ENZYMATIC HYDROLYSIS OF SATURATED AND BY PAUL J. FODOR… · ENZYMATIC HYDROLYSIS OF SATURATED AND...

ENZYMATIC HYDROLYSIS OF SATURATED AND UNSATURATED TRIPEPTIDES

BY PAUL J. FODOR,* VINCENT E. PRICE, AND JESSE P. GREENSTEIN

(From the National Cancer Institute, National Institutes of Health, Bethesda, MaTgland)

(Received for publication, March 1.1949)

Previous studies on the relative rates of enzymatic hydrolysis by rat tissue extracts of analogous saturated and unsaturated dipeptides revealed the generally greater susceptibility of the L form of the former type of substrate (1). Thus, in the presence of liver preparations, glycyl-L-phenylalanine was hydrolyzed about 150 to 250 times faster than either glycyl- n-phenylalanine or glycyldehydrophenylalanine. In the case of certain pairs of analogous compounds, such as chloroacetyl-DL-phenylalanine and chloroacetyldehydrophenylalanine, the L form of the saturated peptide was rapidly attacked, whereas the D form and the dehydropeptide were apparently completely resistant.

It was considered of interest to extend these comparison studies to analogous saturated and unsaturated tripeptides. The saturated tripeptides have two peptide bonds, and the unsaturated tripeptides have one acyl peptide bond and one terminal dehydropeptide bond. In the case of tripeptides containing a racemic amino acid, four peptide bonds may be considered to coexist in the same substrates. We have therefore laid chief emphasis in this study on determining the maximum hydrolysis, under nearly identical conditions, of various related substrates after prolonged incubation periods in the presence of concentrated aqueous extracts of hog or rat kidney. In addition, a few rate studies have been made on selected substrates.

In the case of the dehydropeptides, the hydrolysis was followed either by measurement of the ammonia formed or by ultraviolet spectrophotometry (2), and therefore only the cleavage of the dehydropeptide bond was noted. There is little doubt, however, that, in the susceptible unsaturated tripeptides, both saturated and unsaturated peptide bonds are hydrolyzed. The enzymatic hydrolysis of the saturated dipeptides and tripeptides was followed by the manometric COZ procedure with ninhydrin; and therefore the cleavage of all susceptible peptide bonds in the substrates was noted.1

* Research Fellow, National Cancer Institute; on leave from The Hebrew Uni- versity of Jerusalem.

1 The dehydropeptides yield on hydrolysis not only ammonia but alto the corre- 193

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194 ENZYMATIC HYDROLYSIS OF TRfPEPTIDES

A number of analogous and isomeric peptides were prepared in order to interpret adequately the enzymatic data.

EXPERIMENTAL

Substmtes2

The following compounds were prepared as described: glycyldehydroalanine (3), glycyl-L-alanine (4), glycyl-D-alanine (4), glycyl-DL-alanine (5), chloroacetyldehydroalanine (3), chloroacetyl-L-alanine (4), chloroacetyl-D-alanine (4), chloroacetyl-DL-alanine (5), acetyldehydroalanine (6, 7), acetyl-DL-alanine (4), glycyldehydrophenylalanine (8), glycyl-DL- phenylalanine (9), chloroacetyldehydrophenylalanine (8), Chloroacetyl-DL- phenylalanine(9)) acetyldehydrophenylalanine (lo), acetyl-Ix-phenylalanine (1 l), acetyldehydroleucine (12)) acetyl-DL-leucine (4)) sarcosyldehydroalanine (13), sarcosyldehydrophenylalanine (14), chloroacetylglycyldehydroalanine (14)) glycyl$ycyldehydroalanine (14)) chloroacetylglycyldehydrophenylalanine (15), glycylglycyldehydrophenylalanine (15), chloroacetyldehydrophenylalanylglycine (8);O glycyldehydrophenylalanylglycine (8) ,a chioroacetylsarcosyldehydroaianine (13)) DL-alanylglycine (16)) DL-phenylalanylglycine (17)) glycylglycine (18), glycylglycylglycine (19)) chloroacetylglycine (20), chloroacetylglycylglycine (20), Chloroacetyl-DL- leucine (16), chloroacetylsarcosine (2l), glycylsarcosine (21): glycyl-DL- leucine (16), DL-leucylglycine (16), and DL-leucinamide (22). All the

sponding a-keto acids. Measurement of the hydrolysis of the saturated peptide bond in the tridehydropeptides by the ninhydrin procedure in the presence of appreciable amounts of a-keto acids presents considerable difficulty because of the small but variable amounts of carbon dioxide produced by decomposition of the keto acids (23).

2 Many chloroacetylated amino acids and peptides do not crystallize from the reaction mixture after acidification with HCI. In such cases we have extracted the mixture several times with ethyl acetate, dried the combined extracts briefly over sodium sulfate, and evaporated the solvent at low temperature and pressure. The residue was washed several times with dry petroleum ether and finally treated with dry ether at -10”. The product invariably crystallized.

