A CLASSIFICATION OF PROTEOLYTIC ENZYMES

BY

MAX BERGMANN

New York, N . Y .

CONTENTS PAGE

I. Introduction ........................................................ 49

Heterospecificity .................................................. 52 111. The Activation of Proteolytic Enzymes.. .............................. 61

Bibliography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

11. Tentative Classification of Proteolytic Enzymes-Homospecificity and

IV. Coupled Reactions aa Induced by Proteolytic Enzymes.. . . . . . . . . . . . . . . . . . . 64

I. Introduction

It is a matter of general agreement that proteolytic enzymes hydrolyze peptide bonds and that this hydrolysis is a reversible process. The fact is also recognized by all authorities that every proteolytic enzyme discrimi- nates between various types of peptide bonds and that this specificity comes into play in the course of the physiological digestion of proteins. Every at- tempt to classify the proteolytic enzymes on the basis of their specificity must suffer from the fact that our knowledge of the characteristics of the specificity of proteolytic enzymes is of a rather recent date and is, a t the present time, still in a state of active development.

Emil Fischer was the first to investigate the specificity of proteolytic enzymes by their action upon several peptides (1). Table I is taken from a paper published by Fischer and Abderhalden in 1905 describing the action of pancreatic juice upon 29 synthetic substrates. These workers concluded from their experiments that the enzymatic sensi- tivity of a peptide is a function of the number, constitution, configuration and sequenc-e of the amino acid residues that are present in the peptide. Fischer expressed the hope that a closer study of the action of enzymes upon peptides might provide a means t o classify the synthetic peptides according to their biological significance. Fischer did not consider, however, the use of the various peptides for the classification of the pro-

1 Some of the material in this paper was presented on June 26, 1941, a t the Protein Research Conference held at Stanford University, California.

49

Advances in Enzymology and Related Areas of Molecular Biology, Volume 2 Edited by F. F. Nord, C. H. Werkman

Copyright © 1942 by Interscience Publishers, Inc.

Hydrolyzed

Alanylglycine Alanylalanine Alanyl-leucine A Leucylisoserine A Glycyl-Lt yrosine Leuc yl-Ltyrosine Alanylglycylglycine Leuc ylglyc ylgly cine Gly cylleucylalanine Alanylleuc ylgl ycine Dialanylcystine Dileucylcystine Tetragl yc ylgl y cine Triglycylglycine ester

Fischer’s results were later rejected by Waldschmidt-Leitz (2) and attributed to the poor and variable quality of the enzyme preparations he employed. Waldschmidt- Leitz concluded from his specificity experiments with purified preparations of proteolytic enzymes that a qualitative differentiation of the peptides of the natural-occurring amino acids on the basis of their sensitivity toward pancreatic enzymes is not possible. In his opinion, proteolytic enzymes do not distinguish between the peptides of different amino acids but rather those of different chain length. This latter specificity theory was further developed in many details by the Willstatter school of thought, especially by Grassmann (3) and Waldschmidt-Leitz and resulted in the classification of the proteolytic enzymes, presented in Table 11. This classification distinguishes between proteinases that are supposed to attack high molecular proteins only, and peptidases that attack the low molecular peptides. The group of peptidases was believed to contain a dipeptidase, an aminopeptidase and two carboxypeptidaaes, in addition to a few special enzymes such ~EI prolinase and prolidase. The peptidases were assumed to combine with a terminal a-amino group or a terminal a-carboxyl group of the substrate (4). It was thought that this combination of the enzyme with one of the aforementioned atomic groups of the substrate rendered the adjacent peptide linkage of the substrate more labile toward hydrolysis. The specificity of the various proteinases was supposed to be adapted to the anionic, cationic or zwitterionic character of the substrates. The proponents of this

Not hydrolyaed

Gly c ylalanine Glyc ylgl y cine Alanylleucine B Leucylalanine Leuc ylgl ycine Leuc ylleucine Aminobut yry lgly cine Aminobutyrylaminobutyric acid A Aminobutyrylaminobutyric acid B Amiioisovalerylgl ycine G1 yc ylphenylalanine Leuc ylproline Diglyc ylglycine Trigly cylgly cine Dileuc ylgly c y lglycine

teolytic enzymes and, as a matter of fact, left open the question of whether the pancreas gland secretes only one or several proteolytic enzymes. It may be anticipated here, that our present knowledge of the specificities of trypsin and chymotrypsin permits un to conclude that neither of these enzymes can be responsible for the enzymatic hydrolysis of peptides observed in Fischer’s experiments. On the other hand, the individual enzymes responsible for most of these splittings are not yet recognized.

TABLE I

ACTION OF PANCREATIC ENZYMES UPON SEVERAL PEPTIDES (E. Fischer and E. Abderhalden, 2. physiol. Chern., 46, 52 [1905])

CLASSIFICATION OF PROTEOLYTIC ENZYMES 51

specificity concept did not consider the possibility that proteolytic enzymes might par- ticipate actively in the physiological synthesis of proteins. It appears incomprehensible, indeed, that enzymes specifically adapted to the chain length of their substrates should be capable of organizing the intricate pattern of a genuine protein.

A. Peptidases Dipeptidase Aminopolypeptidase Tryptic carboxypolypep-

Catheptic carboxypoly- tidase

peptidnse

TABLE I1

(as generally accepted about 1935) CLASSIFICATION OF h O T E o L Y T I C ENZYMES

Dipeptides

Polypeptides

Enzyme I Substrate

B. Proteinases High moleculm

proteins and peptones

Pepsin Trypsin Papain and cathepsin

Specificity

‘VH&HR.CO-~-NH.CHR.COOH VH~CHRCO--~-NH, . . . . .

. . . . . . . } CO----NH.CHR.COOH

Cations Anions Zwitterions

Other enzymes: Prolinase, prolidase, dehydro-dipeptidase, gelatinase, yeast-trypsin.

It has always appeared to the author that the specificity concept sum- marized in Table I1 rested upon too narrow an experimental base with respect to the enzymes investigated as well as with regard to the substrates employed. The enzymes, frequently obtained by glycerin extraction of animal tissues and purified by the adsorption-elution method, in many cases represented only a fraction of the enzymatic activity of the original tissues2 and were, in general, not enzymatically homogeneous. The sub- strates studied contained almost exclusively the three monoamino acids glycine, alanine and leucine, while the majority of the 25 amino acids which are known as constituents of proteins are of greater structural complexity.

The situation with respect to the substrates was improved in 1932 when a method was devised that permitted the synthesis of optically active pep- tides of the more complicated amino acids (6). Enzymatic studies per- formed with the help of such peptides necessitated a revision of the speci- ficity concept and of the classification of proteolytic enzymes. This re- vision is a t present still under way and will be, in all probability, for some

2 Grassmann, Volmer and Windbichler have demonstrated that “dipeptidase” loses the major part of its activity toward leucylglycine when subjected to the usual adsorp- tion routine by adsorption on aluminum hydroxide C y and subsequent elution (5).

52 MAX BERGMANN

time to come. Nevertheless, it is already feasible to give a preliminary outline of the specificity requirements of several enzymes and to tenta- tively classify the proteolytic enzymes on the basis of their specificity re- quirements.

11. Tentative Classification of Proteolytic Enzymes-Homospecificity and Heterospecificity

First, it may be mentioned that the existence of an individual enzyme that is adapted to the hydrolysis of the dipeptides of the natural amino acids and of dipeