ENZYMIC CATALYSIS OF THE KETO-ENOL TAUTOMERIZA- TION OF PHENYLPYRIMC ACIDS*

BY W. EUGENE KNOX AND BURNETT M. PITT

(From the Cancer Research Institute, New England Deaconess Hospital, and the Department of Biological Chemistry, Harvard Medical School,

Boston, Massachusetts)

(Received for publication, August 2, 1956)

The present paper describes a new enzyme from animal tissues which catalyzes the keto-enol tautomeriaation of the phenylpyruvic acids. Pre- liminary accounts of this tautomerase have been published (1, 2). The enzyme has not yet proved to be essential in any biological reaction, but a physiological role may be expected since specific tautomers of a-keto acids are substrates or products in some reactions (3-6), and since the spontaneous tautomerization of the phenylpyruvic acids is slow. The spontaneous reaction, from freshly dissolved aqueous solutions of the crys- talline acid (enol) to equilibrated solutions (largely keto), has been studied by following the increased solubility, decreased ultraviolet absorption, and increased reaction with phenylhydrazine which occur in parallel with the tautomerization to the keto form (7-10).

A new assay was developed to follow the enzymic reaction which de- pended upon the formation of a complex between boric acid and the enol tautomer, and which could be followed spectrophotometrically. Com- plexes of boric acid with a-hydroxy and cY-keto acids were studied by Bee- seken and Felix (ll), but the formation of an enol-borate complex was not recognized in these earlier studies. In aqueous solutions without boric acid 96 per cent of the equilibrium mixture was the keto tautomer (I). The boric acid reacted with the enol tautomer (II) to form an enol-borate (III) with its own ultraviolet absorption. The displacement of the ap- parent equilibrium of the tautomerization with boric acid permitted the rate of tautomerization to be followed in the keto to enol as well as in the enol to keto direction. Since boric acid also suppressed the rate of spon- taneous tautomerization, the reaction in this medium tprovided a con- venient and precise enzyme assay (see the accompanying reactions).

* This investigation was supported by Grant A567 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service, and by the United States Atomic Energy Commission contract No. AT(30-l)-901 with the New England Deaconess Hospital. Preliminary investigations (1952) were supported by the United States Atomic Energy Commission contract No. At(30-l)-1093 under the direction of Dr. William H. Sweet, Massachusetts General Hospital.

675

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676 KETO-ENOL TAUTOMERASE

?H l~)2R-CH=C-C00H+li,i30~~

j+Cd+--~=O (-I 9 P + H’ 4 t3

H 0’ ‘0 +3H,O

R&=&f=0

EXPERIMENTAL

Spectroscopically pure p-hydroxyphenylpyruvic acid (pHPP), free from a strongly absorbing impurity identified as p-hydroxybenzaldehyde,’ was obtained from Homburg Chemiewerk, Frankfort-on-the-Main, or pre- pared by the general method used for the other phenylpyruvates (12). 2,5-Dihydroxyphenylpyruvic acid was prepared by a different procedure (13).

Enzyme Assay The reactions and optical measurements were carried out in 1 cm. quartz

cuvettes in a Beckman spectrophotometer, maintained at 25” f lo by thermal spacers. The standard reaction mixture consisted of 2.8 ml. of a solution containing 0.5 M boric acid and 0.2 M sodium phosphate, pH 6.2, to which were added 0.2 ml. of 0.005 M pHPP dissolved in 0.05 M ace-

tate, pH 6.0, water, and enzyme to a final volume of 3.3 ml. The pHPP solution was used for reactions of the enol tautomer of pHPP immediately after preparation and after the 2nd to the 30th day at 5” for reactions of the keto tautomer of pHPP.

Optical densities of the reaction mixtures were read against blanks con- taining all components except pHPP, at 330 rnp for the reaction of the keto form, and at 336 rnp (to avoid extremely high optical densities) for the reaction of the enol form. Readings were continued until equilibrium was reached, indicated by an unchanged optical density over a period of several minutes. Exactly similar measurements were made of the spon- taneous reaction occurring in the same solutions without enzyme. The rate constants were determined from the slope of log (Et - E,) verse t, where E is the optical density at times t and at equilibrium (a), with the use of semilog paper. The values used are 0.434 (kl + kz) in Equa- tion 1. The activity of the enzyme (Ic,) was determined from the rate constant for its reaction mixture (k,) minus that of the spontaneous reaction without enzyme (k,).