1 The absorption spectrum in the ultraviolet of these compounds at 5 X 10-c M

concentration in water was practically identical with that of glycyldehydrophenylalanine or other aromatic dehydropeptides with an absorption maximum at 2750 A and molar extinction coefficient of 18,000 (13, 34).

4 Our preparation possessed a melting point of 214’ (uncorrected). Found C 40.8, H 7.2, N 18.8; calculated C 41.1, H 6.9, N 19.1 per cent. Levene et al. (21) reported a melting point of 200-201’. This lower melting point value might have been due to traces of ammonium chloride, for we have found that several recrystallizations from hot water-alcohol are.necessary to.free the peptide from this salt. The melting point of the preparation of glycylsarcosine made by the carbobenzoxy method by Bergmann,& ~2. was 220” (corrected) (35)


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FODOR, PRICE, AND GREENSTEIN 195

compounds were recrystallized and characterized; the chloroacetylated compounds weri free from chloride ions, and the aminated compounds free of ammonia and chloride.

Sarcosyl-DL-aZanine-1 mole of chloroacetyl-nL-alanine was treated at 40” with 3 moles of methylamine in a 25 per cent aqueous solution. After 3 days, the solution was evaporated to a syrup in vucuo. The syrup was taken up in a small volume of water, treated with glacial acetic acid to pH 5.0, and a large volume of absolute alcohol was added. The product crystallized in long prisms. On recrystallization from water-alcohol the peptide melted at 182”. N found 17.3, calculated 17.5 per cent.

Sarcosyl-nn-phenylalanine-This compound was prepared as above by the use of chloroacetyl-nn-phenylalanine and methylamine. Prisms. M.p. 191”; N found 11.5, calculated 11.8 per cent.

ChZoroacetyZgZycyZ-n-alanine-From glycyl-n-alanine (4) and chloroacetyl- chloride in NaOH solution. Recrystallized from absolute methanol as needles. M.p. 167”; N found 12.6, calculated 12.6 per cent.

I& = -42.7’ (for 0.740/, solution in water)

ChZoroacetyZgZycyZ-n-uZu&e---From glycyl-n-alanine (4) and chloroacetyl chloride in NaOH solution. Recrystallized from absolute methanol as needles. M.p. 167”; N found 12.3, calculated 12.6 per cent.

[oil: = +42.4” (for 1.09% solution in water)

ChloroacelyZgZycyZ-nn-alanine-From glycyl-nn-alanine and chloroacetyl chloride in NaOH solution. Recrystallized from absolute methanol as needles. M.p. 163”; N found 12.4, calculated 12.6 per cent.

GlycyZgZycyZ-m-alanine-By amination of chloroacetylglycyl-nn-alanine in 10 times the volume of 28 per cent ammonia water for 24 hours at 40”. Recrystallized three times from water-alcohol. Long prisms. M.p. 228’; N found 20.5, calculated 20.7 per cent.

Chloroacetylglycyl-m-phenyZak.znine-From glycyl-nL-phenylalanine and chloroacetyl chloride in NaOH solution. Recrystallized from ethyl alcohol as prisms. M.p. 151”; N found 9.2, calculated 9.4 per cent.

GZycyZgZycyZ-m-phenyZaZanine-By amination of chloroacetylglycyl-m- phenylalanine in 10 times the volume of 28 per cent ammonia water for 24 hours at 40”. Recrystallized twice from water-alcohol as long prisms; m.p. 242’; N found 14.8, calculated 15.1 per cent.

Chloroacetyl-nn-phenyZaZanylgly&e-From nL-phenylalanylglycine and chloroacetyl chloride in NaOH solution. Recrystallized from abso1ut.e methanol as long needles. M.p. 180”; N found 9.1, calculated 9.4 per cent.

GZycyZ-nn-phenyZalanylgZytine-By amination of chloroacetyl-nn-phenylalanylglycine as above. Prisms from water-alcohol. M.p. 235’; N found 14.9, calculated 15.1 per cent.


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196 ENZYMATIC HYDROLYSIS OF TRIPEPTIDES

Chbroacetylsarcosyl-m-a~nine-From sarcosyl-DL-alanine and chloroacetyl chloride in NaOH solution. Needles from absolute alcohol. M.p. 138”; N found 11.4, calculated 11.8 per cent. Attempts to aminate this compound, in the hope of obtaining glycylsarcosyl-DL-alanine, were unsuccessful.

Glycylsarcosyldehydroalanine-Attempts to aminate chloroacetylsarcosyldehydroalanine by use of aqueous ammonia were unsuccessful (13). A good preparation, however, was obtained by amination in ammoniacal absolute methanol. 10 gm. of chloroacetylsarcosyldehydroalanine (13) were dissolved in 100 cc. of absolute methanol which had been saturated at 0” with dry ammonia gas. After standing 5 hours at room temperature, the solution was evaporated in vacua to a white solid mass which was taken up in hot water and treated with hot absolute alcohol in excess. The dehydropeptide was recrystallized twice more from water-alcohol as glistening prisms. Yield 38 per cent. M.p. 161’. Found C 41.2, H. 6.8, N 17.6 per cent; calculated for compound with 1 mole of crystal water C 41.2, H 6.9, N 18.0 per cent. When dried for 4 hours at 78’ and 1 mm. pressure of mercury, the compound lost 7.5 per cent in weight; calculated for 1 mole of crystal water 7.7 per cent.6

Chloroacetyl-DL-atiny&.?ycine-Prisms from absolute methanol. M.p. 146”; N found 12.4, calculated 12.6 per cent.

