The Mechanism of Action of Ethanolamine Deaminase · cobalt-linked carbon atom of the coenzyme...

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 246, No. 7, Issue of April 10, pp. 1755-1766, 1970

Printed in U.S.A.

The Mechanism of Action of Ethanolamine Deaminase

VI. ETHYLENE GLYCOL, A QUASI-SUBSTRATE FOR ETHANOLAMINE DEAMINASE*

(Received for publication, October 20, 1969)

BERNARD M. BABIOR~

From the Thorndike Memorial Laboratory, Harvard Medical Unit, Boston City Hospital, and the Department of Medicine, Harvard Medical Xchool, Boston, Massachusetts 02118

SUMMARY

Incubation of 5’-deoxyadenosylcobalamin with ethylene glycol in the presence of ethanolamine deaminase leads to the cleavage of the coenzyme at the carbon-cobalt bond, with the production of equivalent amounts of 5’-deoxyadenosine and acetaldehyde, together with a new corrinoid which appears to arise by a substitution in the coordination sphere of the cobalt whereby the 5’-deoxyadenosyl residue is replaced by another ligand. This reaction is accompanied by the transfer of a hydrogen from ethylene glycol to S’-deoxyadenosine. After the reaction is over the acetaldehyde dissociates slowly from the enzyme, but both 5’-deoxyadenosine and the new corrinoid remain bound to the enzyme, where the latter is gradually converted to hydroxocobalamin. The cleavage of coenzyme, which is first order in enzyme-Blz complex, obeys saturation kinetics with respect to ethylene glycol, showing a V,,, of about 0.2 set-l and a Km for ethylene glycol of 0.02 M.

The optical spectrum of the new Blz derivative resembles that of an alkyl cobalamin or a thiol cobalamin. However, spectral changes observed on treating the reaction mixture with urea or heat to denature the enzyme suggest that the latter possibility is more likely than the former. The possibility that the new compound is a hitherto unknown type of corrin derivative cannot be excluded.

As a result of the reaction, the activity of the enzyme is substantially reduced, but not abolished. The enzyme is capable of promoting the cleavage of more than 1 mole of coenzyme per mole of active site. Both of these observations indicate that the enzyme participates catalytically in the cleavage reaction, and is not destroyed as a consequence of the reaction.

These results are formulated in terms of a mechanism which relates the observed reaction to the catalytic reaction in which ethanolamine is substrate.

Evidence is accumulating in support of the hypothesis that early steps in coenzyme B12-dependent rearrangements involve

* This research was supported in part by United States Public Health Service Grants AM-09115, FR-0076, and AM-5413. Paper V of this series is Reference 11.

1 Recipient of a research career development award from the National Institute of Arthritis and Metabolic Diseases.

cleavage of the carbon-cobalt bond and transfer of a hydrogen from the substrate to the 5’-carbon of the adenosine residue,of the coenzyme. With regard to the latter step, it has been demon- strated that tritium from substrate can be transferred to the cobalt-linked carbon atom of the coenzyme during the course of the reaction, and that tritium in this position can be transferred to the product (l-3). On the basis of this observation, Frey, Essenberg, and Abeles (1) proposed 5’.deoxyadenosine as an intermediate in the coenzyme B12-dependent rearrangement of ethylene glycol. Additional data supporting this formulation have recently been set forth by Miller and Richards in a kinetic study of the methylmalonyl-CoA isomerase reaction (4).

Evidence for the cleavage of the carbon-cobalt bond has been provided by Babior and Gould, who used ESRr spectroscopy to show the appearance of unpaired electrons during the course of the ethanolamine deaminase reaction (5). However the first direct demonstration of the enzymatic cleavage of the carbon- cobalt bond of DMBC was reported in 1966 by Wagner et al. (6), who showed that the incubation of DMBC with diol de- hydrase in the presence of glycolaldehyde leads to the cleavage of the coenzyme, with the production of 5’-deoxyadenosine; the glycolaldehyde is oxidized to glyoxal. Recently a similar reaction was found to occur when DMBC is incubated with ethanolamine deaminase (ethanolamine ammonia-lyase, EC number to be assigned) in the presence of ethylene glycol. The results of an investigation of this reaction are the subject of the present report.

MATERIALS AND METHODS

Ethanolamine deaminase from Clostridium sp. was prepared and resolved of bound cobamides by the method of Kaplan and Stadtman (7). Enzyme concentration was calculated on the basis of a molecular weight of 520,000 (8). The enzyme has previously been shown to possess two active sites per molecule (S-10).

DMBC was kindly supplied by Professor H. P. C. Hogenkamp. OHBlz was prepared by aerobic photolysis of a Co-methyl cobalamin solution (concentration 2.4 X 1O-3 M) and purified by evaporation of the solution to dryness under reduced pressure to remove volatile compounds derived from the methyl fragment (11). Cyanocobalamin, NADH, pyruvate kinase (rabbit liver, type II), and FAD were obtained from Sigma; DMBC, sodium

1 The abbreviations used are: ESR, electron spin resonance; DMBC, 5’-deoxyadenosylcobalamin; OHBis, hydroxocobalamin.

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1756 Mechanism of Action of Ethanolamine Deaminase. VI Vol. 245, Ko. 7

phosphoenolpyruvate, and yeast alcohol dehydrogenase (lyophilized) were purchased from Calbiochem. 5’-Deoxyadenosine was the generous gift of Professor R. H. Abeles. Acetaldehyde and propionaldehyde were distilled immediately before use. Other materials were reagent grade, and were used without further purification.

r4C-ATP (420 mCi per mmole), YLethylene glycol (2.0 mCi per mmole), and 3H-ethylene glycol (62 mCi per mmole) were purchased from New England Nuclear. Radioactivity was determined in a Nuclear-Chicago Mark I liquid scintillation counter, with Bray’s solution (12) as scintillant.

Optical spectra were taken on a Perkin-Elmer model 350 recording spectrophotometer. A l-ml quartz cuvette with a l-cm light path was used for all spectra. ESR spectroscopy was performed on a Varian V-4500 X-band spectrometer as previously described (5). Photolysis was carried out by exposure of the sample to a loo-watt tungsten lamp at a distance of 10 cm for the period of time noted.

Preparation of (‘4C)-6’-DeoxyadenosylcobalamilZ-DMBC la-

beled specifically in the cobalt-linked adenosyl residue was prepared enzymatically from cyanocobalamin and r4C-ATP, with a crude extract from the organism from which ethanolamine deaminase is obtained. To prepare the extract, 30 g of Clos-

tridium sp. (13) in 75 ml of 0.05 M potassium phosphate buffer (pH 7.4) were homogenized for 2 min in a Waring Blendor. The homogenate was placed in a Rosett cell and sonically dis- rupted at 0” for 5 min at maximum power with a Branson model W14OD cell disrupter. The extract was then centrifuged for 20 min at 26,000 x g, and the supernatant was dialyzed over- night at 4” against two changes of 0.05 M potassium phosphate

buffer (pH 7.4). For the synthesis of radioactive DMBC, a mixture was pre-

pared containing 4 pmoles of mercaptoethanol, 0.4 pmole of

0.1 , ,500

ENZYME 0 IO 20 30

FUACT/ON

FIG. 1. Gel filtration of ethanolamine deaminase, DMBC, and adenosyl derivatives obtained by the photolysis of DMBC. A---& ethanolamine deaminase. Enzyme, 100 pg, in 0.2 ml of potassium phosphate bluffer (pH 7.4) was applied to the column. Elution was performed as described in the text. The protein in each fraction was determined by the method of Lowry et al. (15). 0-0, DMBC. I%-DMBC, 0.14 nmole, in 0.2 ml of water was applied to the column. Elution was performed in dim light as described in the text, collecting the fractions directly into liquid scintillation vials. The radioactivitv in each fraction was then assayed as described in the text. 0- 1 -0, adenosyl derivatives. i4C-DMBC. 0.14 nmole. in 0.2 ml of water was illuminated for 5 min by a lo&watt tungsten lamp at a distance of 10 cm. This solution was then applied to the column, and elution was performed as described in the text, collecting the fractions directly into liquid scintillation vials. The radioactivity in each fraction was then assayed as described in the text.