1 W. Troll, unpublished observations.

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W. E. KNOX AND B. M. PITT 677

The changes in optical density during the tautomerization of the keto forms of pHPP and of phenylpyruvate, occurring spontaneously and cata- lyzed by a crude soluble extract of guinea pig liver, are shown in Fig. 1. Also illustrated is the degradation of pHPP which occurred in crude liver extracts, presumably by pHPP oxidase (14), thus making it necessary to approximate the equilibrium value in such reactions. Phenylpyruvate was not changed in this way, nor was either of the compounds changed by

-.- ,*‘-*

,.C

__-- - PHENYLPYRUVATE --- pHPP

I. WITH ENZYME .I 2. SPONTANEOUS REACTION 0 I 1 I 1

IO 20 30 40 TIME IN MINUTES

FIG. 1. Optical density changes occurring during the incubation of keto pHPP (330 mp), in boric acid-phosphate alone (Curve 1, broken line) and with soluble guinea pig liver extract (equivalent to 0.06 gm. of liver) (Curve 2, broken line), and of keto phenylpyruvate (315 rnp) under the same conditions, alone (Curve 1, solid line) and with the liver extract (Curve 2, solid line).

kidney extracts, which have much less pHPP oxidase activity. Purifica- tion of the liver enzyme removed the pHPP-degrading reaction.

Reactions in Boric Acid

Identity of Species of Compounds-The absorption spectrum of the “keto” solution of pHPP (Fig. 2) differed from the absorption curve of tyrosine only by the presence of a shoulder at 300 to 321 rnp. The more intense absorption curve of the enol form of pHPP was obtained by rapid scanning of the freshly made solution in a Cary recording spectrophotometer and the values were extrapolated to zero time to eliminate the effect of the spontaneous tautomerization. On addition of boric acid to the keto form the curve was not immediately altered, but the absorption at 308 rnM in-

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678 KETO-ENOL TAUTOMERASE

creased slowly with time (Curve A) until an equilibrium value was reached (Curve B). The component formed in boric acid and absorbing at 308 rnp was the enol-borate complex. This was obtainable in pure form imme- diately after solution of the crystalline (enol) compound in boric acid solu- tion. The curve of the enol-borate complex was shifted to longer wave lengths, but was otherwise similar to the curve of the enol form of pHPP

--- ENOL

220 240 260 280 300 320 340

my FIG. 2. Absorption spectra of p-hydroxyphenylpyruvate solutions (e = optical

density of 1 M solution, 1 cm. in depth). Errol, spectrum of crystalline acid in 0.05 M

acetate, pH 6.0, determined on a Cary recording spectrophotometer within 5 min- utes after solution and corrected to zero time. Enol-borate, same as enol, dissolved in 0.43 M boric acid, 0.17 M phosphate, pH 6.4. Keto, acetate solution equilibrated for at least 24 hours. Tautomerized solutions in boric acid-phosphate: keto solution 96 minutes after addition of boric acid (Curve A); keto (or enol) solution at equi- librium, 24 hours or more after the addition of boric acid (Curve B).

in the absence of boric acid. Similar absorption curves were obtained for the enol and enol-borate of phenylpyruvate (maxima at 284 and 298 rnp, respectively). The absorption curves of model compounds with the same unsaturated structures, a-acetoxycinnamic acid and p-hydroxy-cr-acetyl- aminocinnamic acid, were very similar to those of the enols of phenylpyru- vate and pHPP, respectively. Additional confirmation of the identities of the enol, the keto, and the enol-borate species was provided by their measurement at 334 rnp in 0.02 N HCl, as used by Biicher and Kirberger (lo), and by the determination of the keto compound with 2,4-dinitro-

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W. E. KNOX AND B. M. PITT 679

phenylhydrazine (15), a reaction not given by either the enol (9) or the enol-borate species. The interconversion of the tautomers under appro- priate conditions was demonstrated by this chemical reaction in parallel with the spectrophotometric changes.

