THE ENZYMATIC CONDENSATION OF PYRUVATE AND FORMALDEHYDE*

BY HELEN HIFTt AND H. R. MAHLER

(From the institute for Enzyme Research, University of Wisconsin, Madison, Wisconsin)

(Received for publication, April 4, 1952)

An enzyme has been obtained from beef liver which condenses formalde- hyde and pyruvate in equimolar amounts to yield a-keto-y-hydroxybutyric acid; the enzyme appears to be highly specific for both substrates.

Preparation and Characterization of Enzyme

The presence of the enzyme could be demonstrated in whole tissue ho- mogenates of rat kidney and liver but not in heart, diaphragm, spleen, or brain. It was concentrated in the mitochondrial fraction of rat, rabbit, hog, and beef liver; the sedimentable debris and nuclear fraction showed only slight activity and the microsomal and soluble fractions were inactive.

The enzyme was prepared in large amounts from beef liver. The par- ticulate preparation was made according to the procedure of Sarkar et al. (1) : the tissue was homogenized in 8.5 per cent sucrose and the mitochon- drial fraction obtained by differential centrifugation and precipitation in isotonic KCl. After repeated freezing and thawing, the enzyme was ob- tained in soluble form (not sedimentable at 30,000 X g in 30 minutes) (Fraction A). It was precipitated between 25 and 40 per cent saturated ammonium sulfate (between 17.5 and 28 gm. of ammonium sulfate per 100 ml.). The buff-colored residue (Fraction B) dissolved readily in 0.025 M

phosphate buffer at neutral pH (15 to 20 mg. of protein per ml.), and was dialyzed against the same buffer in the cold room overnight. At this point the enzyme could be stored frozen for several months without loss in ac- tivity. Treatment with an equal volume of tricalcium phosphate gel (ap- proximately 1 mg. of gel solids per 2 mg. of protein in contact for 1 minute) yielded the enzyme in the supernatant (Fraction C). At this point the preparation contained about 3 to 4 mg. of protein per ml., but lost activity rapidly, and was, therefore, prepared fresh daily. Lyophilization stabilized the enzyme so that it retained activity for several days and could be sub- jected to the gel treatment for a second time, followed again by lyophiliza-

* A preliminary report of this work was presented at the meeting of the American Society of Biological Chemists, New York, 1952 (Hift, H., Federation Proc., 11, 230 (1952)).

t Postdoctorate trainee of the National Heart Institute, National Institutes of Health.

901

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902 ENZYMATIC CONDENSATION

tion. This final preparation (Fraction D), showed a specific activity 60 times that of the original soluble preparation (Fraction A). When ex- amined in the analytical ultracentrifuge (at 160,000 X g), Fraction D was found to sediment slowly, but essentially as one peak. Pertinent data for the purification are given in Table I.

The usual two-step purification procedure yielding Fraction C accom- plished the removal of lactic dehydrogenase and of Racker’s aldehyde de- hydrogenase (2), both of which are enzymes requiring diphosphopyridine nucleotide (DPN) and attack pyruvate and formaldehyde respectively. Gel treatment, furthermore, removed an inhibitor which could be eluted with phosphate buffer and which decreased the activity of the purified

TABLE I Purijkation of Enzyme

Starting material, 2300 gm. of fresh beef liver. 1 unit = the micromoles of py- ruvate disannearing in 35 minutes at 37”. Protein determined by the biuret method of Weichseibaum (19).

Fraction

A, supernatant of frozen and thawed mito- chondrial fraction

w.

15,096

w.

27 0.14

B, precipitated between 25 and 4070 satu- 3,747 17 0.41 rated (NHI)zSO~

C, supernatant from first gel treatment 2,301 3.8 1.36 “ after lyophilization 1,440 18 1.53 D, supernatant from 2nd gel treatment 640 4.0 10.9

Total protein

-

?rotein per ml.

preparation when added to it. Most of the studies reported below were carried out with Fraction C.

The enzyme shows the usual protein peak at 276 to 280 rnp and an addi- tional peak at 406 to 408 rnp which, however, is probably due to an accom- panying hematin impurity. The absorption spectrum of the enzyme in the visual and the ultraviolet regions was not altered in the presence of the substrates. Microbiological assays did not reveal unusual amounts of either folic acid or vitamin B,z.

