The mechanism of action of ethanolamine ammonia-lyase, a B12-dependent enzyme. Evidence for two intermediates in the catalytic process.

Abstract Ethanolamine ammonia-lyase, an enzyme catalyzing the adenosylcobalamin-dependent deamination of ethanolamine, also catalyzes the conversion of l-2-aminopropanol to propionaldehyde and ammonia. In this reaction, tritium is transferred from enzyme·[5'-3H]adenosylcobalamin to the C-1 position of l-2-aminopropanol, as well as to the α carbon of the product aldehyde. The labeling pattern is consistent with the mechanism of hydrogen transfer deduced with other substrates. Tritium transfer also occurs between enzyme·[5'-3H]adenosylcobalamin and propionaldehyde in the presence of NH4+. Unlike the deamination of ethanolamine, the conversion of 2-aminopropanol to propionaldehyde and NH4+ is reversible, since tritiated 2-aminopropanol was isolated from reaction mixtures originally containing only propionaldehyde, ammonia, and enzyme·[5'-3H]adenosyl cobalamin. The partitioning of tritium derived from enzyme·[5'-3H]-adenosylcobalamin between l-2-aminopropanol and propionaldehyde was determined in reactions begun with l-2-aminopropanol as well as in reactions begun with propionaldehyde and ammonia. In the first case, the ratio [3H]propanolamine to [3H]propionaldehyde is 1.8:1; in the second case, the ratio is 0.3:1. This difference in the partitioning of tritium from the tritiated enzyme complex is consistent with the notion that there are at least two intermediates in the catalytic process, each exchanging tritium with coenzyme, which interconvert slowly with respect to the rate of tritium exchange.

ammonia-lyase, an enzyme catalyzing the adenosylcobalamin-dependent deamination of ethanolamine, also catalyzes the conversion of L-2-aminopropanol to propionaldehyde and ammonia. In this reaction, tritium is transferred from enzyme' IS'-W]adenosylcobalamin to the C-l position of L-2-aminopropanol, as well as to the (Y carbon of the product aldehyde.
The labeling pattern is consistent with the mechanism of hydrogen transfer deduced with other substrates.
The partitioning of tritinm derived from enzyme. [5'-aH]adenosylcobalamin between L-2-aminopropanol and propionaldehyde was determined in reactions begun with L-Zaminopropanol as well as in reactions begun with propionaldehyde and ammonia.
In the first case, the ratio [3H]propanolamine to [3H]propionaldehyde is 1.8:1; in the second case, the ratio is 0.3: 1. This difference in the partitioning of tritiurn from the tritiated enzyme complex is consistent with the notion that there are at least two intermediates in the catalytic process, each exchanging tritium with coenzyme, which interconvert slowly with respect to the rate of tritium exchange. This is Paner 11 in this series. The pre&ous paper is Ref. sp., an adenosylcobalamin-requiring enzyme, catalyzes the conversion of ethanolamine to acetaldehyde and NH,+ (2). It has been reported that L%aminopropanol is a competitive inhibitor for that reaction (3). In the course of our studies on the mechanism of action of ethanolamine ammonia-lyase, we have confirmed this fact, but found, in addition, that L-2-aminopropanol is also a substrate for that enzyme.
This substrate displays some unique properties which enabled us to obtain further evidence in support of the mechanism previously proposed for adenosylcobalamin-dependent rearrangements (4-7). The results obtained with L-2-aminopropanol are reported in this and a subsequent communication.

MATERIALS AND METHODS
Enzyme, Coenzyme, and Substrate-Ethanolamine ammonialyase from Clostridium sp. (3) was purified and resolved of bound cobamides as previously described (8,9). Enzyme concentration was calculated on the basis of a molecular weight of 520,000 (8) and the highest specific activity reported of 45 units per mg (10). The enzyme possesses two active sites per molecule (11)(12)(13).
The enzyme was made substrate-free by dialysis against 0.01 M potassium phosphate buffer, pH 7.4.
AdoCbl' was obtained from Glaxo Laboratories. Ethanolamine, L-2-aminopropanol, and propionaldehyde were obtained from commercial sources and were redistilled before use. Both ethanolamine and L-Zaminopropanol were converted to their respective hydrochlorides.
All other reagents were of the highest purity obtained commercially.
L-2-Amino[ UJ4C]propanol was synthesized by reducing the trifluoroacetate salt of I,-[ U-Wlalanine wit,h diborane (14). The trifluoroacetate was prepared by adsorbing 171 pmoles (7.7 x lo5 dpm per pmole) of L-[ U-14C]alanine (New England Nuclear) onto a column (5 x 40 mm) of Dowex 50-X8 resin (H+ form). After washing the column with 20 ml of water, L-[Wlalanine was eluted with 1.5 N trifluoroacetic acid. The eluate was evaporated to dryness on a rotary evaporator, and the glassy oil remaining was further dried in a vacuum desiccator over PZOs for 2 hours. The reduction was carried out by the addition of 3 ml of 1 M diborane in tetrahydrofuran to Q4C]alanine trifluoroacetate; the reaction was allowed to proceed at room temperature for 2 hours. For isolation of the product, tetrahydrofuran was evaporated under a stream of nitrogen.
The residue was dissolved in 2 ml of anhydrous methanol containing 1 drop of trifluoroacetic acid and allowed to stir for 1 hour. The resulting solution was taken to dryness on a rotary evaporator, and the residue was twice dissolved in 3 ml of methanol and immediately evaporated to dryness each time. This procedure served to remove borate as its volatile methyl ester. The residual material was dissolved in 0.2 ml of HzO. L-2-Amino[14C]propanol was purified by chromatography on a column (0.9 x 36.5 cm) of Dowex 50-X8 resin using 0.2 M pyridinium acetate buffer (pH 3.5) as the developing solvent (15). The aqueous solution of radioactive propanolamine was diluted to 2 ml with 0.2 M pyridinium acetate buffer (pH 3.5), the pH adjusted to 2.2 with 3 N HCl, and the resulting solution applied to the Dowex column.
The radioactive fractions, which were eluted between 180 and 210 ml, were pooled and lyophilized.
The lyophilized material was dissolved in 1 ml of Hz0 and passed through a column (5 x 30 mm) of Dowex l-X8 resin (OH-form).
The eluate was neutralized with 0.05 N HCl.
The over-all yield was 26oi,, and the specific activity of the product was 7.4 X lo5 dpm per pmole.
Assays-Ethanolamine ammonia-lyase was assayed by measuring the rate of conversion of ethanolamine to acetaldehyde and ammonia.
The enzyme was diluted for assay to 0.2 to 0.8 unit per ml in 0.05 M potassium phosphate buffer, pH 7.4, containing 0.05 M ethanolamine hydrochloride.
The assay mixtures contained 0.2 ml of diluted enzyme, 50 pmoles of potassium phosphate buffer (pH 7.4), 100 pmoles of ethanolamine hydrochloride, and 0 or 0.013 pmole of AdoCbl in a total volume of 1.0 ml. The reaction was initiated by the addition of coenzyme and was incubated at 37" for 4 min. All reactions in which coenzyme was used were carried out in the dark. The reaction was stopped by the addition of 0.1 ml of 2 N HCl, and the amount of aldehyde produced was measured calorimetrically (16). Sodium pyruvate was used as a standard for the assay. One unit was defined as the amount of enzyme catalyzing the formation of 1 pmole of acetaldehyde per min. Protein was determined by the method of Lowry et al. (17) with appropriate correction (3). The concentration of adenosylcobalamin was assayed spectrophotometrically at 367 nm after conversion to dicyanocobalamin with KCN, using 30.4 x lo3 M-l cm-' as the extinction coefficient (18), L-2-Aminopropanol was measured by oxidation with periodate and subsequent calorimetric determination of the formaldehyde produced (19). Aldehyde concentrations were determined as in the enzyme assay. n-Alanine was quantitated with ninhydrin cw .