The small amount of the enol form of pHPP existing in the equilibrated keto solution (Fig. 2) can be estimated either from the extinction at 308 mP (eketo = 500, ho1 = 13,000), with the assumption that the pure keto pHPP, like tyrosine, does not absorb at this wave length, or from the imme- diate small decrease in absorption at 276 rnp when boric acid is added to the equilibrated keto solution. These measurements confirmed the esti-

TABLE I

E$ect of Boric Acid Concentration on Enzyme-Catalyzed pHPP-Borate Complex Equilibrium

The reactions were run to completion in 0.17 M phosphate, pH 6.55, with 0.075 ml. of enzyme. The equilibrium constants for reaction (b) were calculated for enol- borate complexes of 4 (EB.$, 1 (EB), and 2 (EzB) molecules of enol pHPP per mole- cule of boric acid.

I

I

Boric acid Equilibrium density at 330 rn~

Equilibrium constants calculated for complexes

EB2 EB Ed3 X 10-e

24

0.085 0.17 0.30 0.43 0.64 0.85

0.820 890.0 76.0 4.0 1.33 430.0 73.0 4.5 1.65 198.0 59.0 4.2 1.96 133.0 56.5 4.6 2.20 75.4 48.0 4.4 2.40 52.0 44.2 4.6

mate made on kinetic grounds that about 4 per cent of the enol form of pHPP was present at equilibrium in the range pH 4 to 7 (10).

Equilibrium in Boric Acid-The relation between the equilibrium amount of enol-borate and the boric acid concentration agreed closely with that expected for the reaction of 1 molecule of boric acid with 2 enol molecules as formulated in reaction (6). With [enolJ/[keto] = 0.04/0.96 as meas- ured in water solutions, the equilibrium constant for reaction (b), K = [(enol)zborate]/[enol]2[borate], fits the observed values better than did the calculations based on the reaction of + or 1 enol per molecule of boric acid (Table I).

The extinction coefficient of the enol-borate, measured in fresh solutions of crystalline pHPP, and the equilibrium concentration of the enol-borate were not significantly changed over the range pH 5.0 to 7.0. At pH 8.1 the equilibrium concentration was about 25 per cent less than that at the

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680 KETO-ENOL TAUTOMERASE

lower pH. The constancy of these values in the pH range used, plus the large equilibrium constant which indicated that virtually all the enol form of pHPP reacted with boric acid, permitted the ultraviolet absorption at equilibrium to be used to determine pHPP quantitatively. In the pres- ence of excess boric acid and in this pH range the equilibrium absorption was directly proportional to the concentration of pHPP.

Kinetics-Biicher and Kirberger (10) demonstrated that the spontane- ous tautomerization of the enol form of pHPP followed reversible first order kinetics, increasing in rate with increase in pH. The rate constant

-WITH ENZYME

--- SPONTANEOUS REACTION

1 I 2 3 4 5 6

MINUTES FIG. 3. Comparison of the reaction rates of pHPP starting with the keto tautomer

(K -+ E) and with the enol tautomer ( E -+ K) for both enzyme-catalyzed and spon- taneous reactions. Changes in optical density (Et - E,) are plotted.

i& + kz for reaction (a) was obtained by the relation

0-L - Et) &I + k2)t = In (E, _ E,)

We have confirmed this relationship and shown that it also holds for the spontaneous tautomerism of pHPP in either direction in boric acid solu- tion and for the enzyme-catalyzed tautomerization with or without boric acid.

The applicability of boric acid in rate studies of tautomerization re- quired that the formation and hydrolysis of the enol-borate were not rate- limiting. The formation of the enol-borate was rapid, since only its spec- trum was seen immediately after solution of the crystalline enol form of pHPP in boric acid (within 5 minutes). Direct evidence that these reac-

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W. E. KNOX AND B. M. PITT 681

tions were not limiting, i.e. (kg + Jcq) > (kl + kJ, was obtained by com- parison of the tautomerizations starting with either the enol form of pHPP or with the keto form of pHPP, in which enol-borate hydrolysis and forma- tion, respectively, were the major secondary reactions. The rate constants in both directions were the same within experimental error for the spon- taneous reactions and for the 25-fold greater rates of the enzyme-catalyzed reactions (Fig. 3). No limitation by the formation or hydrolysis of the enol-borate was detected in the most rapid tautomerizations studied with half times of the order of 0.75 minute.

