CATALYSIS OF TRANSAMIDATION REACTIONS BY PROTEOLYTIC ENZYMES*

BY ROBERT B. JOHNSTON, MARY J. MYCEK, AND JOSEPH S. FRUTON

(From the Department of Physiological Chemistry, Yale University, New Haven)

(Received for publication, March 30, 1959)

In a previous communication from this laboratory (l), evidence was presented for the view that proteinases such as papain catalyze the re- placement of one participant in a peptide bond by another, closely re- lated, molecular species (2). Replacement reactions of this type may be termed transamidation or transpeptidation reactions. The experi- ments to be described in what follows are concerned with the enzymatic catalysis of transamidation reactions involving the replacement of the -NH2 group which participates in the amide bond of acylamino acid amides. Studies of replacement reactions involving peptide bonds which link two amino acid residues (transpeptidation reactions) are in progress, and will be reported in a future communication.

The data presented previously (1) dealt with the catalysis, by cysteine- activated papain, of the replacement of the amide N of benzoylglycin- amide (or of carbobenzoxy-L-methioninamide) by N16, introduced into the reaction mixture as isotopic diammonium hydrogen citrate. When papain was allowed to act on the amide at pH 5 until an appreciable fraction of the substrate had been hydrolyzed, the residual amide was found to contain a significant quantity of N’6. The extent of isotope incorporation found was considerably greater than that to be expected by direct synthesis from the hydrolytic products (acylamino acid and ammonia). It was concluded, therefore, that papain had catalyzed a transamidation reaction via a hypothetical transient intermediate in which two -NH2 groups are attached to the carbon atom of the amide bond.

OH .

RCO-NH* + NsHs +

[’ 1

RC-NH2 G= RCO-N”Ha + NH* I N”Ht

R = C6H&O-NHCHz-.

* This study was aided by grants from the Rockefeller Foundation and from the American Cancer Society (on recommendation of the Committee on Growth of the National Research Council).

629

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630 ENZYME-CATA4LYZED TRANSAMIDATION

In order to examine further the mechanism of this enzyme-catalyzed transamidation, a study has been made of the effect of pH on the extent of isotope incorporation and its relationship to the extent of hydrolysis. For this purpose, it was necessary to modify the conditions employed in t,he earlier studies (1). Citrate-phosphate buffers mere used in place of the &rate buffer, and the cysteine concentration was increased. The latter modificat,ion was based on the finding t*hat at pH 7 to 8 the action of

TABLE I Catalysis of Transamidation by Papain

Initial concentration of benzoylglycinamide, 0.05 M; initial concentration of isotopic N&NO3 (63.8 atom per cent excess N’s in NHh+), 0.05 M; enzyme con- centration, 0.26 mg. of protein N per cc. of test solution; initial concentration of activator (cysteine), 0.01 M; 0.3 cc. of methanol per cc. of test solution. -

-

hrs. per cent

.-

i

per cent

-

- ,

-

ztom ficr cent CZCeSS

0.023 0.260 0.186 0.438 0.797 1.127 0.061

‘

15.68 15.78 15.63 15.89 15.69 15.77 15.69

ttom per cent t3xCSI

0.046 0.5205 0.372 0.876 1.594 2.254 0.002

per cent

42 39 11 20 33 41 0

0.07 0.82 0.60 1.4 2.5 3.5 0.003

5.9 7.3 7.9 7.9 7.9

E,, I I -

* Determined by measurement of ammonia liberation in Conway vessels. t Benzoylglycinamide (theory, 15.72 per cent N) was isolated as described pre-

viously (l), and recrystallized to constant isotope concentration. $ Isotope concentration of amide N

Isotope concentration of NHd+ added x 100.

$ Deamidation of a sample of this preparation in a large Conway vessel (3) gave ammonia containing 0.511 atom per cent excess N”.

11 No enzyme present.

Isolated amidef

N’s concentration Replacement$

Compound Amide N

-

papain requires the presence of higher cysteine concentrations than those usuably employed at pH 5; this is presumably due, in large part, to the more rapid oxidation of sulfhydryl groups at alkaline pH values. An additional modification in the procedure previously described (1) was the use of iso- topic ammonium nitrate (63.8 atom per cent excess Nls in the NHh+ N) instead of ammonium citrate of lower isotope content.

As is shown in Table I, repetition of the studies described earlier (1) under the altered experimental conditions gave results that are quali-

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R. B. JOHNSTON, M. J. MYCEK, AND J. S. FRUTON 631

tatively in accord with the earlier data. In all cases in which enzymatic hydrolysis had occurred, the residual amide was found to contain Nls. What is more, when the pH was raised from 5.9 to 7.9, the extent of iso- tope incorporation at the end of a 6 hour incubation period was markedly increased (about 36-fold), while the extent of hydrolysis during the same period decreased slightly with increasing pH. It may be concluded, therefore, that under the conditions of these experiments a change in pH from 5 to 8 has a much more profound effect on the enzyme-catalyzed transamidation reaction than on the enzymatic hydrolysis. The data in Table I suggest that it is the NH3 molecule which reacts with the “ac- tivated” carbonyl group of the amide, since, in going from pH 5.9 to 7.9, there is approximately a lOO-fold increase in the NH3 concentration.

The data presented previously (1) indicated that, under a given set of experimental conditions, there was a proportionality between the extent of enzymatic hydrolysis and the extent of enzymatic transamidation. In order to explore this possibility further, four experiments were per- formed at pH 7.9, at which the greatest isotope incorporation was noted, and each of the enzyme-catalyzed reactions was allowed to proceed until a different fraction of benzoylglycinamide had been hydrolyzed. The residual amide was isolated as before, and, as will be seen from the ap- propriate data in Table I, a roughly proportional relationship between the extent of hydrolysis and the extent of transamidation was found, within the precision of the experimental methods employed. This result supports the suggestion made earlier (1) that the mechanism whereby the proteinase catalyzes the hydrolytic reaction also applies to the catalysis of the transamidation reaction. The enzyme may be considered to “ac- tivate” the carbonyl group of the sensitive amide bond, and a number of molecular species (e.s , NHs, HZO, etc.) may compete for reaction with this activated group. At any given pH, a simple quantitative relationship between the extent of hydrolysis and the extent of transamidation would then be expected. However, with changes in the pH of the solution, or in the molar proportion of the competing reactants, the magnitude of the proportionality factor should change either in favor of hydrolysis or of transamidation .

It will be seen from Table I that the hydrolysis of 41 per cent of the benzoylglycinamide at pH 7.9 is accompanied by a transamidation lead- ing to the incorporation of 2.254 atom per cent excess N15 into the amide N of the residual subst,rate. Under the conditions of these experiments, it may be expected that, as hydrolysis approaches completion, the isotope content of the residual amide will approach (but not necessarily attain) the theoretical maximum of 31.9 atom per cent excess Nls (i.e., 50 per cent replacement). The isotope content actually found after 41 per

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632 ENZYME-CATALYZED TRANSAMIDATIOX

cent hydrolysis at pH 7.9 thus represents approximately 7 per cent trans- amidation. This result attests to the importance of the transamidation reaction in the system under study. What is more, the value of 7 per cent must be considered as a minimum figure for the actual extent of transamidation during the incubation period; clearly, some of the newly formed isotopic amide must have undergone hydrolysis during this period.

In extending the present studies on enzyme-catalyzed transamidation reactions, attention was drawn to the possibility that proteinases may catalyze the replacement of a participant in a peptide or amide bond by the -NHOH group of hydroxylamine.

RCO-NHR’ + NHzOH e RCO-NHOH + NH&i.’

It has long been known that reactive carbonyl groups of acylating agents readily react with hydroxylamine to give hydroxamic acids (4), which form red coordination compounds with ferric ions. These reactions have provided the basis for a calorimetric method which has been applied with signal success by Lipmann and Tuttle (5) in their studies on acetyl phosphate, an acetylating agent comparable to acetic anhydride. More recently, these authors (6) have shown that esterases and lipases present in crude tissue extracts may catalyze hydroxamic acid formation from suitable fatty acids. Chantrenne (7) had previously pointed out that the hydroxamic acid method, under a variety of experimental conditions, may not be specific for the determination of acyl phosphates.

In the experiments reported in the present communication, acylamino acid amides known to be substrates for papain were found to form hydrox- amic acids on incubation with hydroxylamine in the presence of a cys- teine-activated enzyme preparation. In the conduct of these experiments, aliquots of each incubation mixture were removed after various time in- tervals for ammonia determinations in Conway vessels. These analyses served as a measure of the quantity of the amide that had undergone hydrolysis plus that which had reacted with hydroxylamine. Separate aliquots were withdrawn for the calorimetric estimation of the hydroxamic acid present at the end of a given incubation period. In order to trans- late the calorimetric readings into molar quantities of the appropriate hydroxamic acid, it was desirable to have separate calibration curves for each of the hydroxamic acids to be expected in the transamidation reac- tions under study. In view of the number and variety of the acylamino acid amides employed, however, it was decided to assume, as a first ap- proximation, that equimolar quantities of the corresponding hydroxamic acids would give the same intensity of color with ferric chloride under the conditions of the analytical procedure used in these experiments. Ac- cordingly, benzoyl-L-alanylhydroxamic acid was chosen as the standard

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R. B. JOHNSTON, M. J. MYCEK, AND J. S. FRU’CON 633

reference substance; it was prepared by the reaction of the corresponding methyl ester with hydroxylamine. The calibration curve for the ferric salt of this hydroxamic acid is presented in Fig. 1. For comparison, the calibration curves for authentic samples of benzoylglycylhydroxamic acid and of acethydroxamic acid are also given, and it will be seen that the color values of equimolar quantities of the three compounds are quite similar. On the other hand, the slope of the curve for benzhydroxamic acid is considerably steeper (7).

Data are presented in Tables II and III on the catalysis, by cysteine- activated papain, of the replacement of the amide --NH2 group of acyl- amino acid amides by hydroxylamine. In confirmation of earlier stud-

MICROMOLES OF HYDROXAMIC ACID

FIG. 1. Klett-Summerson calibration curves for ferric salts of hydroxamic acids. Curve 1, benzoyl-L-alanylhydroxamic acid; Curve 2, benzoylglycylhydroxamic acid; Curve 3, acethydroxamic acid; Curve 4, benzhydroxamic acid.

ies (S), it was found that hydroxylamine acts as an inhibitor of the proteolytic action of papain; when the cysteine concentration is increased, however, this inhibition can be largely overcome. A similar effect of cys- teine in counteracting the inhibition of papain by the aldehyde reagent phenylhydrazine has been reported previously (9).

The data in Table II show that, as in the experiments on isotope in- corporation presented above, transamidation is favored by a shift in pH from 5 to 7.5. Thus, although the rate of hydrolysis by papain is, in general, decreased at the more alkaline pH value (lo), the extent of hydroxamic acid formation is markedly increased. This result has been found to apply to the action of papain on several of its synthetic sub- strates, i.e., benzoyl-L-argininamide, carbobenzoxy-Gsoglutamine, ben-

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034 ENZYME-CATALYZED TRANSAMIDATION

TABLE II Catalysis of Hydroxamic Acid Formation by Papain

Concentration of substrates, 0.05 M; concentration of NH20H.HCI (adjusted to pH 6 with NaOH), 0.05 M; enzyme concentration, 0.25 mg. of protein N per cc. of test solution; cysteine concentration, 0.025 M.

Benzoyl-n-argininamide . 4.9

“ . . . . . . . . .

“ . . . . . . .

“ . . . .

“ . . . . . . . . . . .

Benzoyl-L-arg.inine . . . . . . . . . . . . Carbobenzoxy-n-isoglutamine . . , .

I‘ . . . . .

“ . . . . .

Carbobenzoxy-L-glutamic acid. . Carbobenzoxy-L-isoasparagine. .

“ . . Carbobenzoxy-n-serinamidet .

“

Benzoylglycinamide$ . . . . 5.1

I‘ . . . . . . . . . . . . . 7.8

“ . . . . . . Carbobenzoxy-L-methioninamides.

“

Carbobenzoxy-n-methioninamides. None. . . . . . . . . . . . S . . . . . . . . . . . . .