Radiochemical assays were carried out by liquid scintillation counting with a solvent system consisting of 7 g of 2,3-diphenyloxazole, 300 mg of p-bis[2-(9phenyloxazolyl)]benzene, and 100 g of naphthalene in 1 liter of dioxane solution. Radioactivity measurements were made using a Nuclear Chicago Mark I or Packard Tri-Carb (model 3320) liquid scintillation spectrometer. The measurements of radioactivity of the 2,4-dinitrophenylhydrazone derivatives were corrected for quenching by internal To isolate the tritiated propionaldehyde and n-2-aminopropanol produced in these experiments, the following procedure was employed. All procedures involving aldehydes were carried out at 04". After the addition of carrier propionaldehyde and L-2-aminopropanol, the pH was adjusted to pH 5.5 to 6.0 with 0.2 M KzHPO~.
The solution was then treated twice with 10 to 20 mg of charcoal (Darco G60, Matheson, Coleman and Bell) to remove [3H]AdoCbl.
The charcoal was removed by centrifugation, and the supernatant was passed through a column of Dowex 50-X8 resin (5 X 50 mm or 10 X 100 mm, depending on the amount of carrier propanolamine added). Small aliquots were taken for measuring propionaldehyde, propanolamine, and radioactivity content before and after separation on Dowex.
Propionaldehyde was eluted with water; and after washing the column exhaustively with H20, LLaminopropanol was eluted with 1.5 ~rj HCI. The propionaldehyde was isolated as the propionaldomethone derivative. This was prepared as described previously (4) and recrystallized to constant specific activity. The mehing point of the recrystallized derivative (156-157") was in agreement with the reported value (21). The acid eluate containing 2-aminopropanol hydrochloride was taken to dryness on a rotary evaporator.
The residue was twice dissolved in 1 ml of water and each time evaporated to dryness to remove any residual HCI. The material was further dried in vaeuo over PZOS. The 0 ,N-di(p-bromobenzoyl) derivative of 2-aminopropanol was prepared by a modification of the procedure of Jeger et al. (22). 2-Aminopropanol (350 to 1000 pmoles), dissolved in 1 ml of dry pyridine, was mixed with 3 ml of a suspension of 0.6 g of p-bromobenzoyl chloride in pyridine and stirred for 21 hours at room temperature.
Ice water was then added to the reaction mixture.
The precipitate which immediately appeared was collected and dissolved in 150 ml of benzene. This solution was extracted three times with equal volumes of a saturated solution of sodium bicarbonate to remove p-bromobenzoic acid. After being dried over anhydrous sodium sulfate, the organic layer was concentrated until crystals appeared. The material was recrystallized from hot benzene (m.p. 158-159" (uncorrected) ; literature, 155" (22)). The specific activity was the same after each crystallization.
The location of tritium in propionaldehyde was determined by oxidation to propionic acid, while in 2-aminopropanol its location was determined by degradation with periodate. Propionaldehyde was separated from propanolamine by ion exchange chromatography as described above. After purification by bulb-to-bulb distillation, the aldehyde was oxidized to propionic acid with KMn04, maintaining the pH at 6.5 with additions of NaOH (23). Propionic acid was isolated by column dhromatography on silicic acid (24) using 4% l-butanol as developing solvent.
The fractions were titrated and assayed for radioactivity as published previously (4). The 2-aminopropanol eluted from the Dowex column (6.4 pmoles) was dissolved in 1 ml of H20 and was oxidized at pH 6.4 with 1 ml of 0.075 M sodium metaperiodate.
The reaction was allowed to proceed for 20 hours in the cold. Formaldehyde and acetaldehyde, formed in the reaction from the alcohol carbon (C-l) and the remainder of the molecule, respectively, were isolated by distillation. The 2,4-dinitrophenylhydrazone derivatives of these aldehydes were then prepared by adding 3 ml of 1 y0 2,4-dinitrophenylhydrazone in 3 N HzS04 to the distillate.