sodium phosphoenolpyruvate, 0.19 pmole of cyanocobalamin, 1.3 pmoles of MnC&, 3 pmoles of NADH, 0.06 pmole of FMN, 0.025 pmole of 14C-ATP, 25 pmoles of potassium phosphate buffer (pH 7.4), and 100 pmoles of Tris chloride buffer (pH 8.0) in a volume of 0.66 ml. Subsequent operations were conducted in dim light. The incubation was begun by adding 0.3 ml of crude extract, and was carried out in absolute darkness at 23”. At + hour, 1 hour, and 14 hours the incubation was supplemented with 0.3 ml of crude extract and 0.04 ml of 0.1 M sodium phosphoenolpyruvate in 0.5 M potassium phosphate buffer (pH 7.4). At 2 hours, the incubation was terminated by the addition of 0.6 ml of 1 N NHIOH. Radioactive nucleotides nere then removed by passage of the reaction mixture through a l-ml column of Dowex l-X8 (Cl-). The column was washed with 1 ml of 1 N NHIOH, the washings were added to the original eluate, and 0.05 ml of lop3 M DMBC was added to the pooled washings and eluate. After neutralization with 1 N IICI, the solution was heated in boiling water for 2 min to precipitate proteins and nucleic acids. The sample was then centrifuged, and the supernatant solution containing the radioactive product was set aside. The precipitate was washed once with water, adding the washings to the supernatant from the previous step. The radioactive coenzyme was then purified by phenol extraction and chromatography on Dowex 50-X2 as described by Barker (14). The synthesis yielded 0.33 &i of radioactive DMBC at a specific activity of 8.2 mCi per mmole. Descending chromatography of Whatman No. 1 paper, with water-saturated sec- butanol as the developing solvent, showed that the radioactive material cochromatographed with authentic DMBC. However, exposure of the radioactive material to light before chromatography resulted in a chromatogram in which the radioactive material migrated as a number of spots, all of which ran well ahead of DMBC.

AssaysThe displacement of the (14C)-5’-deoxyadenosyl moiety (hereafter referred to as the cleavage of the coenzyme) was measured by calculating the amount of unreacted DMBC in a given incubation after separation of the DMBC from the

TABLE I

Requirements for cleavage of DMBC

For Experiment 1, the reaction mixture contained 0.12 nmole of ethanolamine deaminase, 0.22 nmole of IE-DMBC, 0.3 pmole of ethylene glycol, and 0.3 pmole of potassium phosphate buffer (pH 7.4), in a total volume of 0.03 ml. The incubation was begun with enzyme, and was carried out in the dark at 23”. At 1 min, the reaction was terminated by immersion in boiling water for 30 sec. The reaction mixture was then diluted to 0.5 ml with water, and the cleavage of coenzyme was determined as described in the text. The remainder of the experiments were conducted in a similar manner, except for the modifications noted in the table.

Experiment and modification Cleavage of coenzyme

nmole

1. None...................................... 0.16 2. Omit ethylene glycol. . 0 3. Boiled enzymea.. . . . . . . . . . . 0 4. Zero time. . . . . . . . . . . . _. . 0.02b

0 Enzyme was heated in boiling water for 30 set before starting the reaction.

b See text.


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Issue of April 10, 1970 B. M. Babior 1757

adenosyl derivative on a 20-ml column of Uio-Gel P-2. Deter- minations of enzyme-bound radioactivity were carried out with the same column. The mobilities of ethanolamine deaminase, DMBC, and the adenosyl derivatives generated by the photolysis of DMBC are shown in Fig. 1. The protein peak also is well separated from acetaldehyde and ethylene glycol (not shown).

For the determination of enzyme-bound radioactivity, the column was eluted with lo+ M potassium phosphate buffer (pH 7.4), collecting l-ml fractions. The fraction of total radioactivity bound to enzyme was determined by measuring the amount of radioactivity associated with the protein peak and dividing this value by the total amount of radioactivity collected from the column. For the determination of the extent of cleavage of coenzyme, the column was eluted with water, and 2-ml fractions were collected. This determination is not as accurate as the determination of protein-bound radioactivity, because the DMBC peak tails rather badly, resulting in a substantial overlap between this peak and the adenosyl peak. It was therefore necessary to base the calculation of the extent of cleavage on the amount of radioactivity in Fractions 4 and 5. These contain about 60% of the radioactivity from DMBC but none of the radioactivity from the adenosine derivatives. In calculating the extent of cleavage of radioactive coenzyme in a given sample, the quantity of radioactivity in Fractions 4 and 5 was multiplied by 1.7, and this figure, which was taken to rep-

FIG. 2. Kinetics of the cleavage reaction. Left, the extent of cleavage of DMBC as a function of time. Reaction mixtures contained 0.24 nmole of ethanolamine deaminase, 0.16 nmole of 14C-DMBC, 0.3 Fmole of ethylene glycol, and 0.3 rmole of potassium phosphate buffer (pH 7.4) in a total volume of 0.03 ml. Incubations were begun by adding enzyme, and were conducted in dim light at room temperature. At the times noted the reactions were terminated by immersion in boiling water for 30 sec. Water (0.47 ml) was then added to the reaction mixture, and the cleavage of coenzyme was determined as described in the text. Right, the rate of cleavage of DMBC as a function of ethylene glycol concentration. Reaction mixtures contained 0.12 nmole of ethanolamine deaminase, 0.08 nmole of 14C-DMBC, 0.15 Mmole of potassium phosphate buffer (pH 7.4), and ethylene glycol as noted, in a total volume of 0.015 ml. Incubations were begun with DMBC, and were carried out in dim light at 23”. Exactly 5 set after adding DMBC, the reactions were terminated by rapidly adding 15 ~1 of 6y0 trichloracetic acid by means of a micro- liter syringe. The addition was rapid enough so that mixing was virtually instantaneous. Immediately thereafter, 0.17 ml of water was added to the sample and the cleavage of the coenzyme was determined as described in the text. To test the adequacy of the experimental conditions, zero time and 1-min incubations were carried out with the reaction mixture containing 1.5 pmoles of ethylene glycol. No cleavage was observed when trichloracetic acid was added prior to the addition of 14C-DMBC. When the incubation was terminated at 1 min rather than at 5 set, the cleavage of DMBC was quantitative.

resent the amount of unreacted radioactive DMBC remaining in the sample, was divided by the total amount of radioactivity collected from the column to obtain the fraction of coenzyme present in the sample at the end of the reaction. In this determination, all operations were conducted in dim light.

Radioactive ethylene glycol, acetaldehyde, or ethanol which was present in a reaction mixture was identified and quantitatively determined by adding to the mixture a known amount of nonradioactive carrier, preparing an appropriate derivative (ethylene glycol dibenzoate, the dimedon adduct of acetaldehyde, and ethyl 3,5-dinitrobenzoate, respectively), and recrystallizing to constant specific activity. From these data and from the total amount of radioactivity present in the sample to be assayed, the fraction of total radioactivity represented by the compound in question was calculated by the usual method. Ethyl 3,5- dinitrobenzoate and acetaldehyde dimedon adduct were prepared as previously described (11). Ethylene glycol dibenzoate was prepared by the Schotten-Baumann procedure (16), adding 50 ~1 (55.8 mg) of nonradioactive ethylene glycol to the sample and esterifying with 0.30 ml of benzoyl chloride. The ester was purified and crystallized by the method used for the purification and crystallization of ethyl 3,5-dinitrobenzoate. Ethanolamine deaminase activity was determined spectrophotometrically as described by Kaplan and Stadtman (7).

IO 20 30

FRACTION

FIG. 3. Binding of the reaction products to ethanolamine deaminase. Top, from 14C-ethylene glycol. The reaction mixture contained 0.19 nmole of ethanolamine deaminase, 1.0 nmoles of DMBC, 0.13 rmole of ‘4C-ethylene glycol, and 0.08 pmole of potassium phosphate buffer (pH 7.4) in a total volume of 0.01 ml. The reaction, which was begun by the addition of enzyme, was carried out in the dark at 23”. One minute after starting the reaction, 0.19 ml of 10V2 M potassium phosphate buffer (pH 7.4) was added. The mixture was then applied to the column of Bio-Gel P-2 (see Fig. 1) and subjected to chromatography as described in the text, collecting the fractions directly into scintillation vials for assay of radioactivity. Bottom, from XLDMBC. The reaction mixture contained 0.19 nmole of ethanolamine deaminase, 0.054 nmole of 14C-DMBC, 1.0 pmole of ethylene glycol, and 0.08 pmole of potassium phosphate buffer (pH 7.4) in a total volume of 0.011 ml. The experiment was conducted as described above.


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1758 Mechanism of Action of Ethanolamine Deaminase. VI Vol. 245, No. 7

RESULTS

Cleavage of Coenzyme-Incubation of DMBC with an equivalent amount of ethanolamine deaminase in the presence of ethylene glycol leads to cleavage of the coenzyme at the carbon- cobalt bond (Table I). This cleavage appears to be almost quantitative, and takes place rather rapidly. For the reaction to take place, all three components of the incubation mixture must be present; no cleavage is observed if either ethylene glycol or active enzyme is omitted. The small amount of cleavage observed in the zero time control (Table I, Experiment 4) probably represents the extent to which the reaction proceeds in the interval between the addition of the enzyme and the termination of the incubation (approximately 5 set).