The enzyme was found to withstand formaldehyde concentrations up to 0.2 gm. per cent (0.073 M) without loss in activity; it did not, however, survive treatment with 4 per cent formaldehyde.

The enzyme was not active in the presence of tris(hydroxymethyl)amino- methane or histidine buffer, was only partially active in citrate, but seemed to be essentially unaffected by glycylglycine, pyrophosphate, and versenate. It was concluded, therefore, that a metal activator was prob- ably not involved in the reaction.

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H. RIFT AND H. R. MAHLER 903

Assay and Analytical Methods

The enzyme did not require adenosinetriphosphate or any other external “activating” mechanism. The assay mixture consisted of 30 ,UM each of formaldehyde (prepared according to Boyd and Logan (3), from paraform- aldehyde which was redistilled and standardized by titration) and lithium pyruvate (recrystallized), 40 PM of sodium bicarbonate, a suitable amount of enzyme, and water to a final volume of 3.2 ml. For convenience, the reaction was set up in Warburg vessels; the mixture was gassed with 5 per cent COZ in nitrogen and incubated at 37”.

There was no release or uptake of CO2 at any time during the incubation, which was sometimes allowed to proceed for as long as 3 hours. Additions of DPN were without effect. The reaction with the purified enzyme, when buffered with phosphate, proceeded as well in air as in Ns-C02. These observations led to the conclusion that we were dealing with a condensation reaction rather than with an oxidation-reduction or a DPN-linked dis- mutation.

The final pH of the incubation mixture was about 7.5. The reaction was stopped by the addition of 0.4 ml. of 2 M metaphosphoric acid. The deproteinized supernatants were analyzed for formaldehyde by the chro- motropic acid procedure of Boyd and Logan (3), with slight modifications, and for pyruvate and total a-keto acids by the method of Friedemann and Haugen (4). Under our experimental conditions, the methods proved to be accurate within ~t3 per cent. The reaction could be followed by ascer- taining the difference in the initial and residual amounts of either formalde- hyde or pyruvate. The product of the reaction behaved as an a-ket#o acid, and after incubation, the total cu-keto acid recoverable was considerably in excess over the residual pyruvate: the difference (total a-keto acid minus pyruvate) was a measure of the product formed.

A unit of enzyme activity was defined as the number of micromoles of pyruvate disappearing in the standard test system during 35 minutes in- cubation at 37”. The specific activity equals the number of units per mg. of protein.

Kinetics and SpeciJicity

The dependence of the reaction rate on enzyme concentration is shown in Fig. 1. Prolonged incubation of equimolar amounts of pyruvate and formaldehyde results in the disappearance of nearly equimolar amounts of the two substrates; a plateau is reached when one-half to two-thirds (about 15 to 20 PM) of the initial amounts have reacted (Fig. 2, A). The recoveries in total cr-keto acids vary between 70 and 100 per cent after incubation, since the reaction product reacts as an cr-keto acid. The results are essentially the same when pyruvate is present in excess (Fig. 2, A). If,

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904 ENZYMATIC CONDENSATION

however, an excess of formaldehyde is added (Fig. 2, B), the reaction rate is significantly increased; an excess of formaldehyde disappears and the total cy-keto acid recovery tends to be lower. It would seem, therefore, that the formaldehyde concentration is rate-determining and that excess formaldehyde is capable of initiating side reactions, possibly with the reac- tion product itself.

Neither formaldehyde nor pyruvate disappears when incubated singly

I I I I 0.5 1.0 2.0 3.0 I

mcj. PROTEIN

FIG. 1. Dependence of reaction on enzyme concentration. Incubation mixture, 30 PM of formaldehyde, 30 PM of pyruvate, 40 PM of sodium bicarbonate, enzyme at the protein levels indicated, and water to final voIume of 3.2 ml. Incubation at 37” for 90 minutes. Gas phase, 5 per cent CO2 in Nz. Final pH 7.5.

with the enzyme. Both can be recovered fully when they are incubated together in equivalent amounts in the absence of the enzyme, or with de- natured enzyme, or with beef albumin, or with active enzyme in the pres- ence of one of the inhibiting buffers, such as tris(hydroxymethyl)amino- methane. Prolonged incubation of excessive amounts of pyruvate with formaldehyde (ratio 4: l), in the absence of enzyme, may result in some disappearance of pyruvate which, in magnitude, however, never approaches the enzymatic values and for which a correction factor can be applied. Excess of formaldehyde does not seem to lead to any such non-enzymatic reactions.