Glycyl-DL-alanylg.?ycine--By amination of the above in aqueous ammonia. Prisms from water-alcohol. M.p. 242”; N found 20.6, calculated 20.7 per cent.

Chloroacetyl-mAeucylglycine-Needles first from absolute methanol and then from water. M.p. 144’; N found 10.5, calculated 10.6 per cent.

Chloroacetylglycyl-Dbleucine-Needles first from absolute methanol and then from water. M.p. 160’; N found 10.6, calculated 10.6 per cent.

Glycylglycyl-DL-leucine-By ainination of the above in aqueous ammonia. Prisms from water-alcohol. The compound did not melt when heated up to 240”. When heated at 78” and 1 mm. pressure of mercury for 4 hours, the compound lost 6.5 per cent in weight; calculated for 1 mole of crystal water 6.8 per cent. N found 17.1, calculated 17.1 per cent.

Glycyl-DL-leucylglycine -By amination of chloroacetyl-DL-leucylglycine in aqueous ammonia. Prisms from water-alcohol. The compound did not melt when heated to 240”. When heated at 78” and 1 mm. pressure of mercury for 4 hours, the compound lost 6.6 per cent in weight; calculated for 1 mole of crystal water 6.8 per cent. N found 17.0, calculated 17.1 per cent.

6 The absorption spectrum in the ultraviolet of this compound at 1.7 X 1W M concentration in water was practically identical with that of glycyldehydroalaninc or other aliphatic dehydropeptides with an absorption maximum at 2400 A and molar extinction coefficient of 4450 (3,13).


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FODOR, PRICE, .4ND GREENS’I’EKV 197

Chloroacetyl-hr-methyl-m-alar&e-l mole of a-chloropropionic acid was treated with 10 moles of 33 per cent methylamine solution for 2 days at 40”. The solution was evaporated to a syrup in vacua, taken up in a small amount of water, treated with glacial acetic acid to pH 5.0, and finally treated with t,he lo-fold volume of absolute alcohol. After standing for 24 hours at - lo”, the N-methyl-nL-alanine was filtered, washed with alcohol, and recrystallized from water-alcohol. Yield 44 per cent. N found 13.5, calculated 13.6 per cent. On chloroacetylation in the usual manner with chloroacetyl chloride in NaOH solution, chloroacetyl-N-methyl-nn-alsnine was obtained in 64 per cent yield. Long needles from hot absolute alcohol. M.p. 82’; N found 7.8, calculated 7.8 per cent.

G13/cyl-N-methyl-nn-a&m&e-By amination of the above compound in a lo-fold volume of 28 per cent aqueous ammonia for 3 days at 40”. After evaporation of the ammonia solution in vacua, the residue was taken up in the minimum amount of cold water and treated with an excess of alcohol. On standing 1 week at -lo”, long prisms separated. Yield 28 per cent. The product was recrystallized from hot water-alcohol. M.p. 155’; found C 45.0, H 7.8, N 17.3, calculated C 45.0, H 7.5, N 17.5 per cent.

Chloroacetyks-leucinamide-2.6 gm. of nn-leucinamide were dissolved in 15 cc. of water and chilled. The amide was then chloroacetylated in the usual manner with 2.6 gm. of chloroacetyl chloride, and 10 cc. of 2 N NaOH were added alternately with shaking and with chilling. The product which crystallized during the reaction was filtered at the pump and washed with cold water. It was recrystallized from the minimum amount of absolute methanol from which it separated as flat plates. M.p. 155’; found C 46.5, H 7.6, N 13.3, calculated C 46.5, H 7.3, N 13.5 per cent.

Enzymatic Procedure

The digests were composed of 1 cc. of either hog or rat kidney aqueous extract (prepared by grinding the tissue with sand, taking up the paste in distilled mater, and lightly centrifuging), 1 cc. of 0.15 M borate buffer at pH 8.0 or 1 cc. of 0.07 M phosphate buffer at pH 7.2, and 1 cc. of either water or neutralized substrate solution. A concentration of 0.05 M was employed for the racemic substrates, and 0.025 M for all the others. The concentration of extract and the period of incubation at 37” were varied to suit the criterion of nearly maximum hydrolysis. No activators were employed at this time with the crude extract preparations. In all cases the determinations were continued either until (a) the hydrolysis had practically reached an end-point, (b) the titer was so negligible as to raise doubt as to whether any hydrolysis at all had occurred, or (c) until the hydrolysis of relatively resistant bonds in certain substrates was measurable but so


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198 ENZYMATIC HYDROLYSIS OF TRfPEJ?J?IDES

slow as to render impracticable further incubation of the digest. The longest period of incubation for any substrate was 8 hours.”