The method for synthesis of these derivatives, their separation by paper chromatography, and their assay have been described previously (4).

RESULTS
The data presented in Fig. 1 show that ethanolamine ammonialyase catalyzes the conversion of n-2-aminopropanol to propionaldehyde and ammonia.
In the experiment shown, 210 nmoles each of propionaldehyde and ammonia were produced for every nanomole of active site present in the reaction mixture.
The turnover number of about 60 min-' per active site indicates that this catalysis is considerably less efficient than that occurring with ethanolamine, the natural substrate (turnover number 8600 minP (11)). When L-2-aminopropanol was added to the enzyme.
[jH]AdoCbl complex, tritium was transferred from coenzyme to both the product, propionaldehyde, and the starting material, 2-aminopropanol (Table I). Essentially all of the tritium lost from the coenzyme (i.e. not taken up by charcoal) was found in either propionaldehyde or propanolamine. The location of the tritium in each of the two compounds was established by the method described above.
The results, summarized in Table II, showed that 2-aminopropanol was labeled exclusively at C-l, while propionaldehyde was labeled in the a! or p position. Based on earlier enzymatic reactions (4, 7, 25, 26), we conclude that the tritium was located solely on the a! position.
Therefore, the action of ethanolamine ammonia-lyase on rZaminopropano1 is very similar to its action on ethanolamine, except that with (specific activity, 7.4 X 10' dpm per &mole), and 1.5 pmoles of potassium phosphate buffer, pH 7.4, in a total volume of 0.2 ml; (0) 20 units (approximately 1.7 neq in "active sites") of ethanolamine ammonia-lyase, (0) no enzyme. The enzyme was allowed to react with coenzyme for 3 min before 2-amino['%]propanol was added to initiate the reaction. All incubations were carried out at 22". Aliquots of 0.04 ml were removed from the reaction and quenched in 0.5 ml of 0.05 N HCl. Propionaldehyde carrier (5.6 rmoles) was added to each time point. After neutralization, the reaction was distilled, and propionaldehyde isolated as the 2,4-dinitrophenylhydrazone derivative as described under "Materials and Methods." 1685 ethanolamine reversible hydrogen transfer between substrate and coenzyme does not occur (26).
The results of Table III show that incubation of enzyme.
[3H]AdoCbl complex with propionaldehyde, the reaction product, results in the transfer of radioactivity from the coenzyme to a compound or compounds not adsorbed by charcoal, provided ammonia is also present.
In the absence of either ammonia or propionaldehyde, there is no loss of tritium from coenzyme. (In the latter experiments, the tritium which remained in solution after charcoal treatment appears to represent a contaminant of the coenzyme. since the same fraction of tritium remained in solution after charcoal treatment of reaction mixture in which ethanolamine ammonia-lyase was replaced by albumin ("control," Table I).) As expected from the results of Table I, trit- The reaction mixture contained 30.5 units (approximately 2.6 neq of "active sites") of ethanolamine ammonia-lyase, 6.7 nmoles of [3H]AdoCbl (specific activity, 2.95 X 104 cpm per nmole), 53.2 pmoles of L-2-aminopropanol-HCl, 50 pmoles of potassium phosphate buffer (pH 7.4), and 1.1 mmoles of glycerol in a total volume of 1 ml.
The reaction was started by the addition of enzyme and was allowed to proceed for 5 min at 37". A control in which 100 pg of bovine serum albumin were substituted for enzyme was also  ium was also lost from coenzyme when propionaldehyde and ammonia were replaced by LZaminopropanol.
Identification of the compounds to which tritium was transferred in the experiments with propionaldehyde led to the surprising discovery that the deamination of propanolamine was a reversible reaction.
