzyme in that it could use any of the other nucleoside triphosphates equally as well as CTP.

Acknowledgments-The author wishes to express his thanks to Miss Anne Koch for technical assistance in these investigations and to Dr. H. A. Harris of Merck and Company for a generous gift of a-methyl cysteine.


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REFERENCES

1. BROWN, G. M., Federatzon Proc., 1’7, 197 (1958). 13. CRAIG, J. A., AND SNELL, E. E., J. Bacterial., 61, 283 (1951). 2. BROWN, G. M., J. Am. Chem. Sot., 80, 3161 (1958). 14. BROWN, G. M., IKAWA, M., AND SNELL, E. E., J. Biol. Chem., 3. PIERPONT, W. S., AND HUGHES, D. E., Rtkumds des Communi- 213, 855 (1955).

cations, Congrks International de Biochimie, 2‘ Congrbs 15. KING, T. E., AND STRONG, F. M., J. Biol. Chem., 189, 315 Paris, 1952, Masson et Cie., Paris, 1952, p. 91. (1951).

4. BROWN, G. M., AND SNELL, E. E., J. Am. Chem. Sot., ‘76, 2782 (1953).

16. WARBURG, O., AND CHRISTIAN, W., Biochem. Z., 310, 384

5. BROWN, G. M., AND SNELL, E. E., J. Bacterial., 67,465 (1954). (1941).

6. KING, T. E., AND CHELDELIN, V. H., Proc. Sot. Exptl. Biol. 17. N OVELLI, G. D., AND SCHMETZ, F. J., JR., J. Biol. Chem., 192,

Med., 84, 591 (1953). 181 (1951).

7. BROWN, G. M., J. Biol. Chem., 226,651 (1957). 18. BROWN, G. M., J. Biol. Chem., 234, 379 (1959).

8. HOAGLAND, M. B., AND NOVELLI, G. D., J. Biol. Chem., 207, 19. THOMPSON, R. Q., AND BIRD, 0. D., Science, 120, 763 (1954).

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(1954). 21. RASMUSSEN, R. A., SMILEY, K. L., ANDERSON, J. G., VAN

10. PIERPONT, W. S., HUGHES, D. E., BADDILEY, J., AND MATHIAS, LANEN, J. M., WILLIAMS, W. L., AND SNELL, E. E., Proc.

A. P., Biochem. J., 61, 368 (1955). Sot. Exptl. Biol. Med., 73, 658 (1950).

11. WARD, G. B., BROWN, G. M., AND SNELL, E. E., J. Biol. Chem., 22. BROWN, G. M., Ph.D. Thesis, University of Wisconsin, 1953.

213, 869 (1955). 23. MAAS, W. K., Abstracts 3” Congrks International de Biochimie,

12. ATKIN, L., WILLIAMS, W. L., SCHULTZ, A. S., AND FREY, Brussels, 1955, Societb Belge de Biochimie, Libge, Belgium,

C. N., Znd. Eng. Chem. Anal. Ed., 16, 67 (1944). 1955.


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Gene M. BrownThe Metabolism of Pantothenic Acid

1959, 234:370-378.J. Biol. Chem.

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