Formaldehyde did not react enzymatically with acetaldehyde, benzalde-

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H. HIBY! AND H. R. MAHLER H. HIBY! AND H. R. MAHLER 905 905

hyde, crotonaldehyde, acetone, or folic acid, nor with acetoacetic, malonic, hyde, crotonaldehyde, acetone, or folic acid, nor with acetoacetic, malonic, mesoxalic, cr-ketoisocaproic, or P-ketoisocaproic acid. mesoxalic, cr-ketoisocaproic, or P-ketoisocaproic acid. a-Ketoglutaric acid a-Ketoglutaric acid was also essentially inactive. was also essentially inactive. Oxalacetic acid brought about a non-enzym- Oxalacetic acid brought about a non-enzym- atic reaction which was not significantly increased by the presence of the atic reaction which was not significantly increased by the presence of the enzyme. enzyme. Of all the substances tested, only phenylpyruvic acid was capable Of all the substances tested, only phenylpyruvic acid was capable of reacting with formaldehyde enzymatically, although it was only about of reacting with formaldehyde enzymatically, although it was only about

10-20-3040-5080-70-80-90 10-20-3040-5080-70-80-90 10-20-30-40-50-60-7080-90 10-20-30-40-50-60-7~8~90 MINUTES INCUBATION MINUTES INCUBATION MINUTES INCUBATION MINUTES INCUBATION

FIG. 2. Dependence of reaction on substrate concentration. X = pyruvate; FIG. 2. Dependence of reaction on substrate concentration. X = pyruvate; 0 = formaldehyde; A = total a-keto acid. 0 = formaldehyde; A = total a-keto acid. A, solid lines, incubation mixture con- A, solid lines, incubation mixture con- taining 30 PM of pyruvate and 30 NM of formaldehyde. Broken lines, incubation taining 30 PM of pyruvate and 30 NM of formaldehyde. Broken lines, incubation mixture containing 126 pM of pyruvate and 30 fiM of formaldehyde. mixture containing 126 pM of pyruvate and 30 fiM of formaldehyde. B, incubation B, incubation mixture containing 30 pM of pyruvate and 120 PM of formaldehyde. mixture containing 30 pM of pyruvate and 120 PM of formaldehyde. Other conditions Other conditions as indicated in Fig. 1. Enzyme concentration, 3.4 mg. of protein, specific activ- as indicated in Fig. 1. Enzyme concentration, 3.4 mg. of protein, specific activ- ity 3.6. ity 3.6.

30 to 50 per cent as effective as pyruvate. 30 to 50 per cent as effective as pyruvate. Similarly, pyruvate would not Similarly, pyruvate would not react with either acetaldehyde, benzaldehyde, crotonaldehyde, or acetone. react with either acetaldehyde, benzaldehyde, crotonaldehyde, or acetone. The enzyme, thus, appears to be quite specific for both formaldehyde and The enzyme, thus, appears to be quite specific for both formaldehyde and pyruvate. pyruvate.

Approximate K, (Michaelis-Menten constant) values were determined Approximate K, (Michaelis-Menten constant) values were determined in both short term (35 minutes) and long term (90 minutes) incubations, in both short term (35 minutes) and long term (90 minutes) incubations, and found to be of the order of 5 to 6 X 10B6 M for pyruvate and about and found to be of the order of 5 to 6 X 10B6 M for pyruvate and about 9 X 10m6 M for formaldehyde. 9 X 10m6 M for formaldehyde.

A sample of crystalline aldolase was found to be inactive under our ex- A sample of crystalline aldolase was found to be inactive under our ex- perimental conditions. perimental conditions.

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906 ENZYMATIC CONDENSATION

Properties and Xtructure of Product

Preparation-It was discovered quite early in the investigation that, while pyruvate disappeared during the incubation, the recovery of total a-keto acid remained approximately 100 per cent, even after the formalde- hyde had been removed by steam distillation; the reaction product, thus, appeared to be a non-volatile oc-keto acid.

Large scale experiments were, therefore, set up under experimental con- ditions which had been found to be optimal in the experiments described above. About 8 mM of pyruvate and 20.5 mM of formaldehyde were in- cubated for 2 hours in the presence of 90 ml. of enzyme (= about 300 mg. of protein; specific activity about 4) in bicarbonate buffer (pH about 7.5). The incubation mixture was deproteinized with 2 M metaphosphoric acid (final pH 2.5 to 3), the protein removed by filtration through fluted paper, and the clear, almost colorless solution lyophilized to dryness. In this manner, most of the formaldehyde was removed either by polymerization or by evaporation. Simultaneously, a portion of the pyruvate was dis- tilled into the trap. The dry powder was dissolved in a minimum of water (about 20 ml.) and subjected to continuous ether extraction (with about 200 to 250 ml. of ether) for 24 to 36 hours. The extraction was essentially complete. The ether fraction was dried with solid sodium sulfate, filtered, and subjected to a careful vacuum distillation, with the distilling flask sub- merged in a water bath, at about 35”. When the volume had been re- duced to a few ml., the solution was transferred to a micro still, and the distillation was continued under the same controlled conditions until the residue consisted of a yellow, rather viscous material, a mixture of pyruvic acid and the product. At this point, white crystals began to separate. Crystallization was allowed to continue in the ice box for a number of days. The crystals were washed carefully by decantation with ice-cold ether and dissolved in ether at room temperature; the solution was filtered through sintered glass, a little petroleum ether (b.p. 48-58”) was added, and the mixture concentrated until the crystals formed again. If the distillation was conducted too rapidly, a high melting, insoluble product was obtained, presumably a polymer. The recrystallized product was washed with ice- cold petroleum ether and dried in the cold room over calcium chloride in vacua, for 3 to 4 days; drying at room temperature or over PZOC yielded a yellowish more or less amorphous material, presumably a polymer.

The final product consisted of very hard, white, needle-shaped crystals, which had a tendency to adhere to the walls of the vessel and were slightly hygroscopic. The compound was soluble in water, alcohol, and ether, slightly soluble in petroleum ether and heptane, but insoluble in carbon disulfide. It dissolved in chloroform, but with deterioration, since only a resinous residue could be recovered upon removal of the solvent.

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H. HIFT AND H. R. MAHLER 907

The product was identified as the enol form of the y-la&one of oc-keto- y-hydroxybutyric acid; i.e., the lactone of the aldol condensation product of formaldehyde and pyruvate.

HCHO + CH,-CO-COOH --+ CHT-CHs-CO-COOH -+ I

OH

(1) OH I

CHz-CH=C-CO

1

The compound was found to have the following properties.

C4H40a. Calculated. C 48.05, H 4.03 Found. “ 47.80, ‘I 4.07, m.p. 105” (capillary, corrected)

No COZ was evolved when the compound was subjected to the aniline citrate test for fl-keto carboxylic acids (5). The ceric sulfate reaction for Lu-keto carboxylic acids (6) yielded 7.3 PM of CO2 evolved from a sample calculated to contain 7.36 pM of the la&one. The material gave a positive (reddish orange to brown) ferric chloride reaction. Titration with 0.0090 N NaOH (Fig. 3) yielded a curve characteristic of a very weak acid, with a pK of approximately 7.0. This result is consistent with neutralization of the weakly acidic enolic hydrogen. The lactone ring was preserved under these conditions.

X~ectru-The ultraviolet absorption spectrum is given in Fig. 4. A freshly prepared aqueous solution shows the rapid development of a peak at 226 rnp; a stable maximum is reached within 3 minutes. Upon addition of alkali, the peak at 226 rnp disappears in favor of a peak at 261 mp, which also reaches a stable maximum within 3 minutes. In acid solution only the peak at 226 rnp can be detected. In saturated heptane solution a shoulder is observed at 223 rnp, while in dilute heptane solution there is a peak at 254 rnp. The data indicate that the compound exists in the enol form in the solid state. In neutral or acid solution, the keto form pre- dominates, while the enolate anion is formed in alkaline solution. In con-

ii @-)

OH- I (2) CH2--CHZ-C-C=0 ;=t CH2-CH=C-C=O + H+

1,-J H+ IO--l

centrated hydrocarbon solution the compound exists as the keto form, which changes into the enol upon dilution. The observed displacement of the peaks is due to solvent effects. These data are in complete agree-