The hydrolysis of the dehydropeptides, with the exception of that of glycylsarcosyldehydroalanine, was measured in’ terms of the ammonia produced in the digests (2). The hydrolysis of glycylsarcosyldehydroalanine

HOURS INCUSATION

FIG. 1. Hydrolysis of glycylsarcosyldehydroalanine at 37”. The digests con- sisted of 1 cc. of hog kidney aqueous extract equivalent to 660 mg. of fresh tissue, 2 cc. of 0.15 M borate buffer at pH 8.1, and 1 cc. of 0.025 M substrate. The reaction was followed spectrophotometrically at 2400 A; the cells were used with a 1 cm. path. 0, substrate solution in the presence of the buffer.

could not be readily measured by this procedure, for, unlike all the other dehydropeptides studied, this compound was unstable in the presence of the saturated potassium carbonate solution employed for the aeration of the evolved ammonia,. The alternative spectrophotometric procedure was

6 It is probable in several instances that, had the incubation period been extended well beyond the period chosen, the relatively resistant. bonds would have been completely hydrolyzed, and perhaps even those apparently completely resistant bonds in certain substrates might have been at least measurably attacked. It is sometimes judicious, however, to set some sort of a limit to what an enzyme might reasonably be expected to do, and we are reluctant to ascribe authentic enzymatic activity to a reaction which, under otherwise optimum conditions, requires an undue amount of time. We have generally set, as the lower limit of enzymatic activity in hydrolytic reactions involving the saturated or unsaturated peptide bond, a rate value of 0.1 PM of substrate hydrolyzed per hour per mg. of protein N. Any apparent rate of cleavage less than this we consider dubious. Since relatively dilute extract concentrations and short incubation periods were used with highly susceptible racemic substrates from which n-amino acids were hydrolyzed, no concern over oxidase action on these latter acids was taken. The quantitative carboxyl nitrogen titer in such digests confirmed the lack of appreciable oxidase action.


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therefore employed to follow the hydrolysis of this substrate (progressive decrease in absorption at 2400 A) (2) ; (Fig. 1). The hydrolysis of the saturated peptides was measured in terms of the CO2 evolved during the ninhydrin procedure of Van Slyke et al. (23).

All the determinations were corrected for extract and substrate blanks. Under the conditions used, the latter were usually quite negligible. The liberation of each amino acid from the optically active peptides and of ammonia from the dehydropeptides gives rise to 25 j.tM of COOH-N and

I I

I e

HOURS INCUBATION

FIG. 2. Hydrolysis of glycyl-DL-phenylalanylglycine at 37”. The digests con- sisted of 1 cc. of rat kidney aqueous extract equivalent to 33 mg. of fresh tissue, 1 cc. of 0.15 M borate buffer at pH 8.1, and 1 cc. of either water or 0.05 M substrate solution.

of NHI-N respectively. Thus, the complete hydrolysis of glycylglycyl- nn-alanine and of glycylglycyldehydroalanine yields 150 PM of COOH-N and 25 PM of NH,-N respectively. The procedure is illustrated by the hydrolysis of glycyl-nL-phenylalanylglycine given in Fig. 2. A maximum of 100 PM of COOK-N is liberated from 50 PM of this substrate. It may be presumed that the glycyl-L-phenylalanylglycine component of the race- mate is completely hydrolyzed, accounting for 75 PM of COOH-N, while the other 25 jcM are derived by hydrolysis of the acyl glycine from glycyl- n-phenylalanylglycine. The residual n-phenylalanylglycine is relatively resistant to enzymatic hydrolysis (see below).

Resulfs

The effect of pH on the enzymatic hydrolysis of several types of saturated and unsaturated peptides is described in Figs. 3 and 4. The pH


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200 ENZYMATIC EIYDROI,YSIS OF TRTPEPTIDES

Fro. 3:Effect of pH on hydrolysis of dehydropeptides at 37”. The digests con- sisted of 1 cc. of hog kidney aqueous extract, 2 cc. of puffer, and 1 cc. of either water or 0.025 M neutralized substrate. X, chloroacetylglycyldehydroalanine, 0, glycyldehydroalanine, 0, chloroacetyldehydroalanine. Extract concentrations were adjusted for each substrate so as to yield a linear rate of hydrolysis. The Verona1 buffer was used below pH 8.0, the glycine-NaOH buffer above pH 8.0.

4 5 6 7 8 9 IO II

PH Fm. 4. Effect of pH on hydrolysis of saturated peptides at 37”. Experimental

conditions were similar to those described in Fig. 3, except that 0.05 M neutralized substrate solutions and only 1 cc. of buffer were used. Cl, chloroacetylglyoyl-DL- alanine, 0, glycyl-nn-alanine, 0, chloroacetyl-on-alauine, A, chloroacetyf-nn- alanylglycine. The curve for chloroacetylglycine resembles that for chloroacetyl- nL-alanine.