7.7* 6.5 7.8 7.5 7.5

--

-

PH

6.8

7.5

*

kit 7.6 4.9 7.3

7.2* 7.5 4.9 7.4 5.2 7.7

--

-

Time

min. 20 60 60

120 20 60

120 60 60

120 60 60

120 180 180 186 180 120 60

126 90

180 90

186 180 180 180 180 120

?-

--

-

Ammonia liberation

pY )CI cc.

17 31 15 22 7

17 34 0 0 0

21 11 24 0 0 1 2 6 2 5 1 2 3 6 0 5 7 0 0

Eydroxamic rdd present

p* pbl CC.

0.5 0.7 2.9 3.4 1.35 3.0 4.3 0.05 0.1 0.06 0.7 1.4 2.2 0.01 0.1 0.2 0.4 0.2 0.8 1.4 0.3 0.4 1.2 2.2 0.05 0.3 1.3 0.03 0.03

* No enzyme present. t No cysteine present. $0.3 cc. of methanol added per cc. of test solution to dissolve the substrate.

In the presence of 30 per cent methanol, the rate of hydrolysis of synthetic sub- strates by papain is decreased.

Q This 8ub8tmm? was not completely in solution at the start of the experiment even with the addition of 0.3 CC. of methanol per CC. of b38t solution,

zoylglycinamide, carbobenzoxy-L-serinamide, and carbobenzoxy-L-me- thioninamide (11). It is of. interest that carbobenzoxy-L-isoasparagine,

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R. B. JOHNSTON, M. J. MYCEK, AND J. S. FRUTON G35

which has been found to be relatively resistant to the hydrolytic action of papain at pH 5 (ll), exhibits only slight transamidation with hydroxyl- amine in the presence of the enzyme preparation. Also, carbobenzoxy- n-methioninamide was unable to participate in the enzyme-catalyzed re- placement reaction. The conclusions as to the specificity of the enzymatic components of papain, drawn from studies of the hydrolysis of synthetic substrates, also apply, therefore, to the catalysis of transamidation reac- tions by these enzymes. This may be taken as additional evidence for a fundamental similarity, if not identity, in the mechanism of enzymatic catalysis of hydrolysis and replacement, as suggested earlier in this com- mtmication and elsewhere (1).

It will be noted from Table II that, with benzoyl-L-argininamide as the substrate, a shift in pH from 6.8 to 7.5 does not increase the extent of transamidation with hydroxylamine to an appreciable degree. This result may be contrasted with the effect of pH on the transamidation with isotopic ammonia. The explanation for this difference in the behavior of these two replacement agents appear to lie in the fact that the pK of the NH*+ ion is about 9.4, while that of the NH30H+ ion is about 6. From this it would follow that it is the uncharged NHzOH molecule which participates in the enzyme-catalyzed transamidation reaction.

Since benzoyl-n-arginine and carbobenzoxy-L-glutamic acid do not show appreciable hydroxamic acid formation under conditions in which the corresponding amides react extensively, the possibility of prior hydrolysis of the amide to the acylamino acid and subsequent synthesis of the hy- droxamic acid appears to be ruled out. Also, in the absence of a suitable substrate, the enzyme preparation, after incubation with hydroxylamine, gives only a negligible color with ferric chloride. The fact that substitu- tion of the citrate-phosphate buffer by Verona1 buffer did not alter the results to a significant extent indicates that the added phosphate is not specifically involved in the transamidation reaction (Table III). A specific r61e of cysteine also is unlikely, since transamidation with hydroxylamine could be demonstrated in experiments in which this a.ctivator was replaced by cyanide.