TABLE II

Distribution of Tautomerase in Tissues

Rabbit kidney. Rat liver. . . . . . Rabbit liver. . . Beef kidney.. Dog liver. . . . Beef “ Pig “ Guinea pig liver. Pig kidney.. Rabbit heart. .

“

. . .

. . . .

erythrocytes, washedt. “ muscle.................

Horse serumI. . . . .

. . ............. 2.6 . . ............. 1.7

. . . . ............. 1.3 ............. 0.67

. . ............. 0.51 ............. 0.45-1.3 ............. 0.43-1.1 ............. 0.334.90 ............. 0.30-0.55 ............. 0.26 ............. 0.21 ............. 0.072 ............. 0.03

Tissue k, per ml.*

* The activity is given for the supernatant fraction of 33 per cent tissue homog- enates. The ranges of values represent measurements made on several animals.

t The activity is given per ml. of whole blood. $ Undiluted.

Preparation of Tautomerase

Tautomerase activity was first detected incidentally by the dramatic increase in optical density when the keto form of pHPP was incubated with liver extracts in a borate buffer. Evidence that this activity was referable to a particular enzyme protein was obtained from its distribution, its heat lability, and its association with a particular protein fraction during partial purification.

The distribution in various tissues of the activity demonstrated that this was not a non-specific property of proteins in general (Table II). Be- sides the several animal tissues, activity was found in crude extracts of species of Pseudomonas and Neurospora, but not of Aerobacter. The ac- tivity of liver extracts persisted after dialysis, but was destroyed by heat.

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682 KETO-ENOL TAUTOMERASE

The per cent original activity left after 5 minutes treatment at the following temperatures was as follows: 56”, 100 per cent; 65”, 82 per cent; SO”, 23 per cent; loo”, 0 per cent.

Purification was undertaken to increase the specific activity and thus provide evidence that a particular protein was responsible for the activity. In the course of this fractionation functional separation of the tautomerase from tyrosine transaminase, pHPP and homogentisate oxidases, and catalase was also achieved. Similar methods of purification were applicable to both pig liver (1) and pig kidney. The absence of side reactions in crude extracts of kidney made it the preferable starting material. The soluble extract of a 33 per cent homogenate in 0.15 M KC1 of fresh kidney, or of an acetone powder, was treated successively by heat, two precipitations

TABLE III

Fractionation of Keto-Enol Tautomerase from 53 Per Cent Homogenate of Pig Kidney

Fraction volume Protein per ml.

ml. w.

Solubleextract..................... 1650 64 Heat at 56”, 4 min.. . . . . . . . . 1280 37 1st (NH&S04 ppt., 40-65yo.. . 188 49.3 2nd “ “ 40-50%. . 50 46.5 Alcohol ppt., O--50%. . 56 27.2

“ “ 5&75yo. . . . . . 60 3.74

* The figures are in k, per ml. X volume.

ks er B

Total m. activity* Yield

per cent

0.525 865 100 0.560 717 83 3.0 564 65 5.54 277 32 2.34 131 15 1.4 84 9.7

0.0082 0.015 0.061 0.12 0.086 0.37

between the limits of 40 and 65 per cent saturation with ammonium sul- fate, and, after dialysis against 0.03 M sodium acetate, by alcohol frac- tionation at 0”. The results of such a fractionation are given in Table III. The best fraction contained about 10 per cent of the original activity, with a 45-fold increase in the specific activity per mg. of protein. The purified fractions have been stored in the frozen state at -10” for over a year without loss of activity, despite periodic thawing and refreezing.

Enzyme-Catalyzed Tautomerixation

E$ect of Enzyme Concentration-The rate of tautomerization was a linear function of the enzyme concentration, as shown in Table IV by the con- stant k, per ml. determined with several different enzyme concentrations. The half time and final optical density at equilibrium for each reaction are also given in Table IV to demonstrate the rapidity of the reaction. The attainment of equilibrium, representing an optical density increase of 1.6, was completed in 5 minutes with 0.2 ml. of enzyme.

