12
THE JOURXAL OF BIOLOGICAL CHEMISTRY Vol. 244, No. 6, Issue of March 25, pp. 1434-1444, 1969 Printed in U.S.A. Enzymic Reactions of Enamines with N-Ethylmaleimide (Received for publication, August 23, 1967) MARTIN FLAVIN AND CLARENCE SLAUGHTER From the Laboratory of Biochemistry, National Heart Institute, National Institutes of Health, Bethesda, Maryland 20014 SUMMARY y-Cystathionases catalyze elimination of substituents from the /3- or y-positions of 4-carbon chain length amino acids with formation of cr-ketobutyrate, but when N-ethylmalei- mide is added to reaction mixtures the Lu-ketobutyrate is replaced by a product reflecting addition to the maleimide double bond of an intermediate with carbanion character at carbon 3. The product is a-keto-3-(3’-(N’-ethyl-2’,5’- dioxopyrrolidyl)) butyric acid, or KEDB. The reactive intermediate was postulated to be one of the three enamines: free aminocrotonate, aminocrotonate bound to the enzyme in an unprotonated form, or the Schiff base between amino- crotonate and the coenzyme, pyridoxal phosphate. The cystathionases have now been shown to catalyze stereo- specific reactions with N-ethyhnaleimide, yielding only one of the two possiblediastereoisomers. The sameisomer was formed by a third pyridoxal-P enzyme, cystathionine y- synthase acting on 0-succinylhomoserine, but a threonine dehydraseyielded the other isomer of KEDB from threonine. The last two enzymes differ from cystathionases in that the d-carbon substrates, 0-succinylserine and serine, respec- tively, do react in the presenceof the maleimide to yield the analogueof KEDB less the methyl group, KEDP. This evidence that free enamino acids were not the inter- mediates that reacted with maleimide prompted an investi- gation of whether they were intermediates in any form. The basisfor this study was that if they were, solvent tritium would be introduced into the P-position of newly formed pyruvate or a-ketobutyrate in a spontaneous step, and therefore the kinetic isotope effect would always be the same, regardless of which enzyme was used. Preliminary results suggestthat the isotope effects may differ characteristically with different enzymes. A third study of the reactions of enamines with maleimides involved generating free enamines in the presence of the latter, through the action of hydrolytic enzymes on N-acyl derivatives of cw-aminoacrylate and cu-aminocrotonate. A kidney aminopeptidase produced KEDP from the first and a mixture of isomers of KEDB from the second. However, it is possible that the reactive species was an enzyme-bound enamine in this casealso. The pyridoxal-P enzyme, y-cystathionase, normally catalyzes the y-elimination of cysteine from cystathione, the other products then being ammonia and cw-ketobutyrate. But in the presence of N-ethylmaleimide a different reaction is catalyzed (l-3) in which ar-ketobutyrate is replaced by the product a-keto-3-(3’- (W-ethyl-Z’, 5’-dioxopyrrolidyl)) butyric acid, whose structure is shown in Fig. 1. The enzyme can decompose a wide variety of fi- and y-substituted amino acids (4) in addition to cystathionine. The p-elimination of cysteine from lanthionine, in the presence of NEM,’ did not result in the formation of any a-keto-3-(3’- N-ethyl-2’,5’-dioxopyrrolidyl)) propionic acid (Fig. 1) at the expense of pyruvate (I), suggesting that the intermediate which reacted with NEM was one which was unique to a y-elimination, i.e. Structure II, III, or IV of Fig. 2. Of these, only Structure III appeared to have the high electron density on carbon 3 req- uisite for alkylation of NEM to yield KEDB. However, as mentioned in a preliminary report (5), when sub- strates were discovered which were P-substituted but of 4-carbon chain length (Fig. 2, V), it was found that they did yield KEDB. This result indicated that the intermediate reacting with NEM must be one common to both y- and p-eliminations, i.e. one of the enamines VII, VIII, or IX shown in Fig. 2. A nonenzymic reaction has also been briefly described by which, under conditions much more vigorous than those of the above reaction,” ammonia could catalyze formation of KEDB 1 The abbreviations used are: NEM, N-ethylmaleimide; KEDB, a-keto-3-(3’-(N’-ethyl-2’,5’-dioxopyrrolidyl)) butyric acid, the subscripts 1 and 2 referring to the diastereoisomers of slower and faster mobility, respectively, duringpaper electrophoresis;KEDP, or-keto-3-(3’-(W-ethyl-2’,5’-dioxopyrrolidyl)) propionic acid. 2 The spontaneous reaction is observed when a solution of pH 8.5 and 0.667 M in ammonium a-ketobutyrate and 0.006 M in NEM is heated for 30 to 60 min at 50” (M. Flavin and C. Slaughter. un- published results). Pyruvate also reacted, forming KEDP at one-third the rate of KEDB formation from a-ketobutyrate. The possibility that these nonenzymic reactions were mediated by the formation of ketimines (S&ii bases) between ammonia and the or-keto acids was investigated by two types of experiment. In the first, it was found that ammonia did not accelerate the enolization of a-ketobutyrate, as measured by the exchange of tritium out of the P-position of the latter, under the conditions of the reaction forminn KEDB. Primarv. secondarv. and narticu- larly tertiary amineswere more effecti+e’in catalyzing enoiization. This result indicates that if ketimines are intermediates their enolization, i.e. the formation of the reactive common base of the 1434 by guest on December 27, 2018 http://www.jbc.org/ Downloaded from

Enzymic Reactions of Enamines with N-Ethylmaleimide - Journal of

  • Upload
    others

  • View
    6

  • Download
    0

Embed Size (px)

Citation preview

Page 1: Enzymic Reactions of Enamines with N-Ethylmaleimide - Journal of

THE JOURXAL OF BIOLOGICAL CHEMISTRY Vol. 244, No. 6, Issue of March 25, pp. 1434-1444, 1969

Printed in U.S.A.

Enzymic Reactions of Enamines with N-Ethylmaleimide

(Received for publication, August 23, 1967)

MARTIN FLAVIN AND CLARENCE SLAUGHTER

From the Laboratory of Biochemistry, National Heart Institute, National Institutes of Health, Bethesda, Maryland 20014

SUMMARY

y-Cystathionases catalyze elimination of substituents from the /3- or y-positions of 4-carbon chain length amino acids with formation of cr-ketobutyrate, but when N-ethylmalei- mide is added to reaction mixtures the Lu-ketobutyrate is replaced by a product reflecting addition to the maleimide double bond of an intermediate with carbanion character at carbon 3. The product is a-keto-3-(3’-(N’-ethyl-2’,5’- dioxopyrrolidyl)) butyric acid, or KEDB. The reactive intermediate was postulated to be one of the three enamines: free aminocrotonate, aminocrotonate bound to the enzyme in an unprotonated form, or the Schiff base between amino- crotonate and the coenzyme, pyridoxal phosphate. The cystathionases have now been shown to catalyze stereo- specific reactions with N-ethyhnaleimide, yielding only one of the two possible diastereoisomers. The same isomer was formed by a third pyridoxal-P enzyme, cystathionine y- synthase acting on 0-succinylhomoserine, but a threonine dehydrase yielded the other isomer of KEDB from threonine. The last two enzymes differ from cystathionases in that the d-carbon substrates, 0-succinylserine and serine, respec- tively, do react in the presence of the maleimide to yield the analogue of KEDB less the methyl group, KEDP.

This evidence that free enamino acids were not the inter- mediates that reacted with maleimide prompted an investi- gation of whether they were intermediates in any form. The basis for this study was that if they were, solvent tritium would be introduced into the P-position of newly formed pyruvate or a-ketobutyrate in a spontaneous step, and therefore the kinetic isotope effect would always be the same, regardless of which enzyme was used. Preliminary results suggest that the isotope effects may differ characteristically with different enzymes.

A third study of the reactions of enamines with maleimides involved generating free enamines in the presence of the latter, through the action of hydrolytic enzymes on N-acyl derivatives of cw-aminoacrylate and cu-aminocrotonate. A kidney aminopeptidase produced KEDP from the first and a mixture of isomers of KEDB from the second. However, it is possible that the reactive species was an enzyme-bound enamine in this case also.

