THE RELATION OF CHEMICAL STRUCTURE TO THE RATE OF HYDROLYSIS OF PEPTIDES.

III. ENZYME HYDROLYSIS OF DIPEPTIDES AND TRIPEPTIDES.

BY P. A. LEVENE, H. S. SIMfi’IS, AND MIMOSA H. PFALTZ.

(From 2lle Labo,utories of the Rockefeller Institute for Medical Research, New York.)

(Received for publication, June 30, 1926.)

INTRODUCTIOS.

In two previous papers we have reported on the hydrolysis by acid’ and by enzyme2 (erepsin) of the following dipeptides: glycyl-glycine (GG), sarcosyl-glycine (SG), glycyl-sarcosine (GS), sarcosyl-sarcosine (SS).

It was found that each peptide simultaneously hydrolyzed and

formed anhydride at rates dependent upon the values of IC, ” -

( > Kb where I(, and Kb are the acid and basic dissociation” constants of the groups involved in the respective linkages (i.e., the strength of the peptide bond is determined by amino acid constants, while

1 Levene, P. A., Simms, H. S., and Pfaltz, M. H., J. Biol. Cllel?z., 1924, lsi, 445.

2 Levene, P. A., and Simms, H. S., J. Biol. Chem., 192G25, lxii, 711. 3 In the above mentioned papers the term “dissociat.ion” was used

synonymously with “ionization” of 1)oth acid and basic groups, and we wrote (K&) to represent the product of the corresponding constants.

We have since that time reversed our notation with regard to basic (amino) groups and define “dissociation” as the “effect of ionization of the acid groups and hydrolysis of the salts of basic (amino) groups” (see Simms, H. S., J. Am. Chenz. Sot., 1926, xlviii, 1239, 1251). By this definition

I‘, .

( > Ko 1s proportional to the product of the ionization constants, and rep-

resents the ratio of the acid and basic dissociation constants according to the new definition. This change in definition was necessary in order to deal with titration data of complex substances. We regret the confusion which it may produce.

“53

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Hydrolysis of Peptides. III

the strength of the anhydride bond is determined by the peptide constants).

The data agreed with the conclusions that erepsin attacks the neutral molecule of peptide, and that erepsin has a dissociation constant at about pK’ = 7, being active on the alkaline side.

Other dipeptides and some tripeptides have since been prepared. We desired to determine the following facts.

1. Whether these peptides agree in general with the laws which applied to the dipeptides studied before.

2. As a result of a study of the nature of dissociation constants* it seems probable that the stability of each peptide or anhydride bond should depend not upon the dissociation constants (nor the ionization constants) but rather upon the intrinsic constants of the groups involved. These should be proportional to the ioniza- tion constants in comparing the compounds studied before, but would not be proportional when we compare tripeptides with dipeptides.

3. If it is the neutral molecule which is acted upon, then asparagyl-glycine should not be hydrolyzed at all, since it is negatively charged in the range of action of erepsin.

OBSERVATIONS AND DISCUSSION.

1. The following peptides were hydrolyzed with erepsin at 32°C. : glycyl-glycine (GG) , alanyl-glycine (AG) , glycine-alanine (GA), alanyl-alanine (AA), methylalanyl-glycine (“AG), methyl- alanyl-alanine (“AA), sarcosyl-alanine (SA) , and asparagyl- glycine (ApG), also glycyl-amide (GAm) .

The results may best be observed by inspection of Fig. 1. From the dissociation constants of alanine and of alanyl-

alanine (AA) we calculate for the hydrolysis of AA, that a’ =

4 Dissociation constants represent the steps in dissociation according to the classical conception (providing we reverse our definition for the basic groups). Intrinsic constants! on the other hand, represent the

tendencies of the individual chemical groups to dissociate and are inde- pendent of the electrostatic work between the groups. Dissociation constants involve not only the intrinsic constants but also this electro- st.atic work.

5 For AA, PI<, = 3.17 and pI<c’ = 8.42 (standard, 0.100 11 HCl = pH 1.075).

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Levene, Simms, and Pfaltz 255

‘Oi I I I I I I I I 1

0.9

0.8

0.7

0.8

SW a4

a3

a2

0.1

10 20 Xl 40 50 60 VI 80

FIQ. 1. Hydrolysis curves of dipeptides with erepsin.