The data in Table III describe the extent of papain-catalyzed trans- amidation at pH 7.4 to 7.5 as a function of the initial hydroxylamine concentration. As was to be expected, the highest concentration of hy- droxylamine employed (0.2 M) led to the most extensive transamidation; it is significant, however, that the presence of as low a concentration as 0.01 M hydroxylamine was sufficient for appreciable hydroxamic acid formation. It will be seen from the data in Table III that, at the lower hydroxylamine concentrations, the hydroxamic acid level attained a maxi- mum and then receded. This may readily be explained by the fact that

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636 ENZYME-CATALYZED TRANSAMIDATION

the products of transamidation with hydroxylamine are also substrates for papain, as shown in Table IV, where the rates of the hydrolytic action of papain on benzoylglycylhydroxamic acid and on benzoylglycinamide are compared. In this connection, it may be mentioned that a recent report (12) describes the hydrolysis of benzoyl-L-phenylalanylhydroxamic acid by chymotrypsin. In the transamidation experiments, therefore, when the rate of transamidation exceeds the rate of the enzymatic hydrol-

TABLE III EJect of Hydroxylamine Concentration on Extent of Papain-Catalyzed Hydroxamic

Acid Formation Substrate, benzoyl-n-argininamide, 0.05 M; enzyme concentration, 0.25 mg. of

protein N per cc. of test solution; cysteine concentration, 0.025 M; pH 7.4 to 7.5.

Concentration of hydroxylamine

Y

0.2

0.1

0.05

0.05*

0.03

0.01

Tiie

min.

20

70 145 xl 70

145 20 70

145 225

20 70

145 20 70

145 225 xl 70

145

Ammonia liberation Hydroxamic acid present

#Id pt.? cc. I.rY per cc.

4 2.2 16 5.5 23 8.2 6 2.0

19 5.2 29 8.0 7 1.4

23 4.1 36 4.4 43 3.6 6 1.3

19 3.8 34 4.6 8 1.2

27 2.8 42 2.1 46 1.3 13 0.7 39 1.0 49 0.2

* 0.02 M Verona1 buffer used in place of the citrate-phosphate buffer.

ysis of the newly formed hydroxamic acid, the concentration of hydrox- amic acid increases steadily, as at the higher hydroxylamine concentra- tions (0.1 to 0.2 M) of Table III; when the rate of transamidation falls off to the point at which it is less than the rate of the hydrolysis of hydrox- amic acid, a decrease in the hydroxamic acid concentration may be ex- pected. The data in Table III show that the rate of enzymatic hydrolysis of the amide increases as the hydroxylamine concentration is decreased.

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R. B. JOHNSTON, M. J. MYCEK, AND J. S. FRUTON 637

The hydroxylamine appears, therefore, to be performing a dual role in these experiments, since it acts both as an inhibitor of the enzyme and as a partner in the transamidation reaction.

Preliminary experiments with a preparation of beef spleen cathepsin have shown that enzyme-catalyzed hydroxamic acid formation occurs at pH 6.5 to 7, with benzoyl-L-argininamide as the substrate. As in the case of papain, the extent of transamidation in this pH region is much greater than at pH 5, at which cysteine-activated beef spleen cathepsin II acts optimally in the catalysis of the hydrolytic reaction (13). The catalysis of transamidation reactions by crystalline pancreatic trypsin and chymotrypsin is under investigation.

TABLE IV

Hydrolysis of Benzoylglycylhydroxamic Acid and of Benzoylglycinamide by Papain

Concentration of substrates, 0.05 M; enzyme concentration, 0.25 mg. of protein N per cc. of test solution; cysteine concentration, 0.025 M; methanol, 0.3 cc. per cc. of test solution.

Substrate

Benzoylglycylhydroxamic acid*.

“ ‘I . . . . .

Benzoylglycinamidet . . . . . .

“

-

_ _

-

PR

5.9

7.3

5.8

7.8

-

_-

-

Time Hydrolysis

hrs. QW cent

1 21

3 33 5 39 1 22 3 37 5 42 3 19 5 31 3 19 5 32

* Hydrolysis followed by calorimetric estimation of hydroxamic acid. t Hydrolysis followed by measurement of ammonia liberation.

DISCUSSION

The experiments reported above give support to the view that pro- teolytic enzymes of plant and animal tissues catalyze transamidation reactions. The possible rale of such reactions in the biosynthesis of the peptide bonds of proteins and of naturally occurring peptides has been discussed previously (1). A significant result of the present study is the disparity between the pH optima for hydrolysis and for trans- amidation in the action of several intracellular proteinases. This finding suggests that, at physiological pH values, transamidation may represent a major reaction in living cells; upon death of the cell, and the accompany- ing shift of pH to more acid values, the a&ion of the intracellular proteo-

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638 ENZYME-CAT.4LYZED TRANSAMIDlTION

lytic enzymes may be almost exclusively a hydrolytic one. The further experimental exploration of this working hypothesis should throw valuable light on the r81e of these enzymes in the biosynthesis of peptide bonds.