The pyridoxal-P enzyme, y-cystathionase, normally catalyzes the y-elimination of cysteine from cystathione, the other products then being ammonia and cw-ketobutyrate. But in the presence of N-ethylmaleimide a different reaction is catalyzed (l-3) in which ar-ketobutyrate is replaced by the product a-keto-3-(3’- (W-ethyl-Z’, 5’-dioxopyrrolidyl)) butyric acid, whose structure is shown in Fig. 1. The enzyme can decompose a wide variety of fi- and y-substituted amino acids (4) in addition to cystathionine. The p-elimination of cysteine from lanthionine, in the presence of NEM,’ did not result in the formation of any a-keto-3-(3’- N-ethyl-2’,5’-dioxopyrrolidyl)) propionic acid (Fig. 1) at the expense of pyruvate (I), suggesting that the intermediate which reacted with NEM was one which was unique to a y-elimination, i.e. Structure II, III, or IV of Fig. 2. Of these, only Structure III appeared to have the high electron density on carbon 3 req- uisite for alkylation of NEM to yield KEDB.

However, as mentioned in a preliminary report (5), when sub- strates were discovered which were P-substituted but of 4-carbon chain length (Fig. 2, V), it was found that they did yield KEDB. This result indicated that the intermediate reacting with NEM must be one common to both y- and p-eliminations, i.e. one of the enamines VII, VIII, or IX shown in Fig. 2.

A nonenzymic reaction has also been briefly described by which, under conditions much more vigorous than those of the above reaction,” ammonia could catalyze formation of KEDB

1 The abbreviations used are: NEM, N-ethylmaleimide; KEDB, a-keto-3-(3’-(N’-ethyl-2’,5’-dioxopyrrolidyl)) butyric acid, the subscripts 1 and 2 referring to the diastereoisomers of slower and faster mobility, respectively, duringpaper electrophoresis;KEDP, or-keto-3-(3’-(W-ethyl-2’,5’-dioxopyrrolidyl)) propionic acid.

2 The spontaneous reaction is observed when a solution of pH 8.5 and 0.667 M in ammonium a-ketobutyrate and 0.006 M in NEM is heated for 30 to 60 min at 50” (M. Flavin and C. Slaughter. un- published results). Pyruvate also reacted, forming KEDP at one-third the rate of KEDB formation from a-ketobutyrate. The possibility that these nonenzymic reactions were mediated by the formation of ketimines (S&ii bases) between ammonia and the or-keto acids was investigated by two types of experiment. In the first, it was found that ammonia did not accelerate the enolization of a-ketobutyrate, as measured by the exchange of tritium out of the P-position of the latter, under the conditions of the reaction forminn KEDB. Primarv. secondarv. and narticu- larly tertiary amineswere more effecti+e’in catalyzing enoiization. This result indicates that if ketimines are intermediates their enolization, i.e. the formation of the reactive common base of the

1434

by guest on Decem

ber 27, 2018http://w

ww

.jbc.org/D

ownloaded from

Page 2: Enzymic Reactions of Enamines with N-Ethylmaleimide - Journal of

Issue of March 25, 1969 M. Flawin and C. Slaughter 1435

from a-ketobutyrate (5, 6). This suggested that in all cases the reactive intermediate might be free aminocrotonate, formed by XI + IX in the spontaneous reaction and by I + IX or V + IX in the enzymic reactions. This plausible hypothesis received little support from studies of the spontaneous reaction.2

In the present work we undertook to &amine the role of free aminocrotonate in the enzymic reaction, by generating the latter by hydrolysis of its stable N-acyl derivatives, in the presence of NEM (6). Another insight stemmed from examining other pyridoxal-P enzymes which catalyze reactions of the type shown in Fig. 2. It was found that different enzymes yield different diastereoisomers of KEDB (6).

EXPERIMENTAL PROCEDURE

Materials

We are indebted to Dr. H. Cahnman for a gift of N-chloro- acetyl-a-aminoacrylate, to Dr. S. Guggenheim for 0-succinyl-L- serine (7), and to Dr. D. M. Greenberg for rat liver y-cystathion- ase. The sources of the amino acids used in the experiment of Table I have been given elsewhere (5). Aminopeptidase M was obtained from Rohm and Haas via Henley and Company, New York; venom L-amino acid oxidase from Worthington; porcine kidney acylase from Calbiochem; and catalase, lactic dehydro- genase, and glutamic dehydrogenase (in 50% glycerol) from Boehringer. r,-Cystine-U-l40 was from New England Nuclear, L-threonine-U-“C from Calbiochem, and NEM-2, 5J4C from Schwartz. 2-Chloroacetamide was from Eastman, and tricar- ballylic acid was from Aldrich.

Chemical Preparations

N-Chloroacetyl-cr-aminocrotiic Acid-This compound was pre- pared by the procedure used by Wieland, Ohnacker, and Ziegler (8) to make the acrylic acid derivative. For the removal of water by azeotropic distillation we substituted a Soxhlet con- denser for the U-tube arrangement. A cotton plug was placed in the siphon opening and the Soxhlet space was nearly filled with molecular sieve type 4a (Fisher). Twenty-five grams (250 mmoles) of crystalline a-ketobutyric acid were dissolved in 250 ml of trichloroethylene in a 500-ml round bottom flask containing a magnetic stirring bar. 2-Chloroacetamide (9.4 g, 100 mmoles) was added, and the solution was stirred and refluxed for 26 hours with a drying tube attached to the condenser. The solution was chilled in an ice bath for 3 hours, and the crystalline precipitate was filtered out and washed with 50 ml of cold trichloroethylene. The yield of crude N-chloroacetyl-ar-aminocrotonate was 4.9 g (28 mmoles, 28%). This compound had not been isolated

ketimine-enamine pair, must be slow compared with the rate of reaction of this common anion with NEM. Somewhat stronger evidence against ketimine intermediates came from a second ex- periment, in which it was shown that ethyl- or diethylamine could not replace ammonia as catalyst for KEDB formation. This re- sult is not entirely conclusive, since a hydrogen might be required on the ketimine nitrogen if KEDB formation involved a hydrogen- bonded transition state. The rate of KEDB formation was rea- sonably related to the concentration of or-ketobutyrate and am- monium ions, but the relation to NEM concentration was obscure. The mechanism of the apparently very specific catalysis by am- monia remains obscure. A possible clue was that ammonia was found to decrease the rate of NEM hydrolysis.

8 L-Cystine-U-X? and L-threonine-U-W refer to the uniformly labeled compounds.

H R-C-i-CooH 0

H

0 d 0

N

it

KEDB, R = CH,; KEDP, R q H

FIG. l.‘Structures of KEDB and KEDP

previously (8). After one recrystallization from ethyl acetate- chloroform the melting point was 170-172”.

CeHs03NCl

Calculated: C 46.6, H 4.54, N 7.9, Cl 20.0 Found: C 40.6, H 4.73, N 8.1, Cl 20.0

N-Glycyl-cu-aminocrotonic Acid-This was prepared by treating the above compound with ammonia as described by Levintow et al. (9). Aqueous ammonium hydroxide (21 M) was prepared by passing a slow stream of helium for 30 min through one con- tainer of concentrated NH40H (13.5 M) at 55” and into another container of concentrated NH40H at 0”. Ten milliliters of the latter were added to 0.5 g (2.8 mmoles) of N-chloroacetyl-cr- aminocrotonic acid in a glass-stoppered round bottom flask. After 5 days at 25” the clear solution was evaporated under reduced pressure, leaving a waxy white residue, which was washed with warm 95% ethanol. The crude product was dis- solved in 5 ml of warm water, and filtered. Addition of 10 ml of ethanol to the filtrate, and cooling, yielded 247 mg (56%) of spearhead-shaped crystals which did not melt under 270” (9).

CJLoOaNa Calculated: C 45.52, H 6.37, N 17.7 Found: C 44.92, H 6.35, N 17.4

N-Glycyl-a-aminoacrylic acid was prepared in the same way from N-chloroacetyl-ar-aminoacrylic acid, m.p. 188-190”, re- ported (10) 191”. The ultraviolet absorption spectrum showed the shoulder at 240 rnp previously described (ll), whereas the corresponding crotonate derivative showed only end absorption.