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908 ENZYMATIC CONDENSATION

8.0

=& 7.0

6:O

5.0

4.0 ’ I I I I I I 100 200 300 400 500 600

I. 0 il.7 AL!

FIG. 3. Titration curve pl. of 0.0090 N NaOH requ solid ner ml. Neutral eau

ps” I . “ , VI I

of the r-la&one ‘of a-keto-r-hydroxybutyri ired for 500 ~1. of lactone solution containin

_ ivalent, 122 (calculated 100).

c acid. 504 g 1.1 mg. of

I I I

200 250 2nn WV” 14n

YI”

WAVELENGTH my FIG. 4. Ultraviolet absor -*ption spectrum of the r-lactone of a-keto- .r-hydroxy-

butyric acid. Curve 1, 0.000 11 M aqueous solution, pH 2.3; Curve 2,O.OOOl 1 M aqueous solution, pH 11.8; Curve 3, d dilute solution in heptane. Cell thickness, 1 ( :m. Molar extinction coefficient of ketc ) form,4Onn- nf annl9t0 3f%M , ““, __ yyv.uuv, -Y-V.

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H. HIFT AND H. R. MAHLER 909

ment with the observed behavior of other ket’o-enols which have been de- scribed in the literature (7).

The molar extinction coefficient for the keto form in Hz0 at pH 2.3 at 226 rnp is 4000; for the enolate in Hz0 at pH 11.8 and at 261 rnp, 2600.

The infra-red spectrum is given in Fig. 5, A. The 2,4-dinitrophenylhydrazone melts at 218” with decomposition and

shows the usual peaks at about. 260 and 360 rnp. The compound has not been described in the literature. Plattner has

synthesized the P-methyl derivative which was studied by Schwarzenbach and Wittwer (8) and was found to exist as the enol in the solid state and to have a pK (enol) of 7.77. The data presented above for our unsub- stituted lactone are supported by these findings.

Identijication by Synthesis of Phenyl-Substituted Derivative

The earlier literature (9, 10) cites various attempts at condensing pyruvic acid and formaldehyde under laboratory conditions, but the products all appeared to be polysubstituted derivatives of the acid. Phenylpyruvic acid was condensed with formaldehyde bJi Hall, Hynes, and Lapworth (11) and by Hemmerle (12) ; the product in each case was identified as the y- lactone of a-keto-fl-phenyl-y-hydroxybutyric acid. This compound also was shown to exist as the enol.

Since phenylpyruvic acid showed slight activity with the enzyme de- scribed above, a large scale experiment was set up in the hope of isolating as the reaction product the P-phenyl derivative of our lactone. About 1.8 mM each of formaldehyde and phenyl pyruvate were incubated in the pres- ence of 2.4 mM of NaHC03 and 96 mg. of enzyme (specific activity on pyruvate basis, 10.9) for 5 hours. The method of isolation of the product was analogous to that described for the pyruvate-formaldehyde reaction product except that the water solution after lyophilization was subjected to a continuous extraction with ethyl acetate for about 40 hours. Upon concentration a white, granular precipitate formed. After storage in the ice box overnight, this sediment was repeatedly extracted with hot ether until only an insoluble residue remained, which was discarded. The ether extract was concentrated in vacua to a yellow solution, which was stored in the ice box. After about a week, white crystals had separated which were redissolved in ether; a little petroleum ether was added and the solu- tion concentrated in vacua and again stored in the ice box. The recrystal- lization was repeated two more times in order t,o remove any residual phen- ylpyruvic acid. Finally, the yellowish crystals were washed with petro- leum ether and dried in a desiccator over calcium chloride. The yield WM 3.5 mg.

An authentic sample of the phenyl lactone was synthesized from phenyl-

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910 ENZYMATIC CONDENSATION

pyruvic acid and formaldehyde according to the directions of Hall, Hynes, and Lapworth (11).

Both the synthetic and the enzymatic product melted at 197-198” (cap- illary, corrected), with no depression of a mixed melting point (reported

WAVE NUMBERS IN CM-’ xio-3 .A-54 32.5 2 1.51.41.3121.1 I .9 .8 .7 .6

Baird Associates, Inc., infra-red spectrophotometer, model B; NaCl prism. Spectra partially compensated for absorption of mulling oil. A, -Jactone of a-keto-r-hy- droxybutyrio acid; B, r-lactone of or-keto-p-phenyl-r-hydroxybutyric acid obtained enaymatically; C, the same obtained synthetically; D, -r-lactone of a-ketod-methyl- r-hydroxybutyric acid.

values 197-198” (lo), 202’ (12)). The compounds showed a greenish color with ferric chloride, as described in the literature.