Effect of p&--The spontaneous reaction rate was inversely proportional

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W. E. KNOX AND B. M. PITT 683

to the hydrogen ion concentration over the pH range examined, as shown by the straight line relationship of lc, versus pH on the semilog plot in Fig. 4. The much more rapid reaction catalyzed by 0.075 ml. of a purified enzyme preparation (k,, Fig. 4) was unaffected by the hydrogen ion concentration.

TABLE IV E$ect of Tautomerase Concentration on Reaction Rate

Enzyme

ml.

0.05 0.10 0.15 0.20

ka observed

0.106 0.219 0.326 0.431

Ewif-y;p~yit Reaction hati the ke per ml.

min.

1.75 2.84 2.12 1.75 1.37 2.19 1.65 0.92 2.18 1.70 0.70 2.16

4.0-

3.0 -

2.0 -

F a I.o- 02 -

$0.6- F - ::0.4- W oz -

f /’ ,’

,/’ /’

,A” ,T’

4 /

,/ 4’

t

-ENZYME CATALYZED REACTION 0.2 K, / ML ENZYME

---SPONTANEOUS REACTION X0 X 100

0.1 ’ I I I I I I

6.0 6.5 7.0 7.5 6.0 8.5 PH

FIG. 4. Effect of pH on spontaneous and enzyme-catalyzed tautomerization of pHPP in boric acid-phosphate solutions.

The tautomerization therefore is base-catalyzed in the absence of the en- zyme, while the enzyme exercises a nearly maximal effect over the pH range studied without exhibiting a real pH optimum. The reactions were usually carried out at pH 6.2 because of the large differential at this pH between the spontaneous and enzyme-catalyzed reaction rates and be- cause of the greater stability of pHPP in weakly acidic solutions.

Activation and Inhibition of Reaction-The most significant effects of

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684 KETO-ENOL TAUTOMERASE

added compounds on the enzyme activity were observed with sulfhydryl- reactive compounds (Table V). The reversal with cysteine of the p-chloro- mercuribenzoate and mercuric ion inhibitions suggested that a sulfhydryl group on the enzyme was required for its activity. Certain other sub- stances had minor effects on the rate of the spontaneous or enzyme-cata- lyzed reactions. The metal salts, Fe+++ and Cu++, were less effective

TABLE V

Effect of Various Substances on Tautomerase Activity

Compound added

Dipyridyl ....................................... H&3, ............................................ N&N. .......................................... Versene ........................................ Mercuric acetate .................................

“ <I ................................. “ “ + cysteine (3 X 10m3 M). .......

ICH,COOH. .................................... p-Chloromercuribenzortte ........................

I‘ + cysteine (3 x 10m3 M) Maleic acid ...................................... Cysteine .........................................

“ ............... ......................... ol-Ketoglutarate ................................ a-Acetoxycinnamic acid .......................... Cinnamic acid ..................................

T --

-

Concentration

2d x lo=

1.5 3.0 1.0 3.0 0.015 0.03 0.03

30.0 0.04 0.04

100.0 3.0 6.0

38.0 2.0

10.0

Per cent of control activity

95 75

144* 106 75 30 57t 20 59 w 48

107$ 135$ 34 61 61

* Determination of activity was complicated by a simultaneous reaction of the substrate with cyanide.

t Inhibitor added to enzyme 3 minutes before cysteine. $ Enzyme incubated with 0.05 M cysteine for 30 minutes, then diluted for assay.

accelerators in the boric acid assay than has been reported (10). The en- zyme activity was not significantly altered by reagents forming metal com- plexes, such as cu,a’-dipyridyl or Versene. The effect of other compounds, similar in structure to the substrates, is discussed below.