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FODOR, ,PRICE, AND GREENSTEIN 201

activity curve for chloroacetyldehydroalanine is similar to that obtained in rat liver digests (14), and is quite different in shape from that obtained with chloroacetyl-Dr.,-alanine. The dehydropeptide is hydrolyzed to nearly the same extent from pH 6.5 t.o 9.0. The analogous saturated peptide is

TABLE I Action of Concentrated Hog Kidney Aqueous Extract on Analogous Saturated and

Unsaturated Peptides at Nearly Maximum Hydrolysis

Substrate

Glycyldehydroalanine. .................... Glycyl-L-alanine. ......................... Glycyl-D-alanine .......................... Chloroacetyldehydroalanine ............... Chloroacetyl-L-alanine. ................... Chloroacetyl-D-alanine .................... Acetyldehydroalanine ..................... Acetyl-Dbalanine ......................... Glycyldehydrophenylalanine .............. Glycyl-Dbphenylalanine. ................. Chloroacetyldehydrophenylalanine. ....... Chloroacetyl-DL-phenylalanine. ........... Acetyldehydrophenylalanine .............. Acetyl-Dbphenylalanine .................. Acetyldehydroleucine ..................... Acetyl-DL-leucine ......................... Sarcosyldehydroalanine* .................. Sarcosyl-DL-alanine* ...................... Sarcosyldehydrophenylalanine*. ........... Sarcosyl-r%-phenylalanine*. ............... Chloroacetylglycyldehydroalanine ......... Chloroacetylglycyl-L-alanine .............. Chloroacetylglycyl-D-alanine. ............. Glycylglycyldehydroalanine ............... Glycylglycyl-DL-alanine ................... Chloroacetylglycyldehydrophenylalanine . Chloroscetylglycyl-uL-phenylalanine ...... Glycylglycyldehydrophenylalanine ........ Glycylglycyl-w-phenylalunine ............ Chloroacetyldehydrophenylalanylglycine . Chloroucetyl-obphenylalanylglycine. ..... Glycyldehydrophenylaianylglycine ........ Glycyl-DL-phenylalanylglycine ............

-

:/ ./ .I I/ 1 .i

Extract hcuba- concan- tion tration perkId

tug. N Qcr c‘.

2.0 2.3 2.3 2.1 0.1 2.1 2.1 0.2 2.1 2.3 2.3 0.7 2.1 3.8 3.9 0.3 2.3 2.3 2.3 2.4 2.2 2.2 2.2 1.0 2.1 2.4 2.4 2.4 2.6 3.8 2.9 2.2 2.4

hrs.

1 24 2 50 2 50 4 25 1 25 8 0 4 24 2 25 1 25 4 loo 8 0 2 25 8 0 4 25 6 0 1 25 2 24 4 100 4 25 4 100 8 24 4 50 4 0 1 25 2 150 8 0 4 50 1 25 4 150 6 0 2 50 8 W 4 85

Peptide bonds

vailable EIydro- lyz-sd

1 1

1 1

2

2

2

2

2

2

2

2 2

4

4

4

4

4

1 1

1 0

1

2

1

1

1

2

2

2 0

4

2

4

2

2+19


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202 ENZYBUTIC HYDROLYSIS OF TRIPEPTIDES

TABLE I-Concluded

Extract bdXi- COllCCn- tion tration period

mg. N per CC.

Chloroacetylsarcosyldehydroalanine. ....... 2.4 Chloroacetylsarcosyl-nL-alanine. ........... 2.9 Glycylsarcosyldehydroalanine. ............. 2.4 nn-Alanylglycine ........................... 2.4 nn-Phenylalanylglycine .................... 2.4 Chloroacetyl-DL-alanylglycine .............. 2.9 Glycyl-on-alanylglycine .................... 2.2 Glycylglycine. ............................. 3.0 Glycylglycylglycine ........................ 3.0 Chloroacetylglycine ........................ 0.5 Chloroacetylglycylglycine .................. 2.6 Chloroacetylsarcosine ...................... 3.0 Glycylsarcosine ............................ 3.0 Glycyl-nn-leucine. ......................... 2.3 Chloroacetyl-nn-leucine. ................... 2.3

6 4 8 2 4 4 4 2 2 1 4 4 4 2 2 4 4 4 2 4 4 8

0 0

24 73.1 60.3 50

112 50 75 25 31

0 44

106 25 67.1 35.5 50

150 83

0 25

4

2 2 4 4 1 2 1 2 1 1

2 2 2 4 4 4 4 2 2

nn-Leucylglycine. . . . . . . 2.4 1-l-l Chloroacetylglycyl-DL-leucine ., 2.4 IfI Chloroacetyl-on-leucylglycine 3.8 2 Glycylglycyl-nr,-leucine . . . . 2.4 4 Glycyl-nn-leucylglycine . 3.8 2+t Chloroacetyl-N-methyl-nL-alanine 2.3 Glycyl-N-methyl-nn-alanine. . 3.8 :+I,

A plus sign indicates that the bond is seemingly very slowly hydrolyzed. * These sarcosyl peptides and dehydropeptides were hydrolyzed at a markedly

slower rate than the corresponding glycyl forms. t Not hydrolyzed either by rat kidney extracts. $ Incomplete hydrolysis of another peptide bond. 8 With rat kidney, close to 100 PM of COOH-N is liberated (Fig. 2), or equivalent

to the hydrolysis of three peptide bonds. 11 Incomplete hydrolysis of one peptide bond.

bs.