The infra-red spectra of the two compounds are given in Fig. 5, B and C. A sample of the P-methyl derivative, kindly furnished by Dr. Plattner, was examined at the same time (Fig. 5, 0). It will be seen that the trac- ings of the two phenyl compounds are identical and bear a close resem- blance to the methyl derivative. The picture presented by the unsubsti- tuted la&one (Fig. 5, A) does not exhibit the degree of similarity which

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H. HIBT AND H. R. MAHLER 911

had been expected. However, it is believed that the differences noted may be attributed to the availability of the free p-hydrogens for strong intra- molecular hydrogen bonding. In any case, the spectral analyses furnish corroborative evidence identifying the formaldehyde-pyruvite condensa- tion product as the y-la&one of oc-keto-y-hydroxybutyric acid.

Primary Reaction Product

The condensation product, as it appeared in the incubation mixture, reacted as an or-keto acid under the conditions of the Friedemann-Haugen test; i.e., its 2,4-dinitrophenylhydrazone could be extracted into ethyl ace- tate and thence into carbonate. Yet, we were unable to obtain this reac- tion from the y-la&one which was isolated : its 2,4-dinitrophenylhydrazone could be extracted into ethyl acetate but not reextracted into carbonate. Since phenylpyruvic acid also failed to react under these conditions, it would appear that only fairly simple, open chain a-keto acids respond to the test. These results suggested that our primary reaction product was not the lactone, but the open chain acid (Equation 1). When a complete balance sheet was drawn up at each stage of the isolation procedure, it was observed that the recovery of total cr-keto acid showed a sudden drop after lyophilization and ether extraction, suggesting bonversion of the free acid to the lactone at these stages.

More direct evidence for the formation of the free acid in the reaction mixture was obtained as follows: when the 2,4-dinitrophenylhydrazone of the isolated lactone was exposed to alkali, a product was obtained which reacted in the Friedemann-Haugen test for cr-keto acids; i.e., it was ex- tractable into both ethyl acetate and carbonate. These observations can be explained by the reaction shown in Equation 3; i.e., by an alkali-depend- ent opening of the y-la&one ring. The compound thus formed is believed to be identical with the derivative giving the positive Friedemann-Haugen reaction in the original incubation mixture.

N-R NR 0

II II / CHr-CHz-C-C=0 + OH- + CHr-CHr-C-C

I --01

I \ OH 0 (3

(3)

NOz

In a more direct approach to the problem, the reaction mixtures before and after incubation with enzyme were deproteinized, 2,4-dinitrophenyl- hydrazine in HCl was added, and the hydrazones formed were collected.

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912 ENZYMATIC CONDENSATION

Similarly, the 2,4-dinitrophenylhydrazones of all necessary reference sub- stances were prepared. All the compounds were extracted into ethyl ace- tate and subjected to paper chromatography in a system consisting of 5 parts of tertiary amyl alcohol, 4 parts of water, and 1 part of ethyl alcohol (13). After about 20 hours, the results given in Table II were obtained. No additional spots could be detected.

On the basis of this evidence, Equation 1 is believed to describe the following reaction : formaldehyde and pyruvate condense enzymatically to

TABLE II Results of Paper Chromatography

2,4-Dinitrophenylhydrazone of RF

Reference Formaldehyde

r-Lactone of a-keto-y-hydroxybutyric acid*

Pyruvic acid or-Keto-r-hydroxybutyric acidt

Zero time Formaldehyde Pyruvic acid

After incubation Formaldehyde Pyruvic acid Primary reaction product

0.90

0.85

0.55 0.42

0.90 0.56

0.93 0.53 0.43

Appearance under

Visible

Yellow

‘<

“ ‘I

‘I “

‘L “ “

Ultraviolet

Quenching of paper fluorescence

Fluorescence

Quenching ‘I

‘I “

“ ‘I ‘I

Ascending chromatography; solvents as described in the text. Whatman No. 4 paper; no pretreatment. Spots applied by placing approximately 0.010 ml. of the carbonyl compounds in ethyl acetate solution on the starting line.

* Product isolated from a large scale experiment. t Obtained by alkaline hydrolysis of lactone-2,4-dinitrophenylhydrazone.

form o-keto-r-hydroxybutyric acid which, during subsequent treatment and extraction, is converted into the y-lactone.

DISCUSSION

The enzyme-catalyzed condensation reaction described in this communi- cation constitutes the first such reaction definitely implicating formdde- hyde. It also assigns a new metabolic r61e to pyruvate.

It is proposed to call the enzyme “formaldehyde-pyruvate carboligase.” Mitchell and Artom (14) independently obtained preliminary data sug-

gesting that they were dealing with a similar enzyme system. Bernheim (15) described an enzyme system from rat liver which oxidizes formalde-

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H. HIF’I’ AND H. R. MAHLER 913

hyde to formic acid in the presence of aminoguanidine. Additions of the latter compound had no effect in our system. 1 The possible biological significance of the condensation reaction and of the product formed has not yet been investigated. The reaction involves ‘2 very reactive molecules and results in the formation of a 4-carbon com- podnd with three functional groups, a molecule which could be expected to undergo a great variety of reactions. A metabolic r&e for formaldehyde has been repeatedly suggested in recent years (16-18). The reaction de- scribed may be one way in which this compound may enter the pathways of intermediary metabolism.

SUMMARY

An enzyme was purified from beef liver which condenses formaldehyde and pyruvate to yield cr-keto-y-hydroxybutyric acid as the reaction prod- uct. The compound was isolated from the reaction mixture as the y-lac- tone and its properties were described. Phenylpyruvic acid under the same conditions yielded the y-la&one of a-keto+phenyl-y-hydroxybutyric acid. No other aldehyde, ketone, or ketonic acid tested was active in the system.

This investigation was aided by a grant from the National Heart Insti- tute of the National Institutes of Health.

The authors are indebted to Mr. G. Winestock for carrying out the ( and H microdeterminations, and to Mr. D. Johnson for the extensive infra- red spectrophotometry. We are also grateful to Mrs. C. A. Claus, Jr., for the examination in the ultracentrifuge and to Dr. L. J. Teply for the micro- biological vitamin analyses. The authors wish to thank Dr. D. E. Green for his continued interest and advice during the course of this investigation and Dr. D. R. Sanadi for many helpful discussions.

BIBLIOGRAPHY

1. Sarkar, N., Beinert, H., Fuld, M., and Green, D. E., Arch. Biochem. and Biophys., 37, 140 (1952).

2. Racker, E., J. BioZ. Chem., 177, 883 (1949). 3. Boyd, M. J., and Logan, M. A., J. Biol. Chem., 146, 279 (1942). 4. Friedemann, T. E., and Haugen, G. E., J. Biol. Chem., 147,415 (1943). 5. Edson, N., Biochem. J., 29, 2082 (1935). 6. Bamann, E., and Myrblck, K., Methoden der Fermentforschung, Leipzig, 1,

277 (1941); method of Fromageot, C., and Desnuelle, P., Biochem. Z., 279, 174 (1935).

7. Wheland, G. W., Advanced organic chemistry, New York, 580 (1949). 8. Schwarzenbach, G., and Wittwer, C., Helv. chim. acta, 30,663 (1947). 9. Hosaeus, H., Ann. Chem., 276, 79 (1893).

10. Asalinua, Y., and Terada, S. I., Chem. Zentr., 1, 1818 (1927).

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914 ENZYMATIC CONDENSATION

11. Hall, N., Hynes, J. E., and Lapworth, H., J. Chem. Sot., 107, 132 (1915). 12. Hemmerle, D., Ann. chim., 7, 269 (1917). 13. Altmann, S. M., Crook, E. M., and Datta, S. P., Biochem. J., 49, p. lxiii (1951). 14. Mitchell, P., and Artom, C., Federation Proc., 10, 224 (1951). 15. Bernheim, F., J. Biol. Chem., 186, 225 (1950). 16. Handler, P., Bernheim, M. L. C., and Klein, J. R., .I. Biol. Chem., 138,211 (1941). 17. Ling, K.-H., and Tung, T.-C., J. Biol. Chem., 174, 643 (1948). 18. Mackenzie, C. G., J. Biol. Chem., 186,351 (1950). 19. Weichselbaum, T. E., Am. J. Clin. Path., 16, Tech. Sect., 7, 40 (1946).

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