The view that enzymes usually classified as catalysts of hydrolytic reac- tions also catalyze replacement reactions appears to have validity for the acid and alkaline phosphatases (14, 15) and for intest.inal dipeptidase (16). Also, the enzymatic synthesis of y-glutamohydroxamic acid and of p- aspartohydroxamic acid, from glutamine and asparagine respectively, has recently been reported by Waelsch et al. (17). In their studies, which bear an immediate relationship to the results described in the present com- munication, a cell-free extract of Proteus vulgaris was used as the enzyme preparation; no information was given by them (17) as to the hydrolytic activity of the extract toward the two amides. Stumpf and Loomis (18) also have reported the enzymat,ic synthesis of y-glutamohydroxamic acid from glutamine and hydroxylamine; in their experiments, pumpkin seed- lings served as the source of the enzyme preparation. Furthermore, the studies of Hehre (19), Monod and Torriani (20), Doudoroff et al. (21), and others have demonstrated the biological occurrence of replacement reactions involving glycosidic linkages. It is clear, therefore, that in- creasing attention is being given to the possible physiological importance of enzyme-catalyzed reactions in which one participant of an amide (or peptide) bond, of an ester linkage, or of a glycosidic bond is replaced by another closely related molecular species, with a relatively small change in free energy (1). In the face of the data presented in this communication, and by other investigators, it will be important to examine the possibility that some of the enzymes now considered to function solely as catalysts in hydrolytic reactions may have an important physiological r81e in the catalysis of replacement reactions.

EXPERIMENTAL

In t,he conduct of the enzyme experiments, mixtures of 0.05 M citrate and of 0.05 M phosphate were employed, except as otherwise stated. The temperature was maintained at 38.1”. The papain preparation was ob- tained from crude dried papaya latex by the method of Grassmann (22). The beef spleen cathepsin preparation was obtained by fractional precipi- tation with ammonium sulfate, as described previously (13).

Measurement of Ammonia Formation-41 cc. portions of the incuba- t,ion mixture mere placed in duplicate Conway vessels (A. H. Thomas Company, No. 4427-F), which contained 1 cc. of 2 per cent boric acid reagent (23) in the center well. The ammonia was liberated by mixing the test sample with 1 cc. of saturated potassium carbonate solution.

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R. B. JOHNSTON, M. J. MYCEK, AND J. S. FRUTON 639

The vessels were kept at room temperature (20-25’) for 3 hours, and the contents of the cemer wells were titrated with 0.0033 N sulfuric acid. Control experiments showed that, under these conditions, the scylamino acid amides employed did not undergo deamidation, and that the presence of hydroxylamine in the incubation mixture did not interfere wit,h the accuracy of the ammonia determinations.

Calorimetric Determination of Hydroxamic Acid Concentration-The pro- cedure employed involved a slight modification of the method described by Lipmann and Tuttle (6). 1 cc. of the incubation mixture was added to 1 cc. of 20 per cent trichloroacetic acid; 1 cc. of 5 per cent ferric chloride in 0.1 N hydrochloric acid 11-8s then added, and the volume was adjusbed to 5 cc. The mixture was centrifuged, and the optical density of the clear supernatant solution was read within 15 minutes of color development in a Klett-Summerson calorimeter with a No. 54 filter. Additional hydroxylamine was not introduced during the development of the color (6, 24), since this was not found to increase the constancy of the readings to a significant extent. Control experiments showed that the presence of the amounts of phosphate, cysteine, or methanol used in these studies affected the color development only slightly (2 to 5 per cent). The pro- cedure described above also was employed in the determinabion of t.he calibration curves for the authentic hydroxamic acids.

Benzoylglycylhydroxamic Acid-3 gm. of benzoylglycinamide (25) were heated under a reflux with 30 cc. of methanol which contained 2 gm. of free hydroxylamine (26). After 1 hour, 50 cc. of water were.added, and the solution was acidified to about pH 6 with concentrated hydrochloric acid. On chilling the solution, the product (1.7 gm.) crystallized. After recrystallization from water, the substance melted at 158-159”.