T

0

1+s I+$ 2 3+$ 1 2 1 I+#

& 2 1

-

.k

_-

Peptide bonds

vailablc Hydro- lyzed

hydrolyzed pith an optimum at about pH 7.2. Only chloroacetyldehydroalanine and chloroacetyl-DL-alanine differ in this respect, for glycyldehydroalanine, glycyl-nn-alanine, chloroacetylglycyldehydroalanine, chloroacetylglycyl-nn-alanine, and chloroacetyl-nL-alanylglycine are all hydrolyzed with an optimum rate at about pH 8.0 to 8.2.

The effect of incubating a relatively large series of saturated and unsaturated peptides under nearly optimum conditions, until all of the readily hydrolyzable bonds are cleaved, is described in Table I,


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When it is reasonably clear that saturated peptide bonds are or are not hydrolyzed, the data are given in the form of whole numbers. When one bond is seemingly very slowly hydrolyzed, a plus sign follows the number of hydrolyzable bonds described in order to indicate the presence of this relatively resistant bond. In the case of the dehydropeptides, the hydrolysis of the single dehydropeptide bond occurred either practically completely or not at all.

Rates of Hydrolysis qf Certain Peptides and Dehydropeptides-In Table II are given the values of initial reaction rates of some of the susceptible substrates with hog kidney extracts. In all cases, the rates of hydrolysis for

TABLE II Rates of Hydrolysis of Analogous Peptides and Dehydropeptides with Aqueous

Hog Kidney Extracts* .---~__ --.. _-

Substrate 1 Substrate hydrolyzed --. .~.

/.t.tl per hr. #er mg. 1%’

Chloroacetyldehydroalanine............................... Chloroacetyl-L-alanine.................................... Glycyldehydroalanine. j Gly~yl-~-~lanine..........................................i

Glycyl-~-alanine........................................../ Glycyl-N-methyl-m-alanine.. . _I

33 1lGl 237

2860 120

2 Chloroacetylglycine....................................... 133 Glycylglycine............................................. 1765 Glycylsarcosine........................................... 2 Chloroacetylglycyldehydroalanine, . . 1 Chloroacetylglycyl-L-alanine. . , 25 Glycylglycyldehydroalanine. j 210 Glycylsarcosyldehydroalanine. I 2

* All the reactions were conducted at pH 8.0, except in the cases of chloroacetyl-L- alanine and chloroacetylglycine, which were carried out at pH 7.2.

the respective compounds are considerably higher with hog kidney than with rat kidney extracts (14). Chloroacetyldehydroalanine is hydrolyzed at a rate about 30 times slower t,han chloroacetyl-L-alanine, while glycyldehydroalanine is hydrolyzed about 10 t,imes more slowly than glycyl-n- alanine and about twice as fast as glycyl-n-alanine. Glycyl-N-methyl- nn-alanine and glycylsarcosine are hydrolyzed extremely slowly; the latter compound is completely resistant to the action of intestinal mucosa under conditions whereby glycyl-L-proline is readily hydrolyzed (24). In accord with the relative resistance of the glycylsarcosine bond compared with the glycylglycine bond. the susceptibility of glpcylglycyldehydroalanine is greater than that of glycylsarcosyldehydroalanine. Chloroacetylglycylde-


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204 ENZYMATIC HYDROLYSIS OF TRIPEPTIDES

hydroalanine is hydrolyzed at a rate about 25 times slower than chloroacetylglycyl-L-alanine, a ratio which is of the same order of magnitude as that for the rates of hydrolysis for chloroacetyldehydroalanine and chloroacetylglycyl-L-alar&e.

Rough estimations of the rate of hydrolysis of the isomeric chloroacetylated dipeptides revealed that those substrates were more susceptible which possessed glycine as the terminal residue. Thus, under identical experimental conditions, with a hog kidney aqueous extract equivalent to 166 mg. of fresh tissue per cc., the following values in micrograms of COZ were obtained from digests of 50 PM of substrate after 2 hours of incubation at 37”: chloroacetylglycyl-nL-phenylalanine 234, chloroacetyl-nL-phenylalanylglycine 600 (maximum value) ; and chloroacetylglycyl-nL-alanine 224, chloroacetyl-DL-alanylglycine 505. With a more concentrated extract preparation and an 8 hour period of incubation both chloroacetylglycyl- nL-phenylalanine and chloroacetylglycyl-nL-alanine were hydrolyzed at all peptide bonds.

DISCUSSION

Study of the enzymatic susceptibility of acylated amino acids and peptides possesses a special interest in the present series of investigations. The intracellular N-acylase systems, first described under the designation “histozyme” (25) and applied almost exclusively to N-benzoylated amino acids such as hippuric acid, have received relatively little attention. In- terest in this laboratory was early directed to this topic when it was noted that the dehydropeptides, acetyldehydroalanine and chloroacetyldehydroalanine, were hydrolyzed by kidney and liver extracts, and that the activity toward these substrates could be readily separated from the activity toward glycyldehydroalanine (2).