K HE0

II I”“l”“““” 5 10 I5 20 25

FIG. 2. Hydrolysis curves of tripeptides with erepsin.

0.33 and k = 0.85 that of GG. From the experimental data we find that a’ = 0.33 and lc = 0.4 that of GG (uncorrected for steric hindrance) or k = 0.8 (with a correction factor of 2).

The constants of AG and GA were not determined. It is obvious that they should fall between GG and AA which agrees

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256 Hydrolysis of Peptides. III

with the hydrolysis experiments. ‘AG has a curve slightly below AG as would be expected, and SA is about the same as AA, as we would also expect.

Therefore, these compounds agree with the laws which applied to the peptides previously studied. The behavior of asparagyl- glycine (ApG) will be discussed below.

2. The curves of the following tripeptides, glycyl-glycyl-glycine (GGG), alanyl-glycyl-glycine (AGG), and methylalanyl-glycyl- glycine (“AGG), are plotted in Fig. 2, while sarcosyl-glycyl- glycine (SGG) was too insoluble to be studied.

If we calculate the strength of the bonds of GGG from the

dissociation constants we find, on the basis of KlZ

( > F that it

b

should split 43 times as fast as GG (one bond 37 times as fast and

the other 6 times). On the basis of Ka 2

( > F we find that GGG

b

should split 1500 times as fast as GG. The observed rate of hydrolysis is 9 times as fast as GG. (This value would be a little higher if corrected for the effect of pH upon the enzyme activity.)

We are led to conclude that the stability of each peptide bond is a function of the in!rinsic constants of the groups involved in the linkage. This seems more logical since the intrinsic constants represent the afhnity of the groups for electrons while the dissocia- tion constants involve, in addition, electrostatic work betwen groups as a result of ionization. We cannot determine the intrinsic constants but we predict that pKO* (for the acid groups) in GG is slightly higher than in glycine while pKEB (for the basic groups) is slightly lower in GG than in glycine. If we could determine these constants, we would probably find that the

stability of each bond is proportional to K,A

( > 7 KO

.

This is of significance in the hydrolysis of proteins. A chain of similar amino acids should have equal probability of hydrolysis (with acid) at all peptide bonds (except the two end ones which should be more stable). It should, therefore, be impossible to systematically degrade protein by partial hydrolysis. A chain of dissimilar amino acids would, however, favor hydrolysis more at some points than at others.

It will also be observed that the ratios of the rates of hydrolysis

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Levene, Simms, and Pfaltz 257

of AGG to AG and of “AGG to “AG are roughly the same as GGG to GG.

It is to be expected that the higher simple6 peptides should have about the same tendencies to hydrolyze as the tripeptides since their intrinsic constants should be practically identical. The actual rates would depend upon the pH of the solutions. Higher peptides have lower pKZ’ values, hence their solutions would be less favorable to the action of erepsin.

3. The assumption that the neutral molecule of peptide is attacked by erepsin agrees with the experiments of Dernby and our earlier observations. If this is correct, there should be no hydrolysis of asparagyl-glycine (ApG) since this substance is negatively charged throughout the whole range of action of erepsin.’

Our experiments showed that this is true. A solution of as- paragyl-glycine with erepsin showed a constant titration value over a period of 5 days. That this was not due to simultaneous splitting and anhydride formation was shown by the fact that the final solution showed no change in titration value after standing 24 hours with an equivalent of NaOH. If anhydride had been present it would have hydrolyzed.

This therefore supports the view that erepsin cannot act upon peptide bonds in the proximity of negatively charged (anion) groups. In order to determine whether erepsin can act upon cations we prepared some glycyl-amide. This was found to hydrolyze slowly with erepsin (while a blank without enzyme showed no change).

6 By “simple” peptides we mean those composed only of divalent amino acids and which, therefore, have only two ionizable groups: an amino group on one end of the chain and a carboxyl group on the other.

7 If, as appears to be the case, erepsin is an acid which has a pK at about 7, and is active only in the ionized state (above pH 7) and is active upon amide linkings of neutral or positively charged molecules (as,our experi- ments show) and not upon negatively charged molecules, it can never attack ApG since this substance has a single negative charge above pH 3.53 and two negative charges above 9.07.