The rates of the spontaneous and enzyme-catalyzed reactions of pHPP and phenylpyruvate were both similarly affected by the nature of the buf- fer salts employed. Acetate, glycine, phosphate, and arsenate, in that or- der, increased the spontaneous rate of tautomerization when present in 0.17 M concentration. Arsenate, which had the greatest effect, produced a 20-fold increase in the rate over that in boric acid alone. The rate could

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W. E. KNOX AND B. M. PITT 685

be increased further by raising the arsenate concentration (Table VI). In certain analytical procedures depending upon tautomerization, high arse- nate concentrations can therefore be substituted for the tautomerase. The slight acceleration of the tautomerization by guinea pig urine, observed by Painter and Zilva and used by them to insure equilibration to the keto form (9), was not observed in the boric acid assay. This effect has been

TABLE VI

Effect of Arsenate on Rate of Spontaneous Tautomerization of pHPP

Arsenate (pH 6.5) I Borate I ko

M

0.17 0.35 0.53 0.70 1.7

dl

0.43 0.43 0.43 0.43 0.85

0.021 0.057 0.104 0.152 0.393

TABLE VII

Reactivity of Arylpyruvates with Tautomerase

Substrate 1 Assayed at / ko 1 k, / ;

Phenylpyruvate........................... 315 0.020 3.20 p-Hydroxyphenylpyruvate. . . . 330 0.007 2.62 m-Hydroxyphenylpyruvate. . . . 310 0.023 1.04 p-Methoxyphenylpyruvate. . . . . 320 0.010 0.50 2,5-Dihydroxyphenylpyruvate . . . . . 340 0.036 0.26

160 370

45 50

I 7

attributed to catalysis by polyvalent cations (lo), but it could also be ex- plained by catalysis of the spontaneous reaction by the salts in the urine.

The rate of the enzyme-catalyzed reaction was not increased by addition of the above salts, but was usually somewhat slowed. Glycine and phos- phate inhibited little or none. Acetate had the greatest effect by slowing the enzyme-catalyzed reaction to about half.

Xpecijicity of Reaction-The study of enol-keto tautomerization of Lu-keto acids by the present method is limited to those compounds forming enol- borate complexes with identifiable absorption spectra. The spontaneous and enzyme-catalyzed reactions of such a series of phenylpyruvic acids are given in Table VII. The enzyme showed some degree of specificity, man- ifested by the different relative rates of reaction of the compounds. The

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686 KETO-ENOL TAUTOMERASE

catalyzed reaction rates were different from the relative rates of the spon- taneous tautomerization of the same compounds. 2,5-Dihydroxyphenyl- pyruvate, for example, was tautomerized least rapidly with the enzyme and most rapidly without the enzyme. In addition to the reactions shown in Table VII, the enzyme had only a very weak action on imidazolel-pyruvic acid, formed directly in the reaction mixture by oxidation of n-histidine with ophio-n-amino acid oxidase, and the enzyme had no action on indole- pyruvic acid.

Other compounds, including imidazoleacetol, pyruvate, cr-ketoglutarate, and oxalacetate, were tested without clear-cut results. The first two com- pounds underwent no detectable spectral changes with boric acid. The new absorption spectra formed in boric acid with the last two compounds could not be attributed with certainty to tautomerization.

Oxalacetate showed no significant change with keto-enol tautomerase in the rate of formation or disappearance of the new maximum at 270 rnp in the presence of boric acid, nor did the enzyme affect the rate of formation of the aluminum complex, which has its maximal absorption at the same wave length and which is thought to be the enol oxalacetate-aluminum complex (16). Support for the view that the enol was formed with boric acid (and with aluminum) and that the enzyme did not affect the rate of these reactions was provided by analogy with the reactions of pHPP. The spectrum of the pHPP-aluminum complex was virtually identical with that of enol pHPP-borate complex. The tautomerase catalyzed the formation of this pHPP-aluminum complex from keto pHPP.

a-Ketoglutarate in boric acid increased its absorption at 260 rnp follow- ing reversible first order reaction kinetics at 50 times the rate observed with pHPP, but the rate was not affected by tautomerase. The inhibition by a-ketoglutarate of the tautomerieation of pHPP by the enzyme (Table V) suggested that cr-ketoglutarate in some form did react with the enzyme. This inhibition was proportional to the log of ol-ketoglutarate concentra- tion and appeared to be competitive in nature. cr-Acetoxyphenylpyruvic acid and cinnamic acid also inhibited the enzyme, perhaps because of struc- tural similarity to the substrates.