Similar studies on analogous saturated peptides revealed the presence of enzyme systems in rat and hog kidney which acted asymmetrically upon such substrates as chloroacetyl-nL-alanine (1, 26) and permitted thereby the development of a method of preparing L- and n-amino acids in quantity and in a state of high optical purity (4).

There appear to be systems in hog kidney capable of acting upon tripeptides lacking an a-amino group on the acyl residue, such as chloroacetylglycyldehydroalanine, chloroacetylglycyl-L-alanine, or chloroacetyl-L-alanylglycine (Table I). These enzymes possess at least two apparently absolute specificities: (a) The acyl-saturated peptide bond, whether in the completely saturated tripeptide or in the tripeptide with one acyl-saturated and one terminal-unsaturated peptide bond, must be ordinarily susceptible, e.g. chloroacetylsarcosyl-L-alaninc, chloroacetylsarcosyldehydroalanine, chloroscetyl-D-phenylalsnylglycine, or chloroncctyldehydrophenylalanyl-


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glycine is not attacked; and (b) the acyl and terminal residues in the tripeptide chain must possess such a structure or configuration that, if they were united in dipeptide combination, they would be enzymatically susceptible; e.g., whereas chloroacetylglycyl-L-alanine and chloroacetylglycyldehydroalanine are hydrolyzed, chloroacetylglycyl-D-alanine and chloroacetylglycyldehydrophenylalanine are completely resistant (Table I). Thus, the susceptibility of the acylated dipeptides is governed by the relation between the acyl and intermediate residues and between the acyl and terminal residues, and is not apparently concerned with the relation between the intermediate and terminal residues.7

The susceptibility of certain of the acylated amino acids and peptides by hog kidney invites comparison with the carboxypeptidase system of the pancreas. Both carboxypeptidase and the hog kidney systems act upon peptides lacking a free cr-amino group on the acyl residue. The former acts much more readily on those substrates whose terminal amino acid residues are tyrosine, tryptophan, or phenylalanine, and much less readily when such residues are glycine, alanine, or other aliphatic amino acids (27-30). The reverse is true for the hog kidney systems (2,4). The initial attack of pancreatic carboxypeptidase on tripeptides is on the terminal bond; that for the hog kidney systems is not definitely known. The fact that hog kidney hydrolyzes chloroacetylglycyl-L-alanine but not chloroacetylsar- cosylalanine: would suggest that the primary point of attack is at the acyl peptide bond. On the other hand, the fact that the hog kidney hydrolyzes chloroacetylglycyl-L-alanine but not chloroacetylglycyl-D-alanine would suggest with equal cogency that the initial point of attack was at the terminal peptide bond. Unquestionably, the configuration about each of the bonds in these substrates is important, and the question of the initial point of attack of the hog kidney systems on the acylated dipeptides may well be left open at this time.

Both saturated and unsaturated peptides with a free cu-amino group on the acyl residue are much more susceptible to the action of kidney systems than those corresponding substrates lacking the a-amino group. In the

7 Fractionation studies on hog kidney extracts at present being actively pursued in this laboratory have suggested that the enzymes acting upon certain dipeptides and tripeptides are different entities. A hog kidney aqueous extract from which the nuclear fraction was removed by light centrifugation was subjected to 26,000 X g in a refrigerated International centrifuge for 1 hour. The activity toward chloroacetylglycine, chloroacetyl-L-alanine, chloroacetyldehydroalanine, and glycyl-r,- alanine was found very largely in the supernatant. The activity toward chloroacetylglycyl-n-alanine, chloroacetylglycyldehydroalanine, and glycyldehydroalanine was found very largely in the sediment. Similarly, fractionation studies with alcohol precipitations at very low temperatures have yielded results in the same direction. Thesestudies will form the basis of a future communication.


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206 EXZPM~~TIC IISDROLPSIY OF TRIPEPTIDES

case of the tripeptides, there is little doubt that the primary point of attack is the aminoa& peptide bond, which is the characteristic of the classic aminopeptidase systems. Thus, the order of magnitude of the hydrolysis rate of glycylsarcosyldehydroalanine is close t,o that of glycylsarcosine (Table II).

There are apparently no enzymes in hog kidney capable of acting upon acyla,ted n-amino acids, but there are enzymes capable of acting upon amino- acylat.ed n-a.mino acids as glycyl-n-alanine, etc. On the other hand, those isomeric dipeptides in which the acylamino acid is of t)he D configuration and the terminal residue is glycine, such as n-leucylglycine, etc., are hydrolyzed ext,remcly slowly if at all (31). This latter fact account,s for the failure t’o hydrolyze readily all of the bonds in such tripeptides as glycyl- n-leucylglycine, although the isomeric tripeptide, glycylglycyl-n-leucine, is hydrolyzed completely.