This agrees with the observation in another laboratory that glycyl aspartic acid (GAP) does not hydrolyze with erepsin. GAp should have nearly the same pG values as ApG.

The apparent value of erepsin of pK = 7 is interesting since the only ordinary acid group which ionizes at this point is a secondary phosphoric group. This would suggest that erepsin is a monophosphoric ester.

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258 Hydrolysis of Peptides. III

It has already been shown that erepsin acts upon the neutral molecule of the simple (divalent) dipeptides. We have now shown that it cannot act upon a negatively charged peptide but will hydrolyze a positively charged amide. It would appear that erepsin is itself negatively charged (above pH 7) and is repelled by negative groups but may attack peptide bonds in neutral or positively charged molecules.

EXPERIMENTAL.

The erepsin solution* was prepared as described in the second paper. The method of hydrolysis was the same as used before except that 2 cc. of enzyme solution were used (instead of 5 cc.) and the temperature was 31°C. (instead of 40°C.) for the dipep- tides, except “AA and GAm, while the tripeptides were hydrolyzed at 25°C.

In each experiment GG was also hydrolyzed in order to permit comparison between the data of experiments performed under differing conditions. The results are given in Tables I to III.

Preparation of Materials.

Preparation of Methylalanyl-Glycine.-5 gm. of bromopropionyl-glycine were allowed to stand 2 days at room temperature with 15 cc. of 31 per cent aqueous methylamine and then concentrated under diminished pressure until crystallization began. About 50 cc. of absolute alcohol were added, and the syrup heated on the water bath until crystallization was complete. The yield was 3 gm. The material was purified by dissolving it in about 5 cc. of hot water and adding an equal volume of hot absolute alcohol. A crystalline precipitate was immediately formed. A second crop was ob- tained by adding more absolute alcohol to the filtrate. It melted at 237°C. with decomposition. For analysis it was dried at 100°C. over sulfuric acid under diminished pressure.

0.1000 gm. substance required (Kjeldahl) 12.50 cc. 0.1 M HCl. Ca&O&z. Calculated. N 17.50.

Found. “ 17.50. Preparation of Sarcosyl-Alanine.- gm. of chloroacetyl-alanine were

allowed to stand 24 hours at room temperature with 100 cc. of 31 per cent aqueous methylamine and then concentrated under diminished pressure to a thick syrup. The syrup was heated on the water bath with a large

8 We are indebted to Dr. Kellogg and Prof. Boldyreff of the Battle Creek Sanitarium for the intestinal juice which they generously furnished for the present use.

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Levene, Simms, and Pfaltz

excess of absolute alcohol until crystallization took place. The yield was 16 gm. The material was recrystallized by dissolving it in an equal weight of hot water and boiling with a large excess of absolute alcohol until crystal- lization began. On cooling overnight a precipitate of 12 gm. of the dipep- tide was obtained. It melted at 171-172°C. with decomposition. For analysis it was dried at 100°C. over sulfuric acid, under diminished pressure.

0.1000 gm. substance required (Kjeldahl) 12.62 cc. 0.1 M HCI. CSH~~O~N~. Calculated. N 17.50.

Found. “ 17.67. Preparation of MethyZaZan?/Z-AZccnine.-31.5 gm. of bromopropionyl-

alanine were allowed to stand 24 hours with 100 cc. of 31 per cent aqueous methylamine and then concentrated under diminished pressure to a thick syrup. The syrup was heated on the water bath with about 250 cc. of hot absolute alcohol until crystallization took place. The yield was 16 gm. The material was purified by dissolving it in about 50 cc. of hot water and adding five parts of hot absolute alcohol. 14.5 gm. of recrystallized ma- terial were obtained. It melted at 235°C. with decomposition. For analysis it was dried at 100°C. over sulfuric acid, under diminished pressure.

0.1000 pm. substance (Kjeldahl) 11.50 cc. 0.1 M HCl. CrHi40aNi. Calculated. N 16.10.