CsHloOaNz (194.2). Calculated, N 14.43; found, N 14.42

Benxoyl-L-alunylhydroxamic Acid-l.4 gm. of benzoyl-L-alanine methyl ester were heated with 1 gm. of hydroxylamine in 10 cc. of methanol as described for the glycyl compound. After the solution was diluted with water, acidified, and chilled, 1.0 gm. of the product was obtained. M.p., 169-170”.

ChHl,OaNr (208.2). Calculated, N 13.46; found, N 13.45

Benxoyl-rdeucylhyldroxamic Acid-This substance was prepared from 1.7 gm. of the corresponding ethyl ester in the same manner as the alanyl compound. Yield, 1.5 gm.; m.p., 160-161”.

CLHISO~N~ (250.3). Calculated, N 11.20; found, N 11.11

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640 EhXYMECAT.4LYZED TR4NSAMIDATION

Acethydroxamic Acid-This substance was prepared in the manner de- scribed by Miolati (27).

C2H402N (75.1). Calculated, N 18.66; found, N 18.49

Benzhydroxamic Acid-This substance was prepared in the manner described by Jones and Hurd (28). M.p., 123-124”.

The authors are greatly indebted to Dr. Henry D. Hoberman and to Mr. Joseph Doolittle for their generous cooperation in the conduct of the isotope experiments.

SUMMARY

1. Cysteine-activated papain catalyzes the replacement of the amide N of benzoylglycinamide by N16, introduced into the reaction as isotopic ammonium N . This transamidation is favored by a shift in pH from 5 to 8.

2. Cysteine-activated papain catalyzes the replacement of the amide NH2 group of a number of acylamino acid amides by the -NHOH group of hydroxylamine. The hydroxamic acids so formed have been estimated calorimetrically. As with the isotope experiments, a shift in pH from 5 to 7.5 markedly increases the extent of transamidation. Similar re- sults have been obtained with a preparation of beef spleen cathepsin.

3. The possible significance of these findings for the study of the bio- synthesis of peptide bonds is discussed.

BIBLIOGRAPHY

1. Fruton, J. S., Yale J. Biol. and Med., 22, 263 (1950). 2. Bergmann, M., and Fraenkel-Conrat, H., J. Biol. Chem., 119, 707 (1937). 3. Dekker, C. A., Stone, D., and Fruton, J. S., J. Biol. Chem., 181, 719 (1949). 4. Yale, H. L., Chem. Rev., 33, 209 (1943). 5. Lipmann, F., and Tuttle, L. C., J. Biol. Chem., 169, 21 (1945). 6. Lipmann, F., and Tuttle, L. C., Biochem. et biophys. acta, 4, 301 (1950). 7. Chantrenne, H., Compt.-rend. trav. Lab. Curlsberg, 26, 231 (1948). 8. Bergmann, M., and Ross, W. F., J. Biol. Chem., 114, 717 (1936). 9. Bergmann, M., and Fruton, J. S., Science, 86, 496 (1937).

10. Hoover, S. R., and Kokes, E. L. C., J. Biol. Chem., 167, 199 (1947). 11. Dekker, C. A., Taylor, S. P., Jr., and Fruton, J. S., J. Biol. Chem., 180,155 (1949). 12. Iselin, B. M., Huang, H. T., and Niemann, C., J. Biol. Chem., 183, 403 (1950).

13. Fruton, J. S., and Bergmann, M., J. Biol. Chem., 130, 19 (1939). 14. Axelrod, B., J. Biol. Chem., 173, 1 (1948). 15. Meyerhof, O., and Green, H., J. Biol. Chem., 183, 377 (1950). 16. Zamecnik, P., and Fran@ I. D., Jr., Cold Spring Harbor Symposia Quant. Biol.,

14, 199 (1950). 17. Waelsch, H., Borek, E., and Grossowice, N., Abstracts, Division of Biological

Chemistry, American Chemical Society, 116th meeting, Atlantic City (1949).

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