Enzyme Preparations and Assays

Threonine Dehydrate-Thecatabolic, adaptivebacterial enzyme was prepared from Escherichia coli Crookes (ATCC 8739) grown anaerobically on complex medium (12). The cultures were in narrow necked volumetric flasks, filled with culture medium up into the neck, which also contained, above the sterile plug, a pyrogallol seal and a rubber stopper. A loo-ml culture, inocu- lated from a slant, was incubated overnight at 37” and then used to inoculate a Z-liter culture. After overnight cultivation, 1.2 g (wet weight) of cells were harvested by centrifuging at 25,000 x

g, washed with phosphate buffer, and suspended in 2 ml of 0.1 M

potassium phosphate, pH 8.0, containing 0.003 M each of AMP and reduced glutathione (12). The cells were broken in a Bran- son sonifier (13). The extract was centrifuged for 1 hour at 14,000 x g, passed through Sephadex (14) with 0.05 M potassium phosphate buffer, pH 7.3, containing 0.003 M each of AMP and

by guest on Decem

ber 27, 2018http://w

ww

.jbc.org/D

ownloaded from

Page 3: Enzymic Reactions of Enamines with N-Ethylmaleimide - Journal of

1436 Reactions of Enamines Vol. 244, No. 6

glutathione, and then stored frozen. Threonine dehydrase ac- tivity was measured by the rate of ar-ketobutyrate formation in a coupled assay with lactic dehydrogenase. The assay condi- tions are given in the legend to Fig. 4C, and 1 unit was defined as the amount of enzyme forming 1 pmole of a-ketobutyrate in 1 min under these conditions. Because of the high specific activity of the extract, 4.5 units per mg of protein, no further purification was necessary. Reaction mixtures are usually sup- plemented with glutathione (12), but this could not be done in our experiments with NEM. It was found that omission of glutathione, except for the small amount added with the enzyme solution, did not reduce the reaction rate for the first 5 min.

L-Amino Acid Oxidase-The venom preparation was diluted in 0.1 M KC1 when necessary. The activity was measured by the enolborate procedure (4), more conveniently by the rate of cy-ketobutyrate formation from cY-aminobutyrate in a coupled assay with lactic dehydrogenase. The latter was used to define 1 unit as the amount catalyzing the formation of 1 pmole of a-ketobutyrate in 1 min. This assay was done at 37” in cuvettes of l-cm light path containing, in 1 ml: potassium phosphate, pH 7.5, 100 pmoles; DPNH, 0.2 pmole; lactic dehydrogenase, 50 pg; catalase, 20 pg; and Dn-cr-aminobutyrate, 10 pmoles. Amino acid oxidase was added, and the oxidation of DPNH was followed at 340 rnl.c. A stepwise procedure was used to measure the effect of NEM on amino acid oxidase. The reaction mixtures con- tamed, in 1 ml in open tubes: 100 pmoles of n-alanine, 5 pmoles of NEM, 0.23 mg of amino acid oxidase (specific activity 4.1), and catalase (60 pg were found to be the optimal amount).

Aminopeptidase M-The lyophilized powder (15) was dis- solved in 0.05 M potassium phosphate, pH 7.5, and stored frozen. The ability of the enzyme to hydrolyze leucineamide and glycine- amide was determined by measuring the ammonia liberated by Nessler test after diffusion (16). After discovering that the N-glycyl derivatives of aminocrotonate and aminoacrylate were substrates, a more convenient assay was to measure the cy- ketoacid liberated from the latter in a coupled assay with lactic dehydrogenase. This assay was done at 37” in cuvettes of l-cm light path containing, in 1 ml: potassium phosphate, pH 7.3, 100 pmoles; enamine substrate, 5 pmoles; DPNH, 0.2 pmole; and lactic dehydrogenase, 50 pg. The lactic dehydrogenase, which was a suspension in ammonium sulfate, was diluted and passed through Sephadex for this assay; otherwise the ammonia inhibited the aminopeptidase about 50%. One unit of the latter was defined as the amount liberating 1 pmole of pyruvate in 1 min from N-glycyl-a-aminoacrylate. Another assay measured the rate of formation of ammonia from the latter in a coupled assay with glutamic dehydrogenase. The cuvettes were as above except that lactic dehydrogenase was replaced by 1 mg of glutamic dehydrogenase plus 5 pmoles of a-ketoglutarate. The reaction was started by adding aminopeptidase, after first allow- ing 10 min for the scavenging of ammonia contaminating the other reagents, i.e. after the absorbance of 340 mp had become constant.

A&use-This pig kidney preparation, elsewhere called acylase I (17), was handled like the aminopeptidase, although its sub- strate specificity is different. The lyophilized powder was dissolved in 0.01 M potassium phosphate, pH 7.3. The hydroly- sis of N-acetylvaline was measured by withdrawing reaction mixture aliquots at 20-min intervals, precipitating protein with trichloracetic acid, and assaying free valine in the supernatant with ninhydrin. A more convenient assay was to measure the

pyruvate liberated from N-chloroacetyl-cY-aminoacrylate in a coupled assay with lactic dehydrogenase. The reaction mixtures were the same as used for aminopeptidase except that the enamine substrate concentration was 0.002 M; the unit of activity was also defined in the same way.

Cystathionine y-SynULase-This preparation was the pure enzyme from Salmonella (18), and was prepared in this laboratory by Dr. S. Guggenheim.4 In the absence of cysteine the enzyme decomposes 0-succinylhomoserine to ar-ketobutyrate, and it was measured by coupled assay with lactic dehydrogenase, Assay B previously descri.bed (14).

y-Cystathionases of Liver and Neurospora-The rat liver enzyme was a lyophilized powder which had retained its activity since it was sent to us by Dr. D. 111. Greenberg 5 years ago (1). The Neurospora enzyme was enriched lOOO-fold over the normal extract level by derepression and fractionation (4). The decom- position of various substrates by either enzyme could be meas- ured by coupled assays with lactic dehydrogenase as described above (4). The unit of activity is based on the rate of liberation of cysteine from n-cystathionine, determined in the presence of 5,5’-dithiobis-(2-nitrobenzoic acid) (16).

Effects of NEM on Assay Reagents-Since this paper largely concerns effects of NEM on the above six enzymes, a few miscel- laneous observations on its effects on their assay procedures deserve mention. At a concentration of 0.01 M, in the reaction cuvette, NEM did not interfere with the determination of (Y- ketoacids by lactic dehydrogenase, although it increased the optical density at 340 rnp considerably. NEM did inhibit glutamic dehydrogenase. Catalase was little inhibited by ex- posure to 0.005 M NEM for 30 min at 37” at pH 7.6. NEM did not interfere with the assay of ammonia by diffusion and Nessler test. Finally, it should be noted that KEDB is not a substrate for lactic dehydrogenase, at least under the assay conditions used (3). Under the above conditions sensitivity to NEM is not necessarily evidence for essential sulfhydryl groups; as the pH is increased above 7.5 NEM reacts with other functional groups (19) and inhibits many enzymes.

Analytical Procedures

The procedures for determining pyruvate, a-ketobutyrate, and KEDB have been described (2). The two diastereoisomers of KEDB were separated by paper electrophoresis at pH 2.0 with the 5000-volt Gilson model D apparatus (2). The reaction mixtures and conditions used to study the formation of these products are described in Table III. We now stop the reactions by brief incubation with a mercaptan (Table III) before acid precipitation of protein. This modification was introduced when studies of the nonenzymic reaction2 revealed that some KEDB might be formed spontaneously from ar-ketoacids and NEM when dilute solutions were concentrated in a stream of hot air, while being applied to spots on filter paper preparatory to the electrophoretic separation.

Samples of KEDB which had been formed in novel reactions (from chloroaminobutyrate in Table I, and from a-ketobutyrate2) were positively identified by degradation to the two diastereo- isomers of or-methyl-@-carboxy-glutaric acid (2). KEDP, a new compound, could not be resolved to more than one component by electrophoresis at pH 2, as expected for a structure having only 1

4 We are indebted to Dr. S. Guggenheim for providing us with this enzyme and also with 0-succinyl-n-serine (unpublished syn- thesis).

by guest on Decem

ber 27, 2018http://w

ww

.jbc.org/D

ownloaded from

Page 4: Enzymic Reactions of Enamines with N-Ethylmaleimide - Journal of

Issue of March 25, 1969 M. Flak and C. Slaughter 1437

asymmetric carbon; after 6 hours at 3500 volts it migrated 58 cm, as compared with 61 and 70 for the two isomers of KEDB. The Rp of KEDP was 0.80 in pyridine-water, 4: 1; and 0.77 in tertiary butanol-formic acid-water, 70: 15 : 15. Treatment with hydrogen peroxide followed by strong acid (2) converted labeled KEDP to a compound which cochromatographed with tricarballylic acid, and could be recrystallized with carrier amounts of the latter, from acetone-carbon tetrachloride, to constant specific radio- activity.

RESULTS

The impetus for the present studies came from the discovery that y-cystathionase from Neurospora could form KEDB from p- as well as from y-substituted amino acids. This result has been briefly mentioned before (5) and is shown now in Table I. In accord with earlier results (1) neither cystine nor lanthionine yielded any KEDP, a conclusion based until now on failure to find radioactivity in electropherograms in the area near KEDB. It now became more certain with the availability of an authentic sample of KEDP, first isolated from a nonenzymic reaction mix- ture: and identified as described above. It seemed unlikely that substituting a methyl for a hydrogen at carbon 3 would so alter the course of the enzymic reaction as to allow a reaction with NEM in one case but not the other. However, when it was discovered that y-cystathionase could decompose /I-chloro-cr- aminobutyrate and P-methylcystine, both were found to yield KEDB. The yields of KEDB were also proportionately the same as from homocystine or 0-succinylhomoserine (Table I). These results indicated that the intermediate reacting with NEM must be common to both /3- and y-eliminations, and focussed attention on the enamines VII, VIII, and IX (Fig. 2). The work described here is a preliminary attempt to ascertain whether one of these three can be identified as the intermediate reacting with NEM.

The goal of our first studies was to generate in solution free enamino and ketimino acids of the type shown in Fig. 2, IX and X, under exactly the same conditions under which y-cystathion- ase catalyzed KEDB formation from NEM and 4-carbon amino acids, i.e. the same temperature, pH, NEM concentration, and buffers or other components. These conditions were too mild to encourage the possibility of generating the desired products, from stable derivatives, by any means other than enzymic catalysis. Use of the latter introduces some ambiguity because the reaction mechanisms are poorly understood, and at the least involve generating enzyme-bound as well as free products.

Oxidative, Hydrolytic, and Pyridoxal-P Enzymes Yielding Ketimino or Enamino Acids--To generate the ketimines, (Y- iminopropionate (Fig. 3, Reaction a, R = H) or cw-iminobutyrate (R = CH,) we allowed n-amino acid oxidase to act upon alanine or cr-aminobutyrate. The activity of the venom enzyme toward these substrates, under our reaction conditions, is shown in Table II. There is substantial evidence that enamines are not inter- mediates in these reactions (20), although direct evidence for ketimine intermediates is limited to transient spectral changes observed during the decomposition of n-tyrosine (21), and the possibility of alternative interpretations (20) illustrates the ambiguity mentioned above.

To generate the desired unstable enamino acids we utilized hydrolytic enzymes which could liberate them from their easily synthesized and stable N-acyl derivatives. A commercially available pig kidney acylase had been reported (17) to act

TABLE I Alkylation of NEM by intermediates fomed by action of

y-cystathionase on p- or y-substituted amino acids of d-carbon chain length

The reaction mixtures contained, per ml: 30 #moles of potas- sium phosphate, pH 7.6; 0.1 pmole of pyridoxal-P; 7 rmoles of NEM-2, 5-r4C (l,SOO,OOO cpm) ; and Neurospora -y-cystathionase (specific activity 0.29) and substrate, as indicated. After 120 min at 30” the reactions were stopped and products were measured as described in the text. KEDB was isolated by electrophoresis at pH 3.5, which does not resolve its two isomers.

Additions

Amounl Amino acid substrate of sub-

strate

pmoles

0-Succinyl-on- 5 homoserine

nn-Erythro-& 10 chloro-a- aminobutyrate

L-Homocystine 3.3 &Methylcystine, 3.3

Isomer A L-Cystine 3.3 Lanthionine 6.7

(L + meso)

tA

E

_-

-

UUOUU of

nzyme

0.14

Jmolc

0.55

0.76

0.27 0.27 0.21 0.27 0.053 0.0%

0.14 0.14

- I

or-K&o- mtyratc KEDB

Products

Pyru- vate

pmole

1.35 1.7

-

1

_-

-

KEDP

pmolc

0 0

KEDB as per cent of total

prod- UCtS

% 39

45

44 36

specifically upon only one such derivative (Fig. 3, Reaction c). As shown in Table II, it decomposed N-chloroacetyl-cu-amino- acrylate effectively, compared with a standard substrate at the same concentration. Another enzyme was needed to generate aminocrotonate. We found that a recently described (15) and also commercially available aminopeptidase (also isolated from kidney ribosomes) was reasonably effective in hydrolyzing the N-glycyl derivatives of both aminocrotonate and aminoacrylate (Fig. 3, Reaction b, R = CH3 or H). As expected from studies with other amides (15) it did not hydrolyze the chloroacetyl derivatives (Table I). The decomposition of glycylamino- acrylate was followed by coupling aminopeptidase with lactic dehydrogenase or glutamic dehydrogenase and thus following the rates of appearance of pyruvate and ammonia continuously in companion cuvettes. The observed rates were the same (Table II). No initial lags or bursts were seen.6 Ammonia appeared to be liberated faster when it was assayed by sequential diffusion and Nessler test (Table II), perhaps because of better temperature control in this reaction, or because of inhibition by components of the coupled assay mixtures.

Table II also shows the ability of two pyridoxal-P enzymes to catalyze eliminations from both 3- and 4-carbon chain length amino acids. Threonine dehydrase is progressively inhibited as it decomposes serine to pyruvate (12); the reaction shown in Table II had essentially stopped after 10 min. However, the

6 This pair of assays would have been useful for kinetic studies of this or any other reaction between NEM and generated ena- mines. i.e. bv continuouslv measurine: KEDP as the excess of ammonia formed over pyruvate in the presence of NEM. Unfor- tunately, NEM inhibited glutamic dehydrogenase, so the method could not be used.

by guest on Decem

ber 27, 2018http://w

ww

.jbc.org/D

ownloaded from

Page 5: Enzymic Reactions of Enamines with N-Ethylmaleimide - Journal of

1438 Reactions of Enamines Vol. 244, No. 6

A-CH2-CsHpi-COz-

(HO.:;CH&

A-CH2-&i-C-co2-

iI ‘j) *

A-CH2-y=F-C02- CH2= CH - F - COz-

3

,, ;5 * -AH 1 6

N tH H H H

f: H B I

CH3-CH-kC02- CH,-tH-C-CO,

I: k

CH3-CH=y-C02-

-B- V :H

i VI bH * VII ;, -----t KEDB .

0 (j 0 tH H tH

NH3

XI t

X IX VIII I

CH3-CH2-C-COp- -CH3-CH2-;-C02- -CH,-CH=C-C02- -

bl I I tI(IHg

CH3-CH;f-C02- . . . . . enzyme . . . . . H+

tNH2 i NH2 I I

i 1

KEDB KEDB

FIG. 2. Schematic reaction paths for the pyridoxal-P enzyme-catalyzed reactions: -y-replacement, I + IV + I; r-elimination, I + XI; P-replacement, V --f VII -+ V; and &elimination, V --f XI. The letters A and B are general symbols for an electronegative sub- stituent group. For convenience, the coenzyme ring substituents have been omitted, except in Structure I. The dashed arr~w~ in- dicate the three intermediates that we have considered as possible candidates to react with NEM.

H

R-CHI-LCD2- t FAD - FADH2 + R-CH2-$-COZ- (a)

+kH3 L-Amino acid &dare

+!4H,

R-CH = t-CO*- t Hz0 - R-CH=y-CO*- t glycine 6)

NH-C-CH2-NH3+ II Aminopepfidase M

+NH3

0

CH2 I C-CO*- + I

,Q,, - CH2;C-C02- + Chloroocetote (c)

NH-C-CH2-Cl

bl

tNH, Rend ocybre

FIG. 3. Enzymes used to generate ketimines (Reaction a) or enamines (Reactions b and c). R = CH3 or H.

initial rate during the first 2 min was comparable to that with threonine (Table II), and studies with NEM were possible by making use of this initial phase. Similarly, cystathionine y-synthase catalyzes @elimination from 0-succinylserine (7) as well as y-elimination from 0-succinylhomoserine, but in the former case the reaction progressively slows with time.6 This paper is concerned entirely with the elimination reactions by which the y-synthase continues to decompose these esters when

B S. Guggenheim and M. Flavin, unpublished results.

cysteine is omitted from the reaction mixtures. The wide range of substrates decomposed by y-cystathionase has been listed elsewhere (4).

Susceptibility of Enzymes to Inhibition by NE&f-In studying the ability of various enzymes to alkylate NEM, the second question, after identifying appropriate substrates, was whether the enzymes would be inhibited by NEM. Fig. 4A shows the effects of NEM on the apparent rates of ar-ketobutyrate forma- tion catalyzed by liver y-cystathionase (specific activity 0.16 by standard assay). 0-Succinylhomoserine, not previously reported to be a substrate for this enzyme, was rapidly decom- posed, as was homoserine. Addition of NEM reduced both rates, without lag, from 0.18 pmole mg+ mm-1 to constant ap- parent rates of 0.06 (Fig. U); twice as much enzyme now doubled the “inhibited” rate to 0.12. Data of this type are at least as consistent with diversion of products to KEDB, as with enzyme inhibition. A possible method for differentiating the latter effects is shown by the data of Fig. 4B for y-cysta- thionase of Neurospora, which had earlier been shown to form KEDB from 4-carbon substrates, but not KEDP from a-carbon substrates (1); (the data of Fig. 4B are the first report that succinylserine4 is a substrate for this enzyme). Fig. 4B suggests that with this enzyme NEM causes both inhibition and diversion to KEDB. The proportion of rate reduction from the solid line to the middle dashed line could be interpreted as corresponding

by guest on Decem

ber 27, 2018http://w

ww

.jbc.org/D

ownloaded from

Page 6: Enzymic Reactions of Enamines with N-Ethylmaleimide - Journal of

Issue of March 25, 1969 M. Flavin and C. Slaughter 1439

to 31% inhibition of enzyme activity, and the proportion be- tween the two dashed lines as corresponding to diversion of 23% of the products to KEDB (see “Discussion” for alternate interpretations).