Found. “ 16.08. Preparation of Xarcosyl-GZycyZ-GZycine.-15 gm. of chloroacetyl-glycyl-

glycine were allowed to stand 2 days at room temperature with 45 cc. of 31 per cent aqueous methylamine and then concentrated under diminished pressure to a thick syrup. The syrup was heated on the water bath with about 150 cc. of hot absolute alcohol until crystallization took place. The material was purified by dissolving it in five parts of hot water and adding an equal volume of hot absolute alcohol. 9 gm. of recrystallized material were obtained. It melted at 250-253” with decomposition. For analysis it was dried at 100°C. over sulfuric acid, under diminished pressure.

0.1000 gm. substance required (Kjeldahl) 14.70 cc. 0.1 M HCl. C’IH~~O~NS. Calculated. N 20.69.

Found. “ 20.58. Preparation of MethyZuZunyZ-GZycyZ-GZycine.-26.7 gm. of bromopro-

pionyl-glycyl-glycine (0.1 mol) were allowed to stand 24 hours at room temperature with 82 cc. of 31 per cent aqueous methylamine and then concentrated under diminished pressure to a thick syrup. The syrup was heated on the water bath with about 250 cc. of absolute alcohol until crystallization took place. The yield was 18 gm. The material was pur- ified by dissolving it in 50 cc. of hot water and adding about five parts of hot absolute alcohol. To obtain a good analysis a second recrystalliza- tion was necessary. 12.7 gm. of pure material were obtained. It melted at 252-253” with decomposition. For analysis it was dried at 100°C. over sulfuric acid, under diminished pressure.

0.1000 gm. substance required (Kjeldahl) 13.90 cc. 0.1 M HCl. CsHi604N3. Calculated. N 19.35.

Found. IL 19.46.

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i?ydr

olysis

of

Di

pept

ides

With

Er

epsin

at

3F

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Each

so

lutio

n wa

s 1.

0 m

olar

and

cont

ained

2

cc.

of

enzy

me

solu

tion

(per

10

CC

.).

- -7

SA

“AG

AP

G

Lli

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GA

AA

A =O

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A =

0.33

hrs.

0 0.25

1 2 4 7

24

29

30

48

72

cc.

0.81

0.

83

0.87

0.

90

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cc.

0.02

0.

06

0.09

3.

0 2.

3

1.19

0.

38

1.4

1.52

0.

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2.8

1.54

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2.5

1.63

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1.71

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1.4

Aver

age.

. .

. . .

. 2.

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V cc.

0.62

0.

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0.62

0.

62

0.63

0.

62

0.63

0.

63

100

k V

AV

cc.

0.86

0.

88

0.92

0.

94

cc.

1.3

1.3

0.7

1.0

0.6

0.02

0.

06

0.08

0.96

0.

10

1.04

0.

18

1.05

0.

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0.5

0.5

0.7 0.9

1.10

0.

24

- V cc

.

0.81

0.

83

0.88

0.

94

1.04

1.

19

1.30

1.31

1.

30

V AV

.

cc.

0.75

0.

77

0.83

0.

86

0.88

0.

94

1.19

cc.

0.02

0.

08

0.11

0.

13

0.19

0.

44

V AV

--

cc.

cc.

0.79

(0

.76)

0.80

0.

01

0.81

0.

02

0.81

0.

02

0.84

0.

05

0.88

0.

09

1.29

0.

54

0.89

0.

10

1.40

0.

65

0.94

0.

15

1.50

0.

75

1.02

0.

23

V AV

--

cc.

cc.

0.82

0.

83

0.01

0.

85

0.03

0.

87

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1.01

0.

19

1.20

0.

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1.21

0.

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1.24

0.

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1.25

0.

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cc.

0.02

0.

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0.13

0.

23

0.38

0.

49

0.50

0.

49

-

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Time.

t 1.5 t --

hrs. hrs.

0 0 0.25 0.37 1 1.5 2 3 4 6 7 10.5

24 36 48 72

I.

Levene, Simms, and Pfaltz 261

TABLE Il.

Hydrolysis of “AA an.d Glycyl Amide at &“C.

With 2 CD. of ensyme solution. No enzyme.

GG “AA

V AV V AV -- --

cc. cc. cc. CC.

0.81 0.63 0.83 0.02 0.625 0.005 0.88 0.07 0.65 0.02 0.95 0.14 0.67 0.04 1.08 0.27 0.70 0.07 1.25 0.44 0.75 0.12 1.62 O.Sl 0.90 0.27 1.74 0.93 1.00 0.37

GAm GAm

V AV V AV ---

cc. cc. cc. cc.