Kinetics of Cleavage Reaction-The cleavage of DMBC by ethanolamine deaminase in the presence of a large excess of ethylene glycol obeys first order kinetics. This is illustrated in Fig. 2 (left) which shows the results of an experiment in which the extent of cleavage of the coenzyme was determined as a function of time in the presence of a large excess of ethylene glycol. In this experiment, the enzyme was also present in substantial molar excess, so that essentially all of the DMBC was in the form of the enzyme-coenzyme complex. (Previous investigations have shown that at concentrations of the order used in this experiment, DMBC is quantitatively bound to ethanolamine deaminase up to a ratio of 2 moles per mole of enzyme (9).) The data fall on a straight line, indicating that the cleavage is first order in DMBC; since almost all of the DMBC was bound to the enzyme, the cleavage is first order in complex as well.

To investigate the dependence of the reaction rate on the concentration of ethylene glycol, the extent of cleavage of DMBC was determined as a function of ethylene glycol concentration in a series of 5-set incubations. The rate constant was calculated

TABLE II

Requirements for binding to enzyme of radioactivity from W-ethylene glycol

For Experiment 1, the reaction mixture contained 0.19 nmole of ethanolamine deaminase, 1.0 nmole of DMBC, 0.13 rmole of “C-ethylene glycol, and 80 nmoles of potassium phosphate buffer (pH 7.4) in a total volume of 0.01 ml. The incubation was begun with enzyme, and was carried out in the dark at 23”. At 1 min, 0.19 ml of 10-z M potassium phosphate buffer (pH 7.4) was added. Two minutes later, the diluted reaction mixture was assayed for protein-bound radioactivity by gel filtration as described in the text. The remainder of the experiments were conducted in a similar manner, except for the modifications noted in the table.

Experiment and modiication Protein-bound product

nmole

1. None...................................... 0.17 2. Omit DMBC.............................. 0 3. Omit enzyme.. . . . . 0 4. Boiled enzyme”. . . 0 5. Zero timeb.. . . . . . . . . . . 0.03

0 Enzyme was heated in boiling water for 30 set before starting the reaction.

b Incubation was diluted as soon as possible after adding enzyme (~2 to 3 set) and then immediately applied to the column and subjected to gel filtration.

from the extent of cleavage, assuming first order kinetics, and a reciprocal plot was prepared of the figures thus obtained as a function of the concentration of ethylene glycol (Fig. 2, right). The results indicate that the rate constant obeys saturation kinetics with respect to the concentration of ethylene glycol. The K, for ethylene glycol is approximately 0.02 Y, and the maximum rate constant is in the vicinity of 0.2 set-l.

Binding of Reaction Products to Enzyme-When an incubation under the conditions described above is followed by denaturation of the enzyme, a free adenosine fragment appears in the reaction mixture. However, the results of Fig. 3 show that, if after completion of the reaction the ethanolamine deaminase is not denatured, the adenosine fragment remains bound to the protein. In this experiment, the reaction mixture, initially containing enzyme, ethylene glycol, and radioactive coenzyme, was subjected to chromatography as described above without being heated beforehand. Under these conditions virtually all of the radioactivity remains associated with the protein. Treatment of a similar incubation mixture with urea (6.7 hr urea for 40’sec) after completion of the reaction leads to the release from the enzyme of about four-fifths of the radioactivity; the rest remains associated with the protein.

TABLE III

Transfer of tritium from 3H-ethylene glycol to 5’-deoxyadenosine

The reaction mixtures contained 0.55 nmole of ethanolamine deaminase, 0.054 nmole of i4C-DMBC, 1.0 nmole of unlabeled DMBC, 0.22 pmole of aH-ethylene glycol (Experiments 1 and 2) or unlabeled ethylene glycol (Experiments 3 and 4), and 0.15 pmole of potassium phosphate buffer (pH 7.4) in a total volume of 0.02 ml. Incubations were begun with enzyme, and were conducted in the dark at 23”. At 2 min, 0.22 pmole of unlabeled ethylene glycol was added to incubations originally containing tritiated ethylene glycol, while a similar amount of 3H-ethylene glycol was added to incubations originally containing unlabeled material. Two minutes later, the incubations were diluted with 10-z M potassium phosphate buffer (pH 7.4) to a final volume of 0.2 ml. They were immediately subjected to gel filtration according to the technique used for determining protein-bound radioactivity, except that all fractions were discarded except Fraction 6, the fraction which contained most of the enzyme (see Fig. 1). This fraction was heated for 30 set in boiling water to inactivate the enzyme, and then was lyophilized after the addition of 10 I.LI of authentic 5’-deoxyadenosine (1 mg per ml) and 3 pl of hydroxocobalamin (2.4 X lOma M). The residue was triturated with two 0.5.ml aliquots of methanol. The methanol extracts were pooled, reduced under a stream of nitrogen to about 0.05 ml, applied to Whatman No. 1 paper, and subjected to descending chromatography with n-butyl alcohol-water = 172:28 (v/v). The ultraviolet-absorbing spots of carrier 5’-deoxyadenosine were cut out, placed directly in liquid scintillation vials, and counted in Bray’s solution by the double isotope technique. Counting efficiencies for i4C and 3H were determined by internal standardization with I%- and 3H-labeled toluene of known specific activity.

Experiment 8H I

‘4C ‘H:Y ratio

dfim

1 392 105 3.7 2 361 94 3.8 3 47 120 0.4 4 20 71 0.3


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Similarly, it was found that radioactive ethylene glycol, or the product derived from it, also remains bound to the enzyme after completion of the reaction (Fig. 3). This binding was shown to be dependent on both enzyme and DMBC (Table II). How- ever, in constrast to the situation observed with the adenosine fragment, the amount of radioactivity which remains associated with the enzyme under the experimental conditions represents significantly less than 1 mole of ethylene glycol per mole of active site. Furthermore, this material is quantitatively released from the enzyme by treatment with urea.

Identity of Reaction ProductsBy chromatography of the radioactive material recovered from an experiment conducted with labeled coenzyme, it was possible to show that the adenosine fragment released in the reaction was 5’-deoxyadenosine. The reaction mixture contained 0.48 nmole of ethanolamine deaminase, 0.88 nmole of 14C-DMBC, 1.2 pmoles of ethylene glycol, and 1.2 pmoles of potassium phosphate buffer (pH 7.4), in a total voilume of 0.12 ml. The incubation, which was begun with enzyme, was carried out in the dark at 23”. After 1 min, the react on was terminated by heating for 30 set in boiling water. One-tenth milliliter of 5’-deoxyadenosine (1 mg per ml) was then added, and the reaction mixture was subjected to chromatography on Bio-Gel P-2 as described above, eluting with water. The first 7 ml of effluent were discarded; the next

20 ml were collected and taken to dryness under reduced pressure on a rotary evaporator. The residue was dissolved in a small volume of methanol and transferred to a small centrifuge tube. The solvent was then blown off under a stream of nitrogen, the residue was redissolved in 60 ~1 of methanol, and the solution of product was applied to strips of Whatman No. 3MM paper and subjected to descending chromatography in the following three solvent systems: n-butyl alcohol-water = 172:28 (v/v), n-butyl alcohol-acetic acid-water = 12:3 :5 (v/v), and 1 M ammonium acetate-go% aqueous ethanol = 3:7 (v/v) (10). Each chromatogram showed a single ultraviolet-absorbing spot with the mobility of authentic 5’-deoxyadenosine (6). On radio- autography, each chromatogram revealed a single radioactive spot which coincided with the spot of carrier 5’-deoxyadenosine.