This was indicated by the observation that tritium originally in the coenzyme was found not only in propionaldehyde, as expected, but also in propanolamine (Table  IV). Tritium from the coenzyme is transferred to both propionaldehyde and propanolamine not only in the experiments presented in t.he table, but also in experiments in which 6% trichloroacetic acid had been used to terminate the reactions and no carrier had been added.
(In these experiment.s, aldehyde and amine were separat,ed from each other on small columns of Dowex 50-XS (H+) according to the method of Babior and Li (ll), the tritium content in the aldehyde-and aminecontaining fractions being determined without furt.her purification.) aminopropanol alone, From these results we conclude that the difference in the partitioning of tritium from enzyme.
[3H]-AdoCbl in the presence of substrate and product, respectively, did not arise from modification of the int,ermediate by interaction with either propionaldehyde or ammonia.

DISCUSSION
The distribution of tritium between L-2-aminopropanol and propionaldehyde was found to depend on whet,her the reaction was started with L-2-aminopropanol or propionaldehyde. The data in Table IV show that starting with L-2.aminopropanol, the rat,io of total trit,ium in L-2-aminopropanol to that in propionaldehyde was 1.8. When t.he reaction was started wit.h propionaldehyde, however, the ratio was 0.3. To establish t,hat the change in the ratio was indeed dependent on the nature of the starting material and was not merely due to an allosteric interaction between the catalytic complex and other components of the reaction mixture, exchange of t.ritium into propanolamine (0.13 pmole) was measured as described in Table III, except that in addition to L-2-aminopropanol, propionaldehyde (6.8 pmoles) and NH&l (6.5 pmoles) were also present.
Under these conditions, the partitioning of tritium between propanolamine and propionaldehyde was the same as that observed with 2- The results reported here show that L-2-aminopropanol is a substrate for ethanolamine ammonia-lyase and is deaminated to propionaldehyde and NH4+. When the reaction was carried out in the presence of [aH]AdoCbl, tritium was transferred both to propanolamine and to propionaldehyde. Tritium derived from the coenzyme was found in the C-l position of propanolamine, while in propionaldehyde, the tritium was located cx to the carbonyl group.
This labeling pattern is consistent with previously proposed mechanisms for this reaction (5,26,27). Therefore, we believe that the interaction of x&aminopropanol with enzyme-coenzyme complex results in t'he formation of intermediates similar to those present in the normal catalytic pathway.
The reaction with L-2-aminopropanol, however, differs from that with ethanolamine in two respects.
(a) With L-2-aminopropanol, tritium from the coenzyme is transferred to the substrate.
No tritium kansfer from coenzyme to substrate can be det.ected with ethanolamine.
No evidence for reversibility has been detected in the reaction with ethanolamine.
The over-all reversibility of t.he deamination of L-2-aminopropanol was established by the appearance of tritiated propanolamine in an experiment in which only propionaldehyde and ammonia were incubat.ed with the enzyme. 13H]AdoCbl complex. Though these results show clearly that the reaction is reversible, they give no indication as to the equilibrium constant for the  Table IV show that 25% of the tritium lost from the cofactor appears in propanolamine, the remainder being found in propionaldehyde.
It is likely, however, that the tritiated propanolamine is in equilibrium with the tritium-labeled coenzyme, since the exchange of tritium between coenryme and propanolamine is rapid with respect to the duration of the experiment in Table IV (see Table  I). Under such circumstances, it would be expected that the specific activity of the propanolamine would be the same as the specific activity of the tritiated coenzgme, except for a statistical factor of 3 (assuming that t.he product of the react.ion between propanolamine and adenosylcobalamin is 5'-deoxyadenosine) and whatever small equilibrium isotope effect the reaction may display.
The results of Table IV show, on the other hand, that hydrogen exchange between propionaldehyde and coenzyme is far from equilibrium, since the concentration of aldehyde exceeds the concentration of adenosylcobalamin by a factor of 1.7 x 104, while only 18% of the tritium originally in the coenzyme has been transferred to propionaldehyde.