Cystathionine y-synthase was more severely inhibited by NEM. The inhibition was time-dependent, the reaction rate being nearly normal for the first 2 to 3 min. Studies of KEDB formation could still be done, by adding enough enzyme to yield the desired amount of products in 1 or 2 min, as illustrated in Fig. 4C for threonine dehydrase, which was possibly even more sensitive to NEM inhibition. The time dependence of the inhibition was similar to that for cystathionine-y-synthase. The experiment of Fig. 4C was repeated with serine in place of threonine. The inhibitions by NEM and serine did not seem additive; aft,er the second addition of enzyme (0.1 mg), (Y- ketoacid appeared to be liberated faster from serine than from threonine.

L-Amino acid oxidase and aminopeptidase were very little inhibited by NEM. Acylase was severely inhibited but, by adding 50 times more enzyme than the amount suitable for assay without NEM, suflicient reaction products could be obtained.

Enzymic Alkylation of NEM-The results of tests to determine whether these last three enzymes could catalyze alkylation of NEM, to which the work so far had been a prelude, are shown in Table III. As anticipated, L-amino oxidase did not produce any detectable KEDB from cr-aminobutyrate or KEDP from alanine. Because of its sensitivity to NEM we used a large amount of acylase, enough, if it were not inhibited, to hydrolyze ail of the added chloroacetylaminoacrylate in 1 min; in the experiment of Table II half was hydrolyzed in the course of 60 min, and no KEDP was formed. Of course, up to this point KEDP had not been found as the product of any enzymic reaction. The results with aminopeptidase were entirely different. Hydrolysis of crotonate or acrylate derivatives in the presence of NEM yielded appreciable amounts of KEDB and KEDP, respectively (Table III). Small amounts of enzyme were used because the aminopeptidase was not sensitive to NEM, as shown by the continuing formation of products after 30-min reaction time (Table III). Glycylaminocrotonate yielded both isomers of KEDB in proportions similar to those formed in a nonenzymic reaction (5).2 The apparent failure of KEDB, to increase in parallel with the other products between 30 and 90 min is thought to be within analytical error.

We had planned to expand the number of pyridoxal-P enzymes tested for KEDB formation, and chose threonine dehydrate somewhat arbitrarily. Table IV shows the results that were obtained, as well as those of new experiments with the three enzymes that had been tested before (1, 14). Using large amounts of the NEM-sensitive threonine dehydrase, we found that serine did yield KEDP and threonine KEDB. The significant new feature was that the latter was present almost exclusively as one of the two possible diastereoisomers. Isomer 1 was also obtained in the same yield starting with threonineJ4C and unlabeled NEM. Our results with the three cystathionine- metabolizing enzymes differed from earlier ones (1, 14) in several ways, the foremost being that we now observed in several experiments that these three enzymes formed almost exclusively isomer 2 of KEDB (Table IV). Paper electrophoresis of reac- tion mixture aliquots at pH 1.7, where mobilities are quite different from pH 2.0 (2), confirmed the formation of KEDBz

TABLE II

Rates of decomposition of various substrates by enzymes used to test ability of NEM lo react with transient intermediates

Enzyme and substrate

L-Amino acid oxidase L-Histidine .

nn-cY-Aminobutyrate

L-Alanine Aminopeptidase M

L-Leucine amide. Glycineamide.. N-Chloracetyl-or-amino-

crotonate............... N-Glycyl-a-aminocrotonat

N-Glycyl-a-aminoacrylate. N-Glycyl-a-aminoacrylate. N-Glycyl-cu-aminoacrylate.

Renal acylase N-Acetyl-L-valine . . N-Acetyl-L-valine. . . . N-Chloroacetyl-a-amino-

acrylate................ Threonine dehydrase

L-Threonine . .

L-Serine.................. Cystathionine r-synthase

0-Succinyl-L-homoserine.

0-Succinyl-L-serine .

0 1

--

e

-

M

0.01

0.01

0.01

0.01 0.01

0.01 0.00:

0.00: O.OOE 0.01

0.02E 0.00:

0.00:

0.02

0.02

0.01

0.01

Imidazole- pyruvate

a-Ketobu- tyrate

Pyruvate

Ammonia Ammonia

Ammonia cu-Ketobu-

tyrate Pyruvate Ammonia Ammonia

Valine Valine

Pyruvate

cu-Ketobu- tyrate

Pyruvate

oc-Ketobu- tyrate

Pyruvate

1

2

2

3 3

3 2

2 4 3

5 5

2

2

2

2

2

_-

6

1

-

7.5

4.1

0.35

3.0 0.2

0 0.064

0.64 0.64 0.95

4.0 0.3

0.2

4.5

5*

0

6*

0 The details of the assay procedures, as well as the properties of the enzyme preparations and the reaction conditions, are given under “Experimental Procedure.” The assay procedures were: 1, enolborate; 2, coupled assay with lactic dehydrogenase; 3, sequential determination by diffusion and Nessler test; 4, coupled assay with glutamic dehydrogenase; 5, sequential determination with ninhydrin.

* Initial rates; threonine dehydrase and cystathionine r-syn- thase are both progressively inhibited as these respective 3-carbon substrates are decomposed.

by Neurospora y-cystathionase, KEDBl by threonine dehydrase, and the apparent formation of both isomers by aminopeptidase. Proof of structure was obtained by chromatography after decarboxylation and acid hydrolysis (2) for the KEDP formed by aminopeptidase and KEDP and KEDB formed by threonine dehydrase. A second difference from the earlier results was in the proportion of substrate diverted to KEDB by the three enzymes. These proportions were now relatively similar, in contrast to earlier results (1, 14), in which cystathionine y- synthase formed so little KEDB (14) that it might have arisen secondarily from ar-ketobutyrate. The availability of succinyl- serine (7) as a substrate for the latter enzyme enabled us now also to determine that it did yield KEDP (Table IV). The failure of Neurospura y-cystathionase to form KEDP (1) was

by guest on Decem

ber 27, 2018http://w

ww

.jbc.org/D

ownloaded from

Page 7: Enzymic Reactions of Enamines with N-Ethylmaleimide - Journal of

1440 Reactions of Enamines Vol. 244, No. 6

A t

Add 2.2 enzyme

0.04mg r-l-.

Y -Cystathionose of rot liver

I I , I 1 I 0 4 8 12 16

MINUTES

.3

i \

+

I 1 I 5

MINUTLSO 15 20

FIG. 4. A, effect of NEM on the rates of or-ketobutyrate forma- tion from homoserine or succinylhomoserine catalyzed by liver r-cystathionase (specific activity 0.16). Each cuvette contained,

again confirmed with lanthionine and cystine (Table I), and also with succinylserine (Table IV). The same negative result was obtained by using cystine-r4C and unlabeled NEM. It was considered (see “Discussion”) that the absence of KBDP might be because the amino rather than the methylene group of aminoacrylate had added to the double bond of NEM, yielding a radioactive dipolar ion. In the electrophoretic procedure for detecting KEDP only labeled components which were anions at pH 2 could be detected, cations passing into the buffer com- partment. We therefore looked for cationic components, by reversing the origin, in mixtures from reactions of cystine-14C f NEM and cystine + NEMJ4C. No significant cationic com- ponents were found which were labeled in both cases. The same was true for threonine dehydrase mixtures which had contained the label in either threonine or NEM. Aminopep- tidase reaction mixtures contained unique cationic components, possibly due to addition of the starting dipeptide to NEM, which would be expected because of the low pK, of glycine amino group (19).

Table V summarizes the range of results from all of the experi- ments that we have done on NEM alkylation. In each of two experiments with tbreonine dehydrase only isomer 1 of KEDB

\ \

\ \ \

\ \

‘Lb_ /

DPI;H exhausted

/ 2 4 6 8 MINUTES

in l-ml volume at 30”: 5 pmoles of the indicated substrate; 100 pmoles of potassium phosphate, pH 7.5; 0.1 pmole of pyridoxal-P; 0.2 Fmole of DPNH; and 50 pg of lactic dehydrogenase. The reac- tions were started by adding cystathionase. The rate of DPNH disappearance was followed for 5 min, followed again after adding 5 rmoles of NEM, and again after another addition of cystathi- onase, as indicated by the arrows. At the concentration used NEM absorbs quite strongly at 340 rnp. A control cuvette was followed, containing NEM and all other additions, including a substrate, except lactic dehydrogenase. The very slow decline in absorbance of 340 rnp, due to NEM hydrolysis, was substrated from the curves for the reaction cuvettes. B, effect of NEM on the rate of or-ketobutyrate formation from succinylhomoserine, and of pyruvate formation from succinylserine, catalyzed by Neurospora y-cystathionase (specific activity 0.40). The pro- cedure was the same as described in the legend to A, except that NEM was added at zero time to a cuvette with each substrate, and omitted from another. For convenience in drawing the chart, the optical densities of all four cuvettes were normalized at zero time. The reactions were started by adding cystathionase; larger amounts were added to the succinylserine cuvettes to make the rates, in the absence of NEM, the same for the two substrates. C, effect of NEM on the rate of cu-ketobutyrate formation from L-threonine, catalyzed by threonine dehydrase (specific activity 4.5). The procedure was the same as described in the legend to B, except that the substrate concentration was 0.02 M, and both cuvettes also contained 5pmoles of AMP.

was formed. Neurospora y-cystathionase has yielded only isomer 2 in two experiments, but we have also observed mixtures recently. The relative yields of KEDB are quite variable from one experiment to another (Table V). However, the very low yields from cystathionine y-synthase and liver y-cystathionase were observed, in each case, in single experiments done several years ago (1, 14) and have never recurred in recent experiments. We therefore believe that the negative results with amino acid oxidase and acylase and the failure of the Neurospora y-cys- tathionase to form KKDP deserve some confidence.

To obtain the results in Table V with those enzymes that were severely inhibited after 2- to 3-min exposure to NEM, we had relied on adding a very large amount of enzyme. To test whether KEDB formation was itiuenced by having the rea.ction occur in a short burst, we compared the products formed after serial, compared with single, addition of the same total amount of enzyme. The reactions tested were threonine dehydrase acting on threonine and cystathionine y-synthase acting on succinylhomoserine. In one case enzyme was added all at once and the mixture was incubated 30 min; in the other one-tenth as much enzyme was added every 3 min. Serial addition (a) decreased the total yield of products very slightly,

by guest on Decem

ber 27, 2018http://w

ww

.jbc.org/D

ownloaded from

Page 8: Enzymic Reactions of Enamines with N-Ethylmaleimide - Journal of

Issue of March 25, 1969 M. Flavin and C. Slaughter 1441

TABLE III Alkylation of NEM by ketimines and enamines generated from amino acids and N-a& derivatives by nonpyridoxal enzymes

The reaction mixtures were usually of 0.5~ml volume which j3-mercaptopropionate, 2 moles per mole of NEM originally added. contained 20 rmoles of potassium phosphate, pH 7.5, and 0.1 After allowing 2 min at 25” for complete removal of residual NEM, pmole of pyridoxal-P. The latter was added in order to make the 0.1 volume of 1.5 M perchloric acid was added. The protein pre- reaction conditions identical in all experiments of Tables III and cipitate was discarded after centrifugation, and the supernatant IV, except for the enzyme solutions, which in some cases contained was adjusted to pH 4 to 5 with potassium hydroxide, and then small amounts of stabilizing reagents (see “Experimental Pro- pipetted out from the settled precipitate of potassium per&orate. cedure”). The pH of the solutions was adjusted to 7.5 before the Aliquots were assayed with lactic dehydrogenase for pyruvate final additions of NEM-14C + carrier (to give a specific radioac- and cY-ketobutyrate. The other reaction products were deter- tivity of 50,090 to 120,000 cpm per pmole), and enzyme, readjusted mined by radioassay after paper electrophoresis of aliquots of 0.2 if necessary, and rechecked at intervals during the incubations, ml, at pH 2.0 in the Gilford apparatus as previously described (2). which were at 30” or 37”. The reactions were stopped by adding -

_ i

.-

-

-

I --

Additions (per ml) Products Re-

action time

n&in

90

90

60 30 90 30 90

IEM-“C

pnazes

6.6 6.6 6.6 6.6 6.6 6.6 6.6

- I

-

h

_ -

-

Substrate

unw 0.20 2.0 3.8 0.32 0.32 0.051 0.051

p5?coles

10 10 4

10 10 10 10

umoles/d

0 1.40 1.87

0.50 1.80

4.73 7.6

nn-cu-Aminobutyrate L-Alanine Cl-Acetyl-cr-aminoacrylate Glycyl-a-aminocrotonate Glycyl-a-aminocrotonate Glycyl-a-aminoacrylate Glycyl-a-aminoacrylate

0

0.43 0.55

1.81 4.0

L-Amino acid oxidase

Acylase Aminopeptidase M Aminopeptidase M Aminopeptidase M Aminopeptidase M

0 0

1.14 1.50

- - - a Defined under “Experimental Procedure.”

TABLE IV Proportions of two isomers of KEDB formed from threonine, fl-chloro-a-aminobutyrate, or succinylhomoserine, and of KEDP formed from

serine or succinylserine, by four different pyridoxal-P enzymes

The reaction conditions were the same as those described in Table III.

Additions (per ml) Products

EMJ’C

.- moles

20 20 6

10

6.6 6.6 3.8 3.3

min

30 30 15 30

10 6.6 30

10 5 60

10 7.1 60 10 6.6 30

10 7.1 60

Substrate

L-Serine n-Threonine 0-Succinyl-n-serine O-Succinyl-nn-homo-

serine O-Succinyl-nn-homo-

serine O-Succinyl-nn-homo-

serine 0-Succinyl-n-serine O-succinyl-nn-homo-

serine nn-Erythro-p-chloro-a-

aminobutyrate

Threonine dehydrase

Cystathionine r-synthase

l-

-

unit.+ 20 10 2 4.5

4.5

0.09

0.30 0.26

0.30

2.22 0.48

1.73 0.70

2.31 0

1.96 3.16 0.06

3.32 0 0.28

2.40 0 0.46

0.90 0 0.69

1.44

1.30

0

0

1.58

1.10

y-Cystathionase of rat liver

-,-Cystathionase of Neurospora

6 Defined under “Experimental Procedure.”

by 2% with threonine dehyclrase and 8% with cystathionine gardless of which enzyme was used to generate the intermediate. synthase; and (b) increased the proportion of KEDBr with A few studies were done with some of the other enzymes used threonine dehvdrase from 46 to 60% of the total products, and here. The results7 were similar enough, with different enzvnxs, of KKDBa with cystathionine synthase from 16 to26%.

- . I ,

Neurospora y-cystathionase, although insensitive to NEM at 7 By lowering the pH from 7.5 to 7.3 and the NEM from 6.6 to

pH 7.5 (Fig. 4B), was completely inhibited by it at pH 8 (1). 3.3 pmoles per ml, the proportional yield of KEDBz was reduced

Short of this point, the proportional yield of KElDB increased from 52 to 310/, with Neurospora r-cystathionase, and from 16 to

with increasing NEM and pH (1, 3). I f KEDB arose from a 8~~ with cystathionine r-synthase; by lowering the pH from 7.5 to 7.0 with 6 rmoles of NEM the change for threonine dehydrase

free intermediate (Fig. 2, IX) these effects might be alike re- was 34 to 20’%, for aminopeptidase 48 to 32yo.

by guest on Decem

ber 27, 2018http://w

ww

.jbc.org/D

ownloaded from

Page 9: Enzymic Reactions of Enamines with N-Ethylmaleimide - Journal of

Reactions of EnamGnes Vol. 244, No. 6

TABLE V

Summary of ranges of values obtained under standard condition with various enzymes tested for their ability to catalyze formation of

KEDP or two diastereoisomers of KEDB --

Yield of Yield of KEDP as KEDB as er cent 01 Apparent

Reaction catalyst total ?frt% ,er cent of

prFr%ts Pr;tEE” KEDB

s-carbon &bon ?sE%r “;” ubstrates wbstrates

% %

18 46-60 96-98

29 (r28 C30 0 5-43 0

pproxi- mate no. of

experi- ments

Threonine dehydrase.. . Cystathionine r-synthase r-Cystathionase of rat liver. r-Cystathionase of Neuro-

A

.-

-

t

2 4 2

F

.-

-

spora..................... L-Amino acid oxidaee. . . . Renal acylase.. . . . . . . . Aminopeptidase M.. . . . . . . Ammonia*..................

--

0 36-55 o-29 0 0 0

20 3448 23-46 2640

- a The ammonia-catalyzed alkylation of NEM by cu-ketoacids

(5, 6). Both pyruvate and a-ketobutyrate react, and under the same conditions the yield of KEDB is 3 times that of KEDP.

so as not to require that the intermediate reacting with NEM be enzyme-bound.

Are Free Enamino Acids Intermediate&-According to the scheme of Fig. 2 pyruvate or cw-ketobutyrate formed by @- diGnation should acquire 1 solvent hydrogen at carbon 3 in a nonenzymic step; cr-ketobutyrate formed by y-elimination should have a 2nd solvent hydrogen acquired at carbon 4 by IV -+ VII. But if the enzyme-bound enamines VII and VIII are candidates to alkylate NEM, they might also be candidates to capture a proton at carbon 3, bypassing some intermediates; i.e. VIII would yield directly the free ketimine X, or VII would yield free ar-ketobutyrate + NH3 (XI). If either of the latter paths were correct, free enamines would not be intermediates at all, and so would be ruled out as possible candidates to alkylate NEM.

We studied this question by carrying out all of the hydrolytic and elimination reactions discussed above in 3Hz0, and de- termining the amount of tritium incorporated at carbon 3 of pyruvate or a-ketobutyrate. Since the reaction mixtures were all identical except for the enzyme added, the tritium kinetic isotope effects should be the same if the solvent proton were introduced at carbon 3 in a spontaneous step. The results are not presented in detail here because, since the completion of our study (22), more conclusive evidence has become available that free enamino acids are not intermediates in some pyridoxal-P mediated elimination reactions.8 In two cases we found the discrimination against tritium to be significantly less than in the

* After submitting this manuscript we learned from Dr. R. H. Ageles (personal communication) that he and his colleagues had found a-ketobutyrate formed in DzO from homoserine by rat liver r-cystathionase to be optically active. After decarboxylation the resultant a-n-propionate was found to have the 2(S)-(+)-con- figuration. Since then Dr. S. Guggenheim (personal communica- tion) has done similar studies in this laboratory with a-ketobu- tyrate formed from 0-succinylhomoserine by cystathionine y- synthase, and has obtained deuteropropionate which is also de&o-rotatory. It is interesting to note that these two enzymes formed the same isomer of KEDB (Table IV).

rest of the reactions. These were the formation of pyruvate from serine catalyzed by threonine dehydrase and the formation of pyruvate from chloroacetyl-cr-aminoacrylate catalyzed by acylase.

DISCUSSION

The experiments described in this paper were designed to identify the enamine intermediate, formed in the course of enzymic elimination reactions, which can alkylate NEM, and in particular to determine whether it was free aminocrotonate or an enzyme-bound form such as VII or VIII of Fig. 2. The first question might be whether one of the three candidates shown in Fig. 2 was favored from the outset because it better satisfied the requirement for carbanion character at carbon 3. Free aminocrotonate is too unstable to isolate or study directly.9 The potential reactivities of the other two candidates might be augmented by the enzyme; aside from this unpredictable factor, they have been discussed in detail elsewhere (2). In the Schiff base of aminocrotonate (VII of Fig. 2) the phenol group of the coenzyme donates electrons which can attack NEM at carbon 3 (or capture a proton there to yield Lu-ketobutyrate directly (2). The third candidate (VIII) has not been explicitly included in reaction schemes before now. It is an unprotonated form of aminocrotonate (2), actually the common base of the enamine-ketimine pair, which can in principle equally well accept an enzymic proton on its nitrogen to yield IX or on carbon 3 to yield X. Intrinsically, this appears the most attractive candidate, because of its resemblance to the cr,& unsaturated tertiary amines widely used as carbanions in organic synthesis (23) .l”

Stereospeci$city in Enzymic Synthesis of KEDB-Two asym- metric carbons are introduced when NEM is alkylated by a 4-carbon intermediate (Fig. 1) and KEDB should thus be racemic if formed from a free intermediate, but optically active if formed from VII or VIII. We had previously failed to detect optical activity in the dinitrophenylhydrazone of KEDB formed by y-cystathionase of Neurospora (3). This negative result was entirely inconclusive because of (a) the intense color of the derivative, which required rotational measurements to be made with extremely dilute solutions, and (b) the facile racemization of KEDB, indicated by the fact that when either isomer, isolated by electrophoresis, was subjected to electrophoresis again a near equilibrium mixture resulted containing 40% of the other isomer (3). The isomerization was undoubtedly due (3) to racemization at carbon 3, rather than at carbon 3’, and the only hope for detecting optical activity in the colored derivative lay in the possibility that a cotton effect would result from the interaction between the hydrazone group and carbon 3. Al- though there were already indications that the cystathionase produced more of isomer 2 (3) than was formed in the nonenzymic synthesis (Table V),2 this result was inconclusive prior to the present fortuitous discovery of an enzyme, threonine synthase,

9 Dr. N. E. Sharpless (personal communication) has made some preliminary molecular orbital calculations attempting to compare structures such as VIII, IX, and X (of Fig. 2) with their keto and enol oxygen analogues. The results did not show increased elec- tron density at carbon 3 in the SchifY base series, as compared with the ketoacid series.

lo This common base would also be the reactive species in the case of free aminocrotonate, but binding to the enzyme might give it a longer effective lifetime.

by guest on Decem

ber 27, 2018http://w

ww

.jbc.org/D

ownloaded from

Page 10: Enzymic Reactions of Enamines with N-Ethylmaleimide - Journal of

Issue of March 25, 1969 M. Flavin and C. Slaughter 1443

which yielded the other isomer (Table IV). Although we have now found two other pyridoxal-P enzymes which yield isomer 2 (Table IV), it should be emphasized that occasional experiments still yield mixtures; racemization occurs erratically during the procedure of a first electrophoretic separation, but invariably during the procedure (i.e. drying on the paper of the pyridine- acetate buffer, elution) for a second electrophoresis. It follows also that, in the case of the aminopeptidase which has so far yielded both isomers of KEDB (Table V, see below) in the same proportions that are formed nonenzymically, it cannot neces- sarily be concluded that free aminocrotonate was the reacting intermediate.

The finding that all four pyridoxal-P enzymes examined appear to yield directly only one isomer of KEDB and that this isomer may be a different one with different enzymes (Table IV) strongly suggests that in these cases the intermediate that reacts with NEM is enzyme-bound.

Alkylation of NEM by “Free” Enamines-This conclusion would be re-enforced if aminocrotonate could be obtained by an independent route and shown not to react with NEM under the same conditions used above. There is no information on the lifetime of free aminocrotonate in neutral aqueous solution, although the upper limit can probably be set at less than 1 or 2 min. No initial lag in the appearance of ammonia or a-ketobu- tyrate was observed (Table II) during the enzymic hydrolysis of N-glycyl-cr-aminoacrylate.

As a test of whether free enamines could react with NEM we therefore synthesized N-acyl derivatives which were susceptible to hydrolytic enzymes, and allowed the hydrolytic reactions to proceed in the presence of NEM. Consistently with the stereospecific formation of KEDB described above, renal acylase did not yield any KEDP from an N-acyl aminoacrylate, nor did ketimines, generated with amino acid oxidase in control experi- ments, react with NEM (Table III). However, when the aminopeptidase preparation was used, the postulated free aminoacrylate and aminocrotonate both reacted with NEM (Table III). The proportional yields of KEDB and KEDP were as large as were obtained with the pyridoxal-P enzymes (Table V). In a limited number of experiments, the KEDB formed was a mixture of isomers similar to that formed non- enzymically (Table V) .

Taken by themselves these results with aminopeptidase suggested that in this case we were dealing with reactions of free enamines. If this were so, however, one would expect that KEDP would also be formed in the reaction catalyzed by acylase (see below).

E$ects on /3-Elimination Reactions of Interchanging Methyl for Hydrogen at Carbon S-Two of the pyridoxal-P enzymes could not be shown to yield KEDP from 3-carbon substrates (l), whereas KEDP was formed in the nonenzymic reaction, by aminopeptidase, and from serine by threonine dehydrase and from succinylserine by cystathionine y-synthase (Table V). It is intriguing that the last two enzymes are also inhibited by these substrates (7, 12). The inhibition of threonine dehydrase by serine has been known for some time (12, 24, 25) but not yet clearly explained. There are other indications of some profound and still unforeseen effect on /?-eliminations, of interchanging a methyl group for a hydrogen at carbon 3. Fig. 4B shows the rates of formation of pyruvate and cr-ketobutyrate from the succinyl esters of serine and homoserine, respectively, in the presence and absence of NEM, catalyzed by y-cystathionase.