The 5’-deoxyadenosine produced in this reaction was found to incorporate hydrogen from ethylene glycol. The experiments showing this were conducted by incubating tritiated ethylene glycol with r4C-DMBC and ethanolamine deaminase, and showing that tritium had appeared in the 5’-deoxyadenosine isolated from the incubation at the end of the reaction. In performing these experiments, labeled 5’-deoxyadenosine was separated from the vast excess of tritiated ethylene glycol by chromatography of the incubation mixture on Bio-Gel P-2 prior to inactivation of the enzyme (see Fig. 3), taking advantage

TABLE IV

Identity of enzyme-bound radioactive compound derived from W-ethylene glycol

Reaction mixtures contained 1.9 nmoles of ethanolamine deaminase, 10 nmoles of DMBC, 1.3 pmoles of W-ethylene glycol, and 08pmole of potassium phosphate buffer (pH 7.4) in a total volume of 0.1 ml. Incubations were begun with enzyme, and were carried out in the dark at 23“. At 1 min, the incubations were diluted with lo* M potassium phosphate buffer (pH 7.4) to a final volume of 0.2 ml. They were then subjected to gel filtration according to the technique used for determining protein-bound radioactivity, except that all fractions were discarded except Fractions 5, 6, and 7, which contained most of the enzyme. Identification of the enzyme-bound radioactive compound was then carried out as follows. In Experiment 1, the fractions were diluted to a total volume of 4.4 ml with lo-* M potassium phosphate buffer (pH 7.4). For determination of total radioactivity, 0.200 ml of the diluted material was counted directly. Of the remainder, 2.00~ml aliquots were placed in each of two glass centrifuge tubes. After adding 0.8 ml of 2 N HCl to each aliquot the tubes were allowed to stand for 15 min, one in the dark and the other 10 cm from a loo-watt tungsten lamp. Acetaldehyde, 50 ~1 (39.2 mg), was then added to each tube, and the dimedon derivative was prepared and recrystallized to constant specific activity as described in the text. In Experiment 2, the fractions were diluted to a total volume of 4.4 ml with 10-Z M potassium phosphate buffer (pH 7.4). For determination of total radioactivity, 0.200 ml of the diluted material was counted directly. Of the remainder, 1.50-ml aliquots were placed in each of two glass centrifuge tubes. Next, 4.50 ml of 8 M urea were added to each aliquot. The tubes were then allowed to stand for 5 min, one in the dark and the other 10 cm from a loo-watt tungsten lamp. Finally, the room was dark- ened while 2.4 ml of 2 N HCl were added to each tube, after which the lights were turned on and the samples were allowed to stand for 15 min. Acetaldehyde, 50 ~1 (39.2 mg), was then added to each tube, and the dimedon derivative was prepared and recrystallized to constant specific activity as described in the text. In Experiment 3, all column fractions were discarded except Fraction 6. This fraction was diluted with 8 M urea to a final

volume of 4.3 ml. For determination of total radioactivity, 0.100 ml of the diluted material was counted directly. Of the remainder, 2.00~ml aliquots were placed in each of two glass centrifuge tubes. One sample was illuminated for 5 min with a loo-watt lamp at a distance of 10 cm, while the other sample was permitted to remain in the dark for the same period of time. The lights were then extinguished, and 20 mg of solid NaBHd were added to each tube. Five minutes later, 0.2 ml of 12 N HCl was added to each tube.” Fifteen minutes after the addition of HCI the lights were turned on, and 50 ~1 (39.5 mg) of ethanol were added to each tube. Ethyl 3,5-dinitrobenzoate was then prepared and crystallized to constant specific activity as described in the text. Except where noted, all operations up to but not including the addition of carrier acetaldehyde or ethanol were carried out in the dark. Counting efficiency for 1% was determined by internal standardization with W-labeled toluene of known specific activity.

I Specific activity of derivative Radioactivity in the form of the carrier

compound Experi- ment Observed

gzi Photolyzed Unphotolyzed

Photolyzed “~;;$-

dPrn/?w i Zlfil~~~~i_Ei,i_fH~i:$ i; [ IF

a At room temperature, the half-time for the destruction of &hydroxyethylcobalamin at the concentration of HCI present in the samples was found to be about 3 min (l3. Babior, unpublished data).

b Assuming that 100% of the radioactivity in the reaction mixture is in the form of the carrier.

c Mean f 1 S.D. for the last three crystallizations.


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Mechanism of Action of Ethanolamine Deaminase. VI Vol. 245, No. 7

of the fact that the 5’-deoxyadenosine produced in the reaction remains bound to the enzyme. The results, given in Table III, show that during the cleavage reaction a significant amount of tritium is transferred from ethylene glycol into the nucleoside. In experiments in which the tritiated ethylene glycol was added only after the reaction (conducted in the presence of unlabeled ethylene glycol) had essentially reached completion, there was little incorporation of tritium into 5’-deoxyadenosine, indicating that the appearance of tritium in the nucleoside was dependent on the reaction process. Although a aH:W ratio of 76 would be expected on the basis of the relative quantities of each of these isotopes in the reaction mixture, a much smaller ratio is actually observed. The discrepancy between the calculated and the observed values can probably be attributed to a tritium isotope effect, since both deuterium and tritium isotope effects have been observed in a number of coenzyme Biz-dependent rearrangements (17, 18).

As mentioned previously, gel filtration experiments have shown that radioactivity from “C-labeled ethylene glycol remains associated with the enzyme after the completion of the reaction. In the experiments described below, this radioactive material was identified as acetaldehyde. This identification was a by- product of several unsuccessful attempts to obtain evidence for the formation of a new alkyl cobalamin derived from the corrin moiety of the coenzyme and a a-carbon fragment obtained from ethylene glycol. In these latter experiments, 14C-ethylene glycol was incubated with enzyme and DMBC, after which the radioactive product, isolated as the enzyme-bound complex, was subjected to a procedure in one step of which half of the material was illuminated (to photolyze any alkyl cobalamin present) while the other half was kept in the dark. The identities of the radioactive compounds isolated from the two halves were then compared. It was hoped to obtain evidence for the appearance of a new alkyl cobalamin by showing a difference between the identities of the products obtained from the illuminated and unilluminated samples, on the theory that illumination would lead to a homolytic cleavage of the alkyl cobalamin, followed by oxidation of the radical derived from the radioactive fragment (19), while the product obtained from the dark sample would be at the same oxidation level as ethylene glycol.

The results of these experiments are shown in Table IV.

-

0

I& <

I I,,,,, 1, I I I, , I , I , I / , I , , I 600 550 500 450 400 350

WA EL ENGTH fnml

FIG. 4. Spectrum of X-B12, the product of the enzyme-dependent cleavage of DMBC. A cuvette containing 11 nmoles of ethanolamine deaminase, 50 pmoles of ethylene glycol, and 10 rmoles of potassium phosphate buffer (pH 7.4) in a total volume of 0.975 ml was placed in the spectrophotometer. After taking a spectrum of this mixture (lower curve), the reaction was begun by the addition of 0.025 ml of 1W3 M DMBC (25 nmoles). The incubation was conducted at 23” in the dark for 2 min, after which the spectrum was repeated (upper curve). ing DMBC were conducted in dim light.

All operations involv-

Treatment of the sample with acid, or with urea followed by acid, resulted in the release of radioactive acetaldehyde regard- less of whether or not the sample was exposed to light (Experi- ments 1 and 2). Because of the possibility that illumination of the postulated alkyl cobalamin could lead to a photoionization with the production of acetaldehyde, rather than a homolytic cleavage followed by oxidation of the radical to something other than acetaldehyde, Experiment 2 was repeated, except that the samples were treated with NaBH4 prior to acidification (Experiment 3). This was done in an attempt to reduce any new cobamide in the unilluminated sample to a compound (such as /3-hydroxyethylcobalamin) which would be converted to ethylene by treatment with acid (20). Here too, however, the same compound was obtained from both the illuminated and unilluminated samples. In this case the compound was ethanol, the product of the reduction of acetaldehyde by NaBH4.

Although these experiments failed to show an alkyl cobalamin possessing as the sixth ligand a fragment derived from ethylene glycol, results discussed below indicate that under certain circumstances a new photolabile cobamide appears following the cleavage reaction. Therefore, additional experiments were carried out in an attempt to show such a compound. In one set of experiments, n-propyl alcohol or heat was used to inactivate enzyme from an incubation containing 14C-labeled ethylene glycol, DMBC, and ethanolamine deaminase, and labeled cobalamin was sought by chromatography of the reaction mixture on a 40-ml column of Bio-Gel P-2 (this column was found to provide a clean separation between cyanocobalamin and radioactive ethylene glycol). Fractions which contained cobalnmin were devoid of radioactivity. Finally, in an experiment in which excess hydroxocobalamin was added to the incubation after the reaction was complete, in order to displace other cobalamins from the enzyme in the gentlest possible manner, the new postulated radioactive cobalamin derivative failed to appear. Thus, it appears that during the cleavage reaction ethylene glycol is converted to acetaldehyde without the appearance of a stable alkyl cobalamin derivative.

Spectrum of Enzyme-Blz Complex during Course of Reaction- Of the two products thus far identified, acetaldehyde is at the same oxidation level as ethylene glycol, while 5’-deoxyadenosine can be considered in a formal sense to be reduced with respect to the 5’-deoxyadenosyl residue of the coenzyme. It would be expected therefore that an oxidized product, viz. OHB12, would

FIG. 5. The conversion of DMBC to X-B,, as a function of time. A cuvette containing 11 nmoles of ethanolamine deaminase, 2 pmoles of ethylene glycol, and 10 pmoles of potassium phosphate buffer (pH 7.4) in a total volume of 0.975 ml was placed in the spectrophotometer. The reaction was begun by the addition of 0.025 ml of 10-S M DMBC (2.5 nmoles). Spectra were taken immediately after beginning the reaction, and at 0.5, 1, 2, and 3.5 min. Scanning time was 10 sec. Operations involving DMBC were conducted in dim light.