The possibility was considered that the spontaneous and the enzyme- catalyzed tautomerizations might produce enols of different configurations which would be reflected in the absorption spectra of the enol-borates. Cis and trans enol forms of ethyl phenylpyruvate, separable on the basis of physical properties, have been reported (17). Careful comparisons of the spectra resulting from the spontaneous and the enzyme-catalyzed tau- tomerizations of keto pHPP and of keto phenylpyruvate showed no differ- ences in extinction coefficients or in wave length of the absorptions of the

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W. E. KNOX AND B. M. PITT 687

enol-borates produced. Additional experiments on Lu-keto acids with asymmetric ,&carbon atoms will be necessary to determine the configu- rational specificity of this reaction.

DISCUSSION

The present demonstration of a specific enzymic catalysis of enol-keto tautomerism is the first such enzymic reaction to be reported. The phys- iological role of this enzyme remains to be demonstrated, but it may be based on the quite different chemical reactivities of the enol and keto tau- tomers. It has not yet been determined which tautomer is involved in the oxidation of pHPP to homogentisate with pHPP oxidase or in the trans- amination of pHPP to tyrosine by the specific transaminase, but these reactions are not sensitive to the addition of boric acid (unpublished obser- vations). The metabolic reactions of phenylpyruvate have not been stud- ied in this regard. The availability of the keto-enol tautomerase may serve further to elucidate the mechanisms of reactions in which its sub- strates are involved.

Certain reactions of other cw-keto acids are believed to involve either the keto or the enol form. The carboxylation of phosphoenolpyruvate pro- duced the keto tautomer of oxalacetate (3), and the oxidation of the ster- eoisomers of isoleucine or phenylserine by amino acid oxidases occurred with retention of the p-carbon configuration, presumably by production of the keto tautomer (4, 5). The keto tautomer reacted in the metal-cata- lyzed decarboxylation of dimethyloxalacetate and yielded the enol of c11- ketoisovalerate (6).

Immediate applications of the present findings lie largely in the analyt- ical assay of the phenylpyruvic acids and in the control or identification of a particular tautomeric species undergoing reaction. The conversion of the keto acid to the strongly absorbing enol-borate by addition of excess boric acid in the presence of the tautomerase or of arsenate provides a rapid, specific, and sensitive test for these acids. Aluminum salts can be substituted for boric acid under some conditions. With limiting amounts of tautomerase the particular tautomer reacting or being formed can be identified directly.

Enol-borate complexes of a-keto acids have not previously been reported and were, in fact, specifically excluded (11) as a possible structure of the complexes between boric acid and cr-keto acids which gave rise to increased conductance. Certain cu-keto acids without P-hydrogens, and therefore unable to enolize, reacted in some other way with boric acid, but with only small increases in conductance. A restudy of the conductance experi- ments on cr-keto acids would appear to be indicated, especially if done in

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688 KETO-ENOL TAUTOMERASE

conjunction with spectrophotometric studies which have already given a reasonably exact definition of the enol-borate complex and the kinetics of its formation.

SUMMARY

An enzyme of a new type which catalyzed the keto-enol tautomerization of certain phenylpyruvates has been found in various tissues. The distri- bution, properties, and partial purification of the tautomerase from hog kidney are described.

A new spectrophotometric assay for the phenylpyruvates was developed. This method depended on the formation of a strongly absorbing enol-bo- rate complex with apparent displacement of the usual keto-enol equilib- rium. The rate of formation or hydrolysis of the complex depended on the rate of tautomerization of the substrate. The kinetics of this system have been evaluated for both the enzymic and the spontaneous reactions.

The authors wish to thank Dr. Robert I. Gregerman for his determina- tion of the effect of certain compounds on the activity of the enzyme, Dr. E. C. C. Lin for his assistance in the preparation of hog kidney en- zyme, Miss Lois Walker for her technical assistance, and Dr. Edward J. Modest and Dr. Herbert N. Schlein of the Children’s Cancer Research Foundation for the determinations with the Cary recording spectrophotom- eter.

BIBLIOGRAPHY

1. Knox, W. E., in Colowick, S. P., and Kaplan, N. O., Methods in enzymology, New York, 2, 289 (1955).

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W. Eugene Knox and Burnett M. PittPHENYLPYRUVIC ACIDS

KETO-ENOL TAUTOMERIZATION OF ENZYMIC CATALYSIS OF THE

1957, 225:675-688.J. Biol. Chem. 

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