Since this enzyme forms KEDB but not KEDP, the slowing of pyruvate formation was interpreted above as enzyme inhibition, the additional slowing of ar-ketobutyrate formation as diversion of products to KEDB. The resulting value of 23% for the products comprised by KEDB was less than the average directly measured proportion of the latter formed by this enzyme (Table V). Again, the slowing of pyruvate formation did not show the time lag characteristic of other enzyme inhibitions (Fig. 4C). It was noted some time ago that y-cystathionase of Neurospora appeared to catalyze a mixed reaction with cystathionine, and the proportion of p-elimination was reduced on exposure to NEM (26). There seems a possibility of some unusual role of a sulfhydryl group in this enzyme. Thus a number of the observations mentioned above might be explained if blocking a sulfhydryl group with NEM bad the effect of modifying and slowing the &elimination reactions.

Another possible explanation for the failure of the cystathion- ases to form KEDP, and one suggested by analogous organic reactions (23), is that NEM reacts with aminoacrylate formed from succinylserine, but does so by addition to its double bond of the amino group of the enamino acid. The product would be a dipolar ion, rather than KEDP. We looked for, and failed to find, radioactive components of the expected electrophoretic mobility.11 It is tempting to think that some of the unusual effects of substituting methyl for hydrogen at carbon 3 might be related to alternate transfer of an enzyme proton to nitrogen in some cases (VIII + IX, Fig. 2) or to carbon 3 when the methyl group of VIII is replaced by hydrogen. The experiments done to test for enzyme-catalyzed protonation are preliminary, but the nonuniform isotope effects with a-carbon substrates suggest a positive answer, at lea.st in some cases.*

Conclusions-Although at one time free aminocrotonate seemed the most plausible intermediate to alkylate NEM,2 the stereospecific formation of KEDB isomers appears to rule it out for the reactions catalyzed by pyridoxal-P enzymes. Since only a @-substitued amino acid, threonine, has yielded isomer 1, it is worth noting that y-cystathionase yielded isomer 2 from both y- and P-substituted 4-carbon substrates (Table IV). The preliminary evidence for nonuniformity of kinetic isotope effects when different enzymes mediate protonation at carbon 3 of a pyruvate precursor suggests that at least in some cases free enamines are not intermediates at all, and therefore could not react with NEM. However, no specific correlation is obvious between tritium isotope effects, ability to alkylate NEM, or enzyme inhibition by a-carbon substrates.

Seemingly opposed to the above results was the experiment in which we undertook to generate free enamines, by enzymic hydrolysis of their N-acyl derivatives, and found that with one of two hydrolases utilized NEM was alkylated. This result suggests that intermediate VII of Fig. 2, does not react with NEM, because a pyridoxal-P-bound intermediate would be totally unlike anything formed in the peptidase reaction.

The introduction of Structure VIII into the pyridoxal-P reaction scheme (Fig. 2) was prompted by considering that the escape of enamino acids from the elimination-catalyzing enzymes might follow in reverse order the steps thought to mediate the binding of amino acids to transaminases (2). Thus the bond to

11 If the N-substituted aminoacrylate underwent spontaneous hydrolysis like free aminoacrylate, the products would be pyru- vate and a radioactive aminosuccinimide. The latter should also have been detected by paper electrophoresis.

by guest on Decem

ber 27, 2018http://w

ww

.jbc.org/D

ownloaded from

Page 11: Enzymic Reactions of Enamines with N-Ethylmaleimide - Journal of

1444 Reactions of Enamines Vol. 244, No. 6

coenzyme in VII would be broken through transaldimination by an enzyme lysine amino group to yield the protonated lysine aldimine and the unprotonated enamino acid VIII, which would not escape from the enzyme until it had undergone enzyme- directed protonation.

We propose Structure VIII as the most likely intermediate for alkylating NEM in the case of the pyridoxal-P enzymes. A structure similar to VIII might also be the reactive intermediate in the peptidase-catalyzed reaction. Base- or acid-catalyzed amide hydrolyses both yield an unprotonated amino group initially (27). There is, furthermore, a definite suggestion that free aminoacrylate may not be an intermediate for at least one of the hydrolytic enzymes, since the incorporation of solvent tritium into pyruvate was higher with acylase than with amino- peptidase. Stereospecificity has not yet been detected in the aminopeptidase-catalyzed formation of KEDB (Table V), but this may conceivably be fortuitous.

REFERENCES 1. FLAVIN, M., AND SLAUQHTER, C., Biochemistry, 3, 885 (1964). 2. FLAVIN. M.. AND SLAUGHTER. C.. Biochemistru. 6.1340 (1966). 3. FLAVIN; M.‘, AND TSUNAHA~A, s., J. Biol. Ehek, 24i, 3340

(1966). 4. FLAVIN, M., AND SLAUGHTER, C., Biochim. Biophys. Acta, 133,

406 (1967). 5. FLAVI~, M:, Biochem. Biophys. Res. Commun., 20, 652 (1965). 6. FLAVIN. M.. AND SLAUGHTER. C.. Fed. PTOC.. 26. 837 (1967).

24. NISHIMURA, J. S., AND GREENBERG, D. M., J. Biol. Chem., 236, 2684 (1961).

7. GUGOEI&E&, S., AND FLAV;N, ik, Biochik Ekophis.. A&, 25. DAVIS, L., AND METZLER, D. E., J. Biol. Chem., 237, 1883 161, 664 (1968). (1962).

8. WIELAND, T., OHNACKER, G., AND ZIEGLER, W., Berichte, 90, 26. FLAVIN, M., AND SLAUGHTER, C., J. Biol. Chem., 239, 2212 194 (1957). (1964).

9. LEVINTOW, L., Fu, S. C. J., PRICE, V. E., AND GREENSTEIN, 27. GOULD, E. S., Structure and mechanism in organic chemistry, J. P., J. Biol. Chem., 184, 633 (1950). Holt-Dryden, New York, 1959, p. 327.

10.

11.

12.

13.

14.

15.

16. 17.

18.

19.

20.

PRICE, V. E., AND GREENSTEIN, J. P., J. Biol. Chem., 171,477 (1947).

CARTER, C. E., AND GREENSTEIN, J. P., J. Nat. Cancer Inst., 7, 51 (1946).

PHILLIPS, A. T., AND WOOD, W. A., J. Biol. Chem., 240, 4703 (1965).

DELAVIER-KLUTCHKO, C., AND FLAVIN, M., J. Biol. Chem., 240, 2537 (1965).

KAPLAN, M. M., AND FLAVIN, M., J. Biol. Chem., 241, 4463 (1966).

WACHSMUTH, E. D., FRITZE, I., AND PFLEIDERER, G., Biochem- istry, 6, 169 (1966).

FLAVIN, M., J. Biol. Chem., 237,768 (1962). GREENSTEIN, J. P., in S. P. COLOWICK AND N. 0. KAPLAN

(Editors), Methods in enzymology, Vol. II, Academic Press, New York, 1955, p. 109.

KAPLAN, M. M., AND FLAVIN, M., J. Biol. Chem., 241, 5781 (1966).

SHARPLESS, N. E., AND FLAVIN, M., Biochemistry, 6, 2963 (1966).

M&STER, A., AND WELLNER, D., in P. D. BOYER, H. LARDY, AND K. MYRB~CK (Editors), The enzymes, Vol. 7, Academic Press, New York, 1963, p. 628.

21. PITT, B. M., J. Amer. Chem. Sot., 30,3799 (1958). 22. FLAVIN. M.. AND GTJQOENHEIM. S.. in K. YAMADA. N. KATU-

NUMA; AND H. WADA (Edit&s); Symposium 04 pyridoxal enzymes, Maruzen Company, Ltd., 1968, p. 89.

23. SZMUSZKOVICZ, J., Advan. Org. Chem. Methods Result, 4, 1 (1964).

by guest on Decem

ber 27, 2018http://w

ww

.jbc.org/D

ownloaded from

Page 12: Enzymic Reactions of Enamines with N-Ethylmaleimide - Journal of

Martin Flavin and Clarence Slaughter-EthylmaleimideNEnzymic Reactions of Enamines with

1969, 244:1434-1444.J. Biol. Chem. 

  http://www.jbc.org/content/244/6/1434Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/244/6/1434.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on Decem

ber 27, 2018http://w

ww

.jbc.org/D

ownloaded from