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0.2 t- / Y

III,>/, , I

600 550 500 450 400 350 WA VEL ENG TH /nml

FIG. 6. The conversion of X-Bn to OHBlz as a function of time. Top, time course of spectral change. The incubation was conducted as described in Fig. 3. Spectra were taken at 5, 30, 75, and 180 min. Middle, spectrum of free OHBr2. The sample contained 25 nmoles of OHBlz and 10 rmoles of potassium phosphate buffer (pH 7.4) in a total volume of 1.0 ml. Bottom, spectrum of enzyme-bound OHBi2. The sample contained 7.5 nmoles of ethanolamine deaminase, 14.5 nmoles of OHBlz, and 10 pmoles of potassium phosphate buffer (pH 7.4) in a total volume of 1.0 ml. Previous experiments have shown that, under these conditions, essentially all of the OHBn is bound to the enzyme (11).

be obtained from the corrin moiety of the coenzyme. However, spectra taken shortly after the completion of the reaction show that the corrin moiety is not converted to OHBrz, but rather to a hitherto unknown corrinoid, hereafter called X-Bi2.

The spectrum of X-B12 is shown in Fig. 4. This spectrum was obtained only 2 min after starting the incubation, which was carried out under conditions which assured quantitative cleavage of the coenzyme within that period of time. The most striking feature of this spectrum is the relatively constant absorbance between 340 nm and 370 nm. This feature is characteristic of the spectra of alkyl and thiol cobalamins (9, 21, 22), and is to be contrasted with the sharp peak in the vicinity of 355 nm which is seen in the spectrum of OHBrz.

The rate of appearance of X-B12 was studied by obtaining repeated spectra of the reaction mixture containing ethylene glycol at a concentration low enough so that the cleavage took about 5 min to reach completion.2 Fig. 5 shows that the spectrum of the reaction mixture, which initially resembles that of

DMBC, is converted in 34 min to that shown in Fig. 4. This result indicates that the rate of conversion of DMBC to X-Bn as determined by spectrophotometry is similar to the rate of the cleavage reaction. These changes were not observed in the absence of ethylene glycol. Furthermore, the occurrence in this series of spectra of several well defined isosbestic points

2 This figure was calculated from measurements of the rate of cleavage of i4C-DMBC.

600 550 500 450 400 350

WAVELENGTH /nm j

FIG. 7. Spectrum of the reaction mixture after denaturation of ethanolamine deaminase. Top, denaturation by urea. The mixture containing 11 nmoles of ethanolamine deaminase, 15 pmoles of ethylene glycol, and 3 pmoles of potassium phosphate buffer (pH 7.4) in a total volume of 0.315 ml was placed in a cuvette. Subsequent operations were conducted in dim light. The incubation was begun by the addition of 0.025 ml of IO-3 M DMBC (25 nmoles), and was carried out in the dark at room temperature. Two minutes after the addition of DMBC, 0.66 ml of 8 M urea was added. Thorough mixing was accomplished by repeated inversion of the cuvette. The spectrum was taken 30 set after the addition of urea. Bottom, denaturation by heat. In a test tube, 10 X 75 mm, were placed 11 nmoles of ethanolamine deaminase, 50 pmoles of ethylene glycol, and 10 pmoles of potassium phosphate buffer (pH 7.4) in a total volume of 0.985 ml. Subsequent operations were conducted in dim light. The incubation was begun by the addition of 0.015 ml of 1.3 X 1O-3 M DMBC (20 nmoles) and was carried out in the dark at room temperature. At 1 min, the reaction was terminated by placing the test tube in a beaker of boiling water for 30 sec. The reaction mixture was then transferred to a cuvette and spectra were obtained at 3 min and at 10 min (- - -). The reaction mixture was then irradiated for 5 min as described in the text, following which spectra were obtained at 20 and at 30 min (-).

indicates a direct conversion of DMBC to X-B12 without the appearance of an intermediate absorbing species.

X-Brz, however, is not stable, but is slowly converted to OHBr2. This is shown in Fig. 6, which represents a continuation of the previous experiment. Over the course of 3 hours, the X-Bra

plateau at short wave lengths is replaced by a peak at 355 nm, while the peaks at 535 and 504 nm are shifted to 522 and 492 nm, respectively. The rate of appearance of the peak at 355 nm is not affected by increasing the concentration of ethylene glycol

in the reaction mixture to 0.05 M, but it appears to be slightly increased by a IO-min exposure to a loo-watt tungsten lamp at a distance of 10 cm. The presence of isosbestic points in these spectra implies that X-B12 is converted directly to OHBiz; it

further indicates that the conversion of DMBC to X-B12 is complete before significant amounts of OHBn appear in the reaction mixture. The fact that the a-hour spectrum shows an a-peak at 522 nm and a P-peak at 492 nm suggests that the conversion takes place while the corrinoid is bound to the enzyme, since the spectrum of enzyme-bound OHBrz shows similar CY- and P-peaks, while the spectrum of free OHBn displays only a single a-peak at 538 nm, the P-peak being represented by a shoulder at 505 nm (Fig. 6).


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TABLE V

Identification of corrinoid derived by treatment of X-B12 with cyanide

The experiment was conducted as described in the text.

Corrinoid RF

Solvent I Solvent II Solvent III

From X-Blz. 0.13, 0.25” 0.48 0.32b Cyanocobalamin. 0.13, 0.255 0.48 0.31b

0 Two zones: red leading, orange trailing. b Streaking; RF measured from point of maximum density.

ESR Spectroscopy-To determine whether X-B12 is paramagnetic, a reaction mixture containing enzyme, DMBC, and ethylene glycol was subjected to ESR spectroscopy after a preliminary incubation to cleave the coenzyme. The reaction mixture, which was placed directly in an ESR tube, contained 6.7 nmoles of ethanolamine deaminase, 10 pmoles of ethylene glycol, 13 nmoles of DMBC, and 1.8 pmoles of potassium phosphate buffer (pH 7.4) in a final volume of 0.2 ml. The incubation was begun with DMBC, and was conducted in the dark at room temperature. At the end of 2 min, the incubation was terminated by immersing the ESR tube in isopentane cooled in liquid Nz (77°K). The ESR spectrum of the frozen reaction mixture was then obtained. No signal was observed at a sen- sitivity sufficient to show 0.05 spin per active site, even though the reaction conditions were such that DMBC was quantitatively converted to the new compound in the preliminary incubation. Thus, X-B12 does not appear to be a paramagnetic species.

Spectral Changes upon Inactivation of Enzyme-On the basis of its spectrum, it appeared that the new corrinoid could be either a labile alkyl cobalamin or a thiol cobalamin. If it were the former, its stability under illumination could be ascribed to pro- tection against photolysis by virtue of its association with the enzyme. A precedent for such a situation exists in the case of 6-(9-adenyl)butylcobalamin, which is extremely resistant to photolysis when bound to ethanolamine deaminase, although the free compound is easily destroyed by light (11). To obtain further information concerning the structure of X-B12, spectra were obtained before and after photolysis from reaction mixtures which had been subjected to denaturation by urea or heat to disrupt the binding between the new corrinoid and the enzyme.

Fig. 7 shows the spectrum taken 30 set after the addition of urea to a reaction mixture containing enzyme-bound X-B1z. Even in the dark, the compound was rapidly converted to OHBi2 upon addition of the denaturing agent. The spectrum, however, shows long wave length peaks at 522 nm and 492 nm. Since urea at this concentration has no effect on the spectrum of free OHBIg, this result suggests that the OHBr2 remains bound to protein even in the presence of a concentration of urea sufficient to disrupt the quaternary structure of the enzyme (8). (In this connection, it has been found that ethanolamine deaminase can protect 6-(9-adenyl)butylcobalamin from photolysis even in the presence of 5 M urea.3)

To confirm the finding that disruption of the structure of the enzyme-X-Brz complex leads to the conversion of X-Br2 to OHB1z, similar experiments were performed with heat as the denaturing agent. Unexpectedly, the corrin was converted

3 B. Babior, unpublished results.

T/ME fminl

FIG. 8. Stoichiometry of the cleavage of DMBC with respect to enzyme. Reaction mixtures contained 0.048 nmoles of ethanolamine deaminase, 0.22 nmole of ‘GDMBC, 0.3 pmole of ethvlene glycol, and 0.3 pmole of potassium phosphate buffer (pH 7:4) in a total volume of 0.03 ml. Incubations were begun with enzyme, and were carried out in the dark at 23” for the times noted, terminating the reactions by immersion in boiling water for 30 sec. The reaction mixtures were then diluted to 0.5 ml with water; and the cleavage of coenzyme was determined as described in the text.

only in part to OHB12; the remainder appears in a form which is converted to OHBre by light (Fig. 7, bottom). A similar result was obtained in an experiment in which the reaction mixture contained the same quantities of enzyme, ethylene glycol, and buffer but only 13 nmoles of DMBC (0.6 mole of DMBC per mole of active site). In this experiment the preliminary incubation was carried out for 2 min to ensure quantitative cleavage of the coenzyme. The photolabile product was not DMBC, since the spectra before and after photolysis cross at 375 nm, whereas the spectra of unphotolyzed and photolyzed DMBC cross at 370 nm. The identity of this photolabile product is discussed below.