Three of the four peptide bonds of glycyl-nL-phenylalanylglycine are readily hydrolyzed by rat kidney extracts (Fig. 2). On the other hand, the analogous tripeptide, glycyldehydrophenylalanylglycine, is completely resistant (15).5 It would appear that none of the peptidases capable of acting upon the saturated peptide acts upon the analogous dehydropeptide. Chloroacetyl-L-phenylalanylglycine and acetyl-L-leucine are completely susceptible substrates, whereas chloroacetyldehydrophenylalanylglycine (15) (Table I) and acetyldehydroleucine (32) are completely resistant. Taken together lvith recent evidence on the. separation by high speed centrifugation of the activities toward glycyl-L-alanine, glycyl-n-alanine, and glycyldehydroalanine, respectively (33), it would appear that peptidases and dehydropept’idases were distinct and sometimes separable entities.

The authors are indebted to Mr. Robert Koegel for the nitrogen analyses.

SUMMARY

1. A considerable number of dipeptides and tripeptides, s3me of them containing all-saturated and some containing unsaturated p3ptide bon&, were incubated with hog or rat kidney aqueous extracts under conditions whereby nearly the maximum hydrolysis of the susceptible bmds were achieved.

2. Like chloroacetyldehydroalanine, chloroacetyl+alanine, chloroacetyl-

8 The lower rate of hydrolysis of the acyl bond between glycine and D-phenyl- alnnylglycinc in glycyl-o-phenylalanylglycine by hog kidney is the only example so far noted of a weaker activity in this tissue than in rat kidney. The difference may he due (a) to a natural property of the kidneys of the two species, or (b) to the fact that the hog kidney was frozen before use, whereas the rat kidney was employed fresh.


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L-phenylalanine and chloroacetyl-L-leucine, the tripeptides, chloroacetyl- glycyldehydroa,lanine, chloroacetylglycyl-L-a.lanine, chloroacetylglycyl-I>- phenylalanine, and chloroacetylglycyl-L-leucine, are completely hydrolyzed. Again, like chloroacetyldehydrophenylalanine, chloroacetyl-D-alanine, chloroacetyl-D-phenylalanine, and chloroacetyl-D-leucine, the tripeptides, chloroacetylglycyldehydrophenylalanine, chloroacetylglycyl-D-alanine, etc., are practically completely resistant to enzymatic action of hog kidney. In as much as the dipeptides themselves, such as glycyldehydrophenylalanine and glycyl-D-alanine, are readily hydrolyzed, it would appear that the susceptibility of the chloroacetylated dipeptides and dehydropeptides was governed largely by the nature of the acyl and terminal residues.

3. The pH-activity curve for chloroacetyldehydroalanine shows a broad optimum zone between pH 6.5 and 9. That for chloroacetyl-L-alanine has a relatively sharp optimum at pH 7.2. The pH-activity curves for chloroacetylglycyldehydroalanine, glycyldehydroalanine, chloroacetylglycyl-L-al- ,anine, glycyl-L-alanine, and chloroacetyl-DL-alanylglycine all show an optimum at about pH 8.0.

4. Chloroacetyl-DL-alanylglycine, chloroacetyl-DL-leucylglycine, and chloroacetyl-DL-phenylalanylglycine are half hydrolyzed, and it is presumed t,hat it is the L moieties of these racemic tripeptides which are susceptible. Chloroacet~yldehydrophenylalanylglycine is completely resistant.

5. The glycylglycylated DL-amino acids and glycylglycylated dehydro- amino acids are completely hydrolyzed, probably through the primary action of aminopeptidase on the susceptible aminoacyl bond. Glycyl- sarcosyldehydroalanine is slowly hydrolyzed, in accord with the low degree of susceptibility of the glycylsarcosine bond. The isomeric tripeptides, such as glycyl-DL-alanylglycine, glycyl-DI,-phenylalanylglycine, and glycyl- DL-leucylglycine, are hydrolyzed at three or close to three bonds, instead of the theoretical four. The reason for this presumably lies in the relative resistance of the bond between the D-amino acid and the terminal glycine residue. Such dipeptides as D-alanylglycine are hydrolyzed extremely slowly. Glycyldehydrophenylalanylglycine is completely resistant, a phe- nomenon which at once sets off the dehydropept’idase from the peptidases which act upon the analogous saturated peptides.

6. Approximate rat.e studies were made with several types of substrates. The aminoacylated substrates are hydrolyzed at a faster rut’e than the corresponding chloroacetyluted substrates, whether dipeptides or t’ripep- tides, or containing saturat,ed or unsaturated bonds. The pept’ides are in general hydrolyzed at a faster rat,e than t,he dehydropeptides, except for glycyl-D-alanine which is cleaved at a slower rate than glycyldehydroalanine. Hydrolysis of chloroacetyl-DL-phenylalanylglycine and of chloroacetyl- DL-alanylglycine was considerably fa.ster than that, respectively, of the


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208 ENZYMATIC HYDROLYSIS OF TXIPEPTIDES

isomeric substrates, chloroacetylglycyl-I&-phenylalanine and chloroacetylglycyl-nL-alanine. It is suggested that different enzyme entities act upon dipeptides and tripeptides.

BIBLIOGRAPHY

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GreensteinPaul J. Fodor, Vincent E. Price and Jesse P.

TRIPEPTIDESSATURATED AND UNSATURATED

ENZYMATIC HYDROLYSIS OF

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