Assuming isotopic equilibration between propanolamine and adenosylcobalamin, the quantity of propanolamine in the reaction mixture is calculated to be 0.072 nmole, compared with 6.8 pmoles of propionaldehyde. The similarity in the amounts of radioactivity can be ascribed to differences in specific activity, the propanolamine being much more highly labeled than the propionaldehyde.
The data in Table IV show that the ratio between the amount of tritium transferred from the coenzyme to substrate and that transferred to product depends upon whether the reaction is started with L-2.aminopropanol or with propionaldehyde and NH,+.
When started with propanolamine, t.he ratio of t,ritium in propanolamine to that in propionaldehyde is 1.8, while when started with propionaldehyde and NH4+, it is 0.3. Since the tritium distribution depends upon whether the reaction is started from the product or substrate side, it can be concluded that there must be more than one intermediate species which can exchange tritium with product or starting material.
If there were only one such species (Scheme l), tritium distribution should be the same whether the reaction is started with propanolamine or Tritiated species are denoted by superscript 3. According to this scheme, the partitioning of tritium between starting material and product, represented by the ratio S3H :PaH, will depend only upon the fate of Z.ZFH; i.e. it will be determined by the relative rates of Steps 3 and 4. In such a scheme, SaH:P3H will be the same whether the reaction is started with SH or PH. Since the observed ratios depend on the nature of the starting material, the single intermediate mechanism is not applicable to the reaction investigated here.
A mechanism consistent with the results is the one shown in Scheme 2, involving two intermediates each of which can ex-&H &H SCHEME 2 In such a mechanism there are conditions under which the SaH : PaH ratio would depend upon the nature of the starting material (i.e. upon whether the reaction is started with SH or with PH). If the rate of interconversion of the two intermediates is slower than the rate of transfer of tritium to starting material (i.e. if kz < k4 and k& < k5), then when SH is the substrate, SaH:P3H will depend upon the value k2/k4, while S3H :P3H obtained with PH as substrate will depend upon k-2/ks. If kz/k4 # k--2.1k5, then the partitioning of t.rit.ium will be different for each of the two starting materials.2 Mechanisms of this type have, if fact, been proposed for reactions involving adenosylcobalamin (5,6,25,27). In these mechanisms, (Z.EH)l and (Z.EH)Q correspond to enzyme-bound complexes consisting of a substrate-cobalamin adduct and 5'deoxyadenosine and a product-cobalamin adduct and 5'-deoxyadenosine, respectively.
The results reported here, therefore, is not proportional to the ratio of concentrations of the two species.
In this table are shown  two exchange  experiments, one starting with 0.31 mM propanolamine and the other with 1.0 mM propanolamine.
The same amount of tritium was washed out of the cofactor in each experiment, showing that the amount of propionaldehyde produced in the two experiments was the same, i.e. propanolamine was saturating at both concentrations.
The propanolamine to propionaldehyde ratio in one incubation mixture was therefore 3 times greater than in the other throughout the course of the reaction. Despite this circumstance, the p&itioning of tritium between propanolamine and propionaldehyde was the same in the two incubations.
This finding constitutes strong evidence against the alternative mechanism outlined above. change tritium with the coenzyme. in this hydrogen transfer ((I.EH)I, Scheme 2) by thesubsequent irreversible step would ensure that the reversibility of hydrogen transfer would be undetectable experimentally.
We propose that when L-2-aminopropanol is the substrate, the interconversion of the cobalamin adducts ((Z.EH)l * (I.EH)z in Scheme 2) becomes rate-determining, so t,hat the reversibility of formation of the first. complex can now be observed.
Tritium transfer to both substrate and product can, therefore, occur. A consequence of the proposed change in rate-determining step is that when the substrate is propanolamine the amount of the first intermediate (1. EH) 1, which accumulates under steady state condit,ions, should be larger than when it is et,hanolamine.
In a subsequent communicat,ion, we will present, evidence that this is, in fact,, the case.