Unlike urea treatment, heating appears to release corrinoids from the protein. This is suggested by the observation that the a-band in the spectrum of the reaction mixture after photolysis is at about 535 nm, as expected for free OHBIT.

Identijication of X-B12 as Cobalamin Derivative-It is possible that the conversion of DMBC to X-B12 represents a reaction in which the corrin ring is modified. The observation, however, that X-B12 was converted by several procedures to a compound whose spectrum was identical with that of OHBlz militates against this possibility. Further evidence that the ring is not altered in the formation of X-Br2 is provided by an experiment in which the corrin moiety obtained from a reaction mixture containing X-B12 was identified by chromatography. The reaction mixture contained 7.4 nmoles of ethanolamine deaminase, 16.9 nmoles of DMBC, 25 pmoles of ethylene glycol, and 2.6 pmoles of potassium phosphate buffer (pH 7.4) in a volume of 0.5 ml. After a 2-min incubation in the dark, the reaction was terminated by immersion in boiling water for 30 sec. Sub- sequent steps were performed in the light. Solid NaCN (1 mg) was added to the reaction mixture. After 2 min, 0.5 ml of 1 N

HCl was added and the corrinoids were extracted into 0.2 ml of liquefied phenol (Fisher). After washing the corrinoid-containing phenol layer once with 0.1 N HCl and twice with water, adding additional phenol as necessary to maintain a two-phase system, the corrinoids were identified by descending chromatography on Whatman No. 3MM paper, with authentic cyanocobalamin as standard. The following solvent systems were used: I, set-butanol-acetic acid-water = 100: 1:50 (v/v) ; II, n-butyl


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B. M. Babior Issue of April 10, 1970

TABLE VI

Stoichiometric relationship between production of acetaldehyde and cleavage of DMBC

For the determination of acetaldehyde production, the reaction mixtures contained 0.67 nmole of ethanolamine deaminase, 1.3 nmoles of DMBC, 26 nmoles of W-ethylene glycol, and 0.2 rmole of potassium phosphate buffer (pH 7.4) in a total volume of 0.024 ml. Incubations were begun by adding enzyme, and were carried out in the dark at 23” for the times noted. After terminating the incubation by adding 20 ~1 of 6% trichloracetic acid, the reaction mixture was diluted with 1.3 ml of water, stoppered, and placed in ice. For determination of tota radioactivity, 0.100 ml of the diluted material was counted directly. Acetalde- hyde was determined by adding 50 ~1 (39.2 mg) of unlabeled acetaldehyde to 1.00~ml aliquots of the reaction mixtures, preparing the dimedon derivative, and recrystallizing to constant specific activity. Incubations in which coenzyme cleavage was determined contained 0.67 nmole of ethanolamine deaminase, 0.054 nmole of W-DMBC, 1.3 nmoles of unlabeled DMBC, 26 mnoles of ethylene glycol, and 0.2 pmole of potassium phosphate buffer (pH 7.4) in a total volume of 0.024 ml. Incubations were begun by adding enzyme, and were carried out in the dark at 23’ for the times noted. The reactions were terminated by the addition of 0.08 ml of 8 M urea followed by a 30-set immersion in boiling water. One-tenth milliliter of water was then added, and coenzyme cleavage was determined by gel filtration as described in the text.

Specific activity of diiedon derivative Amount Incubation

time Calculateda Observed Acetal-

dehyde S-Dewy- adenosine

milt &wm n?nole

0 305.17 0.62 f 0.04b 0.05 0 5 294.18 6.82 f 0.02 0.60 0.42

10 280.60 7.96 f 0.19 0.74 0.64 15 281.71 7.90 f 0.02 0.73 0.68

a Assuming that 100% of the radioactivity in the reaction mixture is in the form of the carrier.

6 Mean f 1 S.D. for the last three crystallizations.

alcohol-isopropanol-acetic acid-water = 100:70:1: 100 (v/v) ; III, isopropanol-NH40H-water = 7:1:2 (v/v) (21). As shown in Table V, the chromatographic properties of Brz derivatives isolated from the reaction mixture were identical with those of the standard in all three solvent systems. The corrin ring, therefore, is not modified by the ethylene glycol-dependent cleavage of DMBC.

Xtoichiometry--In terms of its participation in the cleavage reaction, the enzyme acts catalytically; that is, more than 1 mole of DMBC is split per mole of active site. This is shown in Fig. 8, which illustrates the results of an experiment in which samples containing enzyme and DMBC in a ratio of 2.5 moles of coenzyme per mole of active site were incubated for various periods of time. The curve displays an initial burst representing the cleavage of 1 mole of DMBC per mole of active site; rather than stopping at this point, however, the reaction continues so that by the end of 10 min almost 2 moles of DMBC per mole of active site have been split. This result suggests that, even after participating in the cleavage reaction, the enzyme retains at least some of its activity. Further evidence supporting this conclusion is presented below.

In contrast to this finding are the results presented in Table VI, in which the amount of coenzyme consumed in the reaction

1763

I I I

1.50- I I I I I I

Add enzyme

0.75~ I I I I I 0 I 2 3 4

TIME /min/

FIG. 9. Deactivation of enzyme in the presence of ethylene glycol . A mixture containing 0.4 pmole of ethanolamine.HCl, 700 rcmoles of ethylene glycol, 0.01 pmole of DMBC, 0.2 pmole of NADH, 50 pmoles of potassium phosphat,e buffer (pH 7.4), and 750 wg of yeast alcohol dehydrogenase (lyophilized, Boehringer) in a volume of 1.0 ml was placed in a l-ml quartz cuvette with a l-cm light path. At the time indicated by the arrow, the reaction was begun by adding 6.0 pg of ethanolamine deaminase. The reaction was followed spectrophotometrically as previously described (3). - - -, the rate of change in absorbance at the end of the process of deactivation, extrapolated back to zero time. Inset, a plot of the logarithm of the difference between the observed absorbance and the absorbance indicated by the extrapolated line (AA), as a function of time.

is compared with the quantity of acetaldehyde produced under the same conditions. These results show that approximately 1 mole of acetaldehyde is produced per mole of coenzyme split. In the absence of coenzyme, only 0.04 nmole of acetaldehyde was obtained from a 15-min incubation of radioactive ethylene glycol with enzyme under conditions otherwise identical with those given in Table V. In another experiment in which W- ethylene glycol was used as substrate, it was shown that a simultaneous catalytic conversion of ethylene glycol to an unidentified compound is not taking place, since at the end of a 25-min incubation essentially all of the radioactivity originally present as ethylene glycol can be accounted for as either ethylene glycol or acetaldehyde. For this experiment, two reaction mixtures were prepared, each containing 0.48 nmole of ethanolamine deaminase, 1.0 nmole of DMBC, 13 nmoles of lGethylene glycol, and 0.2 pmole of potassium phosphate buffer (pH 7.4) in a total volume of 0.025 ml. Incubations were conducted for 0 and 25 min as described in Table VI. Total radioactivity and acetaldehyde production were determined as described in Table VI. Un- reacted ethylene glycol was determined by adding 50 ~1 (55.8 mg) of unlabeled ethylene glycol to O.lOO-ml aliquots of the reaction mixtures, preparing the dibenzoates, and recrystallizing to constant specific activity as described above. By these determinations the original reaction mixture (incubated for 0 min) was found to contain 13.3 nmoles of ethylene glycol and 0.03 nmole of acetaldehyde, while the reaction mixture which had been incubated for 25 min contained 13.0 nmoles of ethylene glycol and 0.47 nmole of acetaldehyde. Thus, the production of acetaldehyde from ethylene glycol is not catalytic with respect to the coenzyme. This is a remarkable finding in view of the fact that with ethanolamine, the authentic substrate, acetaldehyde is the product of the catalytic reaction.