0.79 0.77 0.79 0.77 0

(0.83) 0.77 0 0.81 0.02 0.77 0 0.82 0.03 0.76 -0.01 0.83 0.04 0.77 0 0.93 0.14 0.77 0 1.04 0.25 0.77 0

Owing to the faster reaction rate resulting from higher temperature, the values of t are multiplied by 1.5 in order that the data of “AA and GAm may be plotted in Fig. 1 and compared with the data in Table I.

TABLE III.

Hydrolysis of Tripeptides With Erepsin at 35°C.

Each solution was 0.75 molar with 2 cc. enzyme solution (per 10 cc.). Due to insolubility GGG was diluted to 15 cc.

GG GGG

Time. c = 0.75 c = 0.50

A = 0.80 A = 0.80 -I- I

t 1 V 1 A+$/lOOk/ VIAV/+OOk

---.-__ -- hrs. cc. cc. cc. cc.

0 0.60 0.40 0.25 0.62 0.02 0.03 6.4 0.50 0.500.100.20 31 1.0 0.66 0.06 0.08 4.6 0.550.150.30 20 2.0 0.70 0.10 0.13 3.9 0.680.280.56 26 4.0 0.79 0.19 0.25 4.1 0.840.440.88 7.0 0.83 0.25 0.31 4.4 0.950.551.10 2.4 1.15 0.55 0.73 4.4 1.15,0.751.50

Average.. . . . 4.3 26

-

( ( ( ( ( ( ( 1

.

AGG “AGG

e = 0.75 e = 0.75

,Io.I-M-I- cc.

I.601 1 IO.601 1 ).650.050.070.700.100.13 ).680.080.110.720.120.16 ).700.100.130.730.130.17 ).760.160.210.740.140.19 ).900.300.400.820.220.29 ).980.380.510:870.270.36 1.220.620.831.060.460.61

For comparison with GG the velocity constant for GGG should be multi- plied by 1.5 (i.e., 26 X 1.5 = 39) since the concentration of enzyme was less, due to dilution.

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262 Hydrolysis of Peptides. III

Preparation of Bromopropionyl-Sarcosine.-21.6 gm. of sarcosine were dissolved in 240 cc. of sodium hydroxide (1 equivalent) and treated alter- nately with 50 gm. of bromopropionyl chloride (1.2 equivalents) and 396 cc. of normal sodium hydroxide, under cooling, each reagent being added in ten equal portions. The solution was then neutralized with 72 cc. of 5 normal hydrochloric acid and concentrated to dryness under diminished pressure. The residue was extracted several times with warm ether. The combined ether extracts (total 400 cc.) were dried over sodium sulfate and concentrated to a thick syrup. The syrup was stirred up with a little benzene. On cooling overnight under diminished pressure the syrup crystallized. It was filtered and washed with benzene. A yield of 18 to 27 gm. was obtained. A second crop was obtained by treating the filtrate with an excess of petrolic ether. The substance is very soluble in water and in alcohol, less soluble in chloroform, in ether, and in benzene, and insoluble in petrolic ether. It melted at 84°C. For analysis it was dried over sulfuric acid, under diminished pressure.

0.2000 gm. substance required (Kjeldahl) 8.80 cc. 0.1 M HCl. CsHloOINBr. Calculated. N 6.25.

Found. ‘( 6.16. Preparation of Methylalanyl-Sarcosine Anhydride.- gm. of bromo-

propionyl-sarcosine were allowed to stand 24 hours at room temperature with 80 cc. of 25 per cent aqueous methylamine and then concentrated under diminished pressure to a thick syrup. The syrup was extracted several times with hot benzene (total 300 cc.). The combined benzene extracts were concentrated under diminished pressure to a small volume and were then taken up with an excess of absolute ether. On cooling and stirring the methylalanyl-sarcosine anhydride crystallized. The yield was 5 gm. The substance is soluble in alcohol, in acetone, in chloroform, and in benzene and insoluble in ether and in petrolic ether. It gave no color reaction on boiling with fresh copper hydroxide. It melted at 7% 80°C. For analysis it was dried over sulfuric acid, under diminished pressure.