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1764 Mechanism of Activn of Ethanolamine Deaminase. VI Vol. 245, No. 7

TABLE VII

Requirements for deactivation of enzyme

The experiment was conducted as described in the text. The preliminary incubation mixtures contained 0.08 nmole of ethanolamine deaminase, 0.2 nmole of DMBC, 20 rmoles of ethylene glycol, and 20 nmoles of potassium phosphate buffer (pH 7.4), with omissions as noted, in a volume of 0.02 ml. The preliminary incubations were begun with enzyme. After 30 set, the ethanolamine deaminase activity in 3 pl of the preliminary incubation mixture was determined by the spectrophotometric assay. The assay mixture contained 10 pmoles of ethanolamine.HCl (pH 7.4), 10 nmoles of DMBC, 0.2pmole of NADH, 50pmoles of potassium phosphate buffer (pH 7.4), and 0.8 mg of yeast alcohol dehydrogenase (lyophilized, Boehringer) in a total volume of 1.0 ml.

Omission Activity

twtdes/min

None.................................. 14.5 Glycol................................. 104.0 DMBC................................ 119.8 Glycol and DMBC.. . . . 110.0

Activity of Enzyme Participating in Cleavage Reaction-A fall in the activity of ethanolamine deaminase occurs when the enzyme is incubated with DMBC in the presence of ethylene glycol. This is illustrated in Fig. 9, which shows the course of the deamination reaction in the presence of ethylene glycol. The rate of production of acetaldehyde decreases in an exponen- tial manner until it reaches a final constant velocity which thereafter remains unchanged. This result is reminiscent of the deactivation of enzyme which is observed when the deamination is carried out in the presence of certain coenzyme Blz analogues (10). From data presented above (Fig. S), it is unlikely that the deactivation of the enzyme is due to consumption of the coenzyme by the cleavage reaction; it appears rather that it is the result of a modification involving the enzyme itself.

The conditions necessary to produce deactivation are presented in Table VII. In these experiments, ethanolamine deaminase was incubated in the dark at room temperature with the constituents noted. At the end of 30 set, an aliquot of this preliminary incubation mixture was added to a cuvette containing the assay mixture, and the rate of deamination of ethanolamine was determined spectrophotometrically by the usual method. The concentration of ethylene glycol is such that, by 30 set, more than 980/, of the enzyme-coenzyme complex in the complete preliminary incubation mixture will have under- gone the cleavage reaction. These results show that deactivation is only observed when the enzyme is incubated with both ethylene glycol and DMBC, suggesting that the process is related to the cleavage reaction. However, despite the fact that under the conditions of the preliminary incubation less than 2% of the enzyme will have remained unaffected by the cleavage reaction, the activity of the enzyme from the complete preliminary incubation mixture is more than 10% that of the control. This confirms the observation in Fig. 9 that the deactivation associated with the cleavage reaction does not proceed to complete loss of activity.

The dependence of the rate of deactivation of enzyme on the concentrations of ethylene glycol and ethanolamine is shown in Fig. 10. The incubations were conducted under the conditions

4- P 0

3 l

c 2-

,+,

GLYCOL

IM

0.7 M 0.4 M

0 1

FIG. 10. Rate of deactivation of enzyme as a function of the concentrations of ethylene glycol and ethanolamine. For each determination, a mixture containing 0.01 pmole of DMBC, 0.2 pmole of NADH, 50 rmoles of potassium phosphate buffer (pH 7.4), 750 rg of yeast alcohol dehydrogenase (lyophilized, Boehringer), and ethanolamine and ethylene glycol as noted in a total volume of 1.0 ml was placed in a l-ml quartz cuvette with a l-cm light path. The reaction was begun by adding a convenient amount of enzyme (depending on the concentrations of ethanolamine and ethylene glycol, this quantity varied from 3.0 to 8.0 rg) and was then followed spectrophotometrically as previously described (3). From the spectrophotometric tracing, the rate constant for the deactivation process was calcldated as described in the text.

described in Fig. 9, except that the concentrations of ethanolamine and of ethylene glycol were varied as shown. For each incubation, the half-time for the deactivation was obtained as described previously (lo), and from the half-time the rate constant was calculated in the usual manner. Although the error in the rate constant is so large that fine mechanistic distinctions cannot be drawn from these results, it is nevertheless apparent in a qualitative way that the rate of deactivation is greater at higher concentrations of ethylene glycol, and that the deactivation process is inhibited by ethanolamine.

DISCUSSION

Nature of X-B12

The conversion of DMBC to X-B12 probably represents a substitution of 5’-deoxyadenosyl by another ligand in the coordination sphere of the cobalt. Such a replacement of 5’- deoxyadenosyl would be expected to lead to definite changes in the cobalamin spectrum, as have been observed in the present study. Further support for this formulation is provided by the observation that the 5’-deoxyadenosyl residue leaves the cobalt at about the same rate that X-1312 appears. That these changes do not appear to be due to a modification of the corrin ring is indicated by the observation that the cyanocobamide prepared from X-B12 is chromatographically identical with authentic cyanocobalamin.

From the spectral characteristic@ of the new corrinoid and from its behavior when the enzyme is denatured with urea, the sixth ligand of X-BIT appears to be a sulfhydryl group. The source of this group is presumably a cysteine residue in the vicinity of the active site. As mentioned previously, the spectrum of X-B12 resembles that of either an alkyl cobalamin or a

* The spectrum of X-B,2 is probably only slightly affected by the fact that it is enzyme-bound, since it has been previously shown with a number of cobamides that the binding of a cobalamin derivative to ethanolamine deaminase results in only slight changes in its spectrum (11).


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thiol cobalamin (that is, the product of the reaction between OHBlz and a mercaptan, such as glutathione or cysteine). Of these two types of cobamide, the observation that X-B12 is rapidly converted to OHBi2 after denaturation with urea is much more consistent with the latter, since an alkyl cobalamin which is completely hydrolyzed in 30 set at room temperature and neutral pH has not been described (20, 22), whereas the reaction

OHBlz + RSH + RSBlz + Hz0

occurs “instantaneously” (23) and, except in the case in which RSH is glutathione, the equilibrium lies well to the left under the usual experimental conditions (23). However, the possibility that X-Br2 represents a previously unreported alkyl cobalamin, or even a hitherto unknown type of corrin derivative, cannot be excluded.

The appearance of the photolabile compound upon denaturation by heat is difficult to explain. It probably represents a compound formed from X-B12 during the denaturation process rather than X-B12 itself, since the rapid appearance of OHB12 under the much milder conditions of denaturation by urea indicates that X-B12 is probably not stable under the conditions of heat denaturation. If X-Br2 is a thiolcobalamin, it may be that the photolabile compound is an alkyl cobalamin produced by reaction of X-B12 at a high temperature with an alkylating agent which is present on the enzyme either in its native form or as a result of the denaturation process. The formation of alkyl cobalamins from thiolcobalamins and alkyl halides has been well documented (21). Another alternative, also consistent with the assumption that X-B12 is a thiolcobalamin, is that the photolabile compound is a cobalamin sulfonate (21) arising when a sulfur compound at a high oxidation state, formed by the oxidation of SH groups at high temperatures under catalysis by cobamides (24), combines with the cobalamin as the reaction mixture cools. However, it must be emphasized that these speculations merely provide a rationalization for the appearance of a photolabile compound in the event that X-B12 is a thiolcobalamin. The appearance of a photolabile compound under these conditions cannot in itself be construed as evidence in favor of the hypothesis that X-B12 is a thiolcobalamin.

Mechanism of Reaction and Its Relationship to Catalytic Reaction

In this report it was shown that ethanolamine deaminase catalyzes the conversion of ethylene glycol to acetaldehyde at the expense of coenzyme Brz, which is split to yield 5’-deoxyadenosine and a new corrinoid which is referred to as X-Br2. It is likely that this reaction represents a portion of the reaction which is ordinarily catalyzed by the enzyme-DMBC complex, viz. the deamination of ethanolamine. With ethylene glycol as substrate, however, the reaction is aborted because one of the intermediates generated from ethylene glycol undergoes a side reaction, terminating the sequence which, if ethanolamine were the substrate, would lead to the catalytic production of acetaldehyde. Among the pieces of evidence favoring the inter- pretation that the observed process represents a partial reaction are the following.

Rate of Reaction-The V,,, for the cleavage reaction is of the order of 0.2 see-l. While this represents only 0.2% of the rate of the enzyme-catalyzed deamination of ethanolamine (18), it is nevertheless an impressive velocity for a “nonphysiological”

reaction. Furthermore, it shows the extent to which the enzyme is capable of facilitating the rupture of the carbon-cobalt bond, a process which is widely regarded as representing an integral part of the mechanism by which coenzyme Biz-dependent rearrangements take place.

The Fact That Hydrogen Is Transferred from Ethylene Glycol to Adenosine Moiety of Coenzyme-In all coenzyme B12-dependent rearrangements so far studied, the adenosine moiety of the coenzyme has been shown to serve as an intermediate hydrogen carrier, accepting a hydrogen atom from the substrate and returning it to the product (2). The transfer of hydrogen from ethylene glycol to 5’-deoxyadenosine therefore supports the idea that the reaction with ethylene glycol is closely related to the catalytic reaction with ethanolamine.

Xtructural Resemblance between Ethylene Glycol and Ethanola- mine-From the close structural resemblance between these two compounds, it would be reasonable to expect that the former might serve as an analogue of the latter in the ethanolamine deaminase reaction. The proposal that ethylene glycol serves as an analogue of ethanolamine is further supported by the observation that the deactivation of enzyme by ethylene glycol is antagonized by ethanolamine.

Reaction Product-The fact that both ethanolamine and ethylene glycol are converted to acetaldehyde is evidence that the reaction mechanisms for these two compounds are likely to be very similar.

The results obtained in this study can be accounted for by the mechanism presented in Scheme I. (In this scheme, R-CH2 represents 5’-deoxyadenosyl, Co represents cobalamin, X represents the side chain of an amino acid in the vicinity of the active site, and dotted lines represent unsatisfied valences. The repre- sentation of the product derived from ethylene glycol as an aldehyde is a matter of convenience, and does not exclude the possibility that this product exists on the enzyme as an aldehyde precursor such as vinyl alcohol or a 1, I-diol.) In the first step, the carbon-cobalt bond of the coenzyme is split. A hydrogen is then transferred from ethylene glycol to the 5’-carbon of the adenosyl residue. Simultaneous with or following this step, the Z-carbon fragment is converted to the protoaldehyde. Finally, the protoaldehyde removes a hydrogen from an amino acid residue lying in its vicinity to form acetaldehyde, whereupon the residue falls into the coordination sphere of the cobalt to form X-Bi2.

From the fact that the ethylene glycol reaction is terminal while the reaction with ethanolamine is catalytic, it is likely

SCHEME I


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that a different hydrogen atom is abstracted from the vicinity of the active site by the protoaldehyde from each of the two substrates. While the present evidence indicates that in the catalytic reaction the protoaldehyde probably takes a hydrogen from the 5’-carbon of the adenosine moiety of the coenzyme (1, 2), it appears that the hydrogen atom taken by the corresponding species in the ethylene glycol reaction is removed from the side chain of an amino acid residue in the vicinity of the active site (-XH: see Scheme I). Moreover, this amino acid residue probably has an electronegative atom from which the hydrogen is removed, since the evidence indicates that a new cobalamin is formed which is very susceptible to hydrolysis and it is likely that the sixth ligand of this new cobalamin is derived from the side chain from which the hydrogen is taken. On the basis of evidence discussed previously, it appears that the hydrogen is removed from a sulfhydryl group, with the formation of a thiol cobalamin. A hydrogen abstracted from a sulfhydryl group would probably be removed as a proton or a hydrogen atom; it would be very unlikely to depart as a hydride ion. The report that the enzyme-DMBC complex appears to be able to generate ESR signals both in the absence and particularly in the presence of substrate (5, 11) suggests that the hydrogen is abstracted as an atom.

The differences referred to above undoubtedly reflect the fact that the reaction paths for the two substrates diverge at some point. Two possible points of divergence are considered here. The first is concerned with the mechanism of conversion of each of the two substrates to the protoaldehyde, and the second deals with possible differences between the protoaldehydes arising from the two substrates.

With regard to the mechanism of conversion of substrate to protoaldehyde, it is likely that the conversion of ethanolamine to acetaldehyde involves the migration of the amino group with the transient formation of 1-aminoethanol as an intermediate in the reaction (18, 25). It is possible, however, that the formation of acetaldehyde from ethylene glycol involves an elimination rather than a rearrangement. According to this mechanism, elimination of water or OH- would accompany or follow the abstraction of hydrogen from ethylene glycol; subsequently, acetaldehyde would be formed by ketonization of the resulting enol. This mechanism would imply that the hydrogen is abstracted from ethylene glycol as a proton.

According to the second alternative, even if the conversion to protoaldehyde occurs in each case via a rearrangement, it is possible that the -CH&H(OH)NH8+ fragment arising from ethanolamine is restricted to a fixed location in the active site, while the -CH2C!H(OH)z group produced from ethylene glycol is relatively mobile. Under these circumstances it is conceivable that the ethanolamine-derived fragment is obliged to remove a

hydrogen atom from the 5’-carbon of the adenosine of DMBC, thereby permitting the coenzyme to be regenerated and the cycle to be repeated, while the glycol-derived fragment is able to take a more easily removed hydrogen from elsewhere in the active site, with the result that the reaction is terminated because the coenzyme cannot be regenerated. Both of these alternatives can account for the differences between the catalytic reaction and the reaction with ethylene glycol. Nevertheless, it is clear that they do not exhaust the possibilities, and that the differences between the two reactions may be based on considerations en- tirely different from the ones discussed above.

Acknowledgments-We wish to thank Earl R. Stadtman, Henry M. Lutterlough, and Maurice Miles for providing facilities and assistance for growing the bacteria from which ethanolamine deaminase is obtained.

1. FREY, P.A., ESSENBERG, M.K., ANDABELES, R.H., J.BioZ., Chem., 242, 5369 (1967).

2. 3. 4.

5.

BABIOR, B.. Biochim. Biophzls. Ada, 167, 456 (1968). RETEY,.J., BND ARIGONI, D.;Ezperie&ia, 22, 783 (1968). MILLER. W. W.. AND RICHARDS. J. H.. J. Amer. Chem. Sot..

91, 1498 (1969 j . ,~ ,

BABIOR, B., AND GOULD, D. C., Biochem. Biophys. Res. Com- mm., 34, 441 (1969).

6. WAGNER.~. W., LEE, H.A., JR.,FREY,P.A..ANDABELES,R.

7. H., J..BioZ. bhem.; 241, i751 .(1966): ’

KAPLAN. B. H.. AND STADTMAN. E. R.. J. Biol. Chem.. 243.

8. 1787 (i968). ’

, I

KAPLAN, B. H., AND STADTMAN, E. R., J. Biol. Chem., 243, 1794 (1968).

9. BABIOR, B. M., AND LI, T. K., Biochemistry, 8, 154 (1969). 10. BABIOR, B. M., J. Biol. Chem., 244, 2917 (1969). 11. BABIOR, B. M., KON, H., AND LECAR, H., Biochemistry, 8,

2662 (1969). 12. BRAY, G. A., Anal. Biochem., 1, 297 (1960). 13. BRADBEER, c., J. Biol. Chem., 240, 4669 (1965). 14. BARKER. H. A.. U. S. Patent 3.037.Oi6 (Mav 29. 1962). 15. LowRY,'~. H., ~OSEBROUGH, N.‘J., FARR: A. ~.,A&DRANDALL,

16. R. J.; J. Biol. Chem., 193; 265 (1951).

SHRINER. R.L.. FUSON. R. C.. AND CURTIN. D.Y.. The sus-

17.

tematic’ identification ‘0-f or&k compounhs, Ed: 5, John Wiley and So&, Inc., fiewkork, 1964, p. 114.

ZAGALAK. B.. FREY. P. A.. KARABATSOS.G. L.. AND ABELES. R. H.,‘J. biol. khem., i41, 3028 (1966). ’

ii: 20.

21. 22. 23.

BABIOR,' B. M., J. Biol.’ Chem., 244; 449. (1969). HOGENKAMP. H. P. C.. Biochemistru. 6. 417 (1969). HOGENKAMP: H. P. C.: RUSH, J. E:; AND SWENSON, C. A.,

J. Biol. Chem., 240, 3641 (1966). DOLPHIN, D., AND JOHNSON, A. W., J. Chem. Sot., 2174 (1965). HOGENKAMP, H. P. C., Fed. Proc., 26, 1623 (1966). ADLER, N., MEDWICK, P., AND POZNANSKI, T. J., J. Amer.

Chem. SOL, 88, 21 (1966). 24. 25.

PEEL, H. L., Biochem. J., 88, 296 (1963). RETEY, J., UMANI-RONCHI, A., SIEBL, J., AND ARIGONI, D.,

Experientia, 22, 502 (1966).

REFERENCES


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Bernard M. BabiorGLYCOL, A QUASI-SUBSTRATE FOR ETHANOLAMINE DEAMINASE

The Mechanism of Action of Ethanolamine Deaminase: VI. ETHYLENE

1970, 245:1755-1766.J. Biol. Chem.

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