0.1000 gm. substance required (Kjeldahl) 12.77 co. 0.1 M HCI. C;Hr20:N. Calculated. N 17.94.

Found. “ 17.88. Preparation of Bromoisovaleryl-Sarcosine.--18 gm. of sarcosine were

dissolved in 200 cc. of normal sodium hydroxide (1 equivalent) and treated alternately with 50 gm. of bromoisovaleryl chloride (1.2 equivalents) and 330 cc. of normal sodium hydroxide under cooling, each reagent being added in ten equal parts. The solution was then neutralized with 60 cc. of 5 nor- mal hydrochloric acid. The bromoisovaleryl-sarcosine immediately precipitated as an oil which crystallized on cooling and stirring. The yield was 39 gm. The substance is soluble in ether, in chloroform, in alco- hol, in acetone, and in benzene and insoluble in petrolic ether. It melted at 7677°C. It was purified by dissolving it in a minimum amount of warm ether, and adding a large excess of petrolic ether.

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Levene, Simms, and Pfaltz 263

0.2000 gm. substance required (Kjeldahl) 7.75 cc. 0.1 M HCl. CaHrd03NBr. Calculated. N 5.56.

Found. ‘I 5.56. Preparatz’on of Methylvaleryl-Sarcosine Anhydride.- gm. of bromo-

isovaleryl-sarcosine were allowed to stand 6 days at room temperature with 35 cc. of 25 per cent aqueous methylamine and then concentrated under diminished pressure to a thick syrup. The syrup was extracted several times with hot benzene. The combined benzene extracts (total 250 cc.) were concentrated under diminished pressure to a small volume and were then taken up in an excess of absolute ether. On cooling and stirring the methylvaleryl-sarcosine anhydride crystallized. The yield was 4 gm. The substance is soluble in alcohol, in benzene, in chloroform, and in acetone and insoluble in ether and in petrolic ether. It gave no color reaction on boiling with fresh copper hydroxide. It melted at 95°C. For analysis it was dried over sulfuric acid, under diminished pressure.

0.1000 gm. substance required (Kjeldahl) 10.68 cc. 0.1 M HCl. CgHlsOzNt. Calculated. N 15.20.

Found “ 14 95.

SUMMARY.

Continuing our study of the hydrolysis of peptides with erepsin, we find that seven other dipeptides give results which agree with our previous conclusions. They hydrolyze and form anhydrides

K 2 at rates dependent upon the values of -Z

( > Kb involving the

dissociation constants of the groups involved in the respective linkages. (Due to change in notation Kb is proportional to the reciprocal of Kb as used in previous papers in this series.)

These results were corroborated quantitatively in the case of alanyl-alanine (AA) and qualitatively for the other peptides. Methyl groups on the carbon atom (as in alanine) have the same effect as on the nitrogen (as in sarcosine) but to a smaller extent.

The values of the dissociation constants (K, and Kb) should be nearly proportional to the intrinsic constants (K,” and KOB) of the respective groups, if we compare dipeptides with each other. However, in comparing dipeptides with tripeptides there should be considerable difference. We have hydrolyzed three tripep- tides and find that the rates of hydrolysis are such as prove that the stabilities of the bonds are functions of the intrinsic constants

K,A

f (3 KoB rather than of the dissociation constants. This is

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264 Hydrolysis of Peptides. III

theoretically to be expected. Tripeptides hydrolyze about ten times as fast as similar dipeptides. Higher peptides should be about the same as tripeptides, if we correct for ionization and change in enzyme activity with pH. A long chain of similar amino acids should be split by acid equally well at all the amide bonds (except the end two).

It is shown that erepsin fails to attack an anion (asparagyl- glycine, ApG, is not hydrolyzed), while neutral molecules (all simple peptides below pGz) and also cations (glycyl-amide) are hydrolyzed by erepsin above pH 7.

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PfaltzP. A. Levene, H. S. Simms and Mimosa H.

DIPEPTIDES AND TRIPEPTIDESENZYME HYDROLYSIS OF

HYDROLYSIS OF PEPTIDES: III.STRUCTURE TO THE RATE OF THE RELATION OF CHEMICAL

1926, 70:253-264.J. Biol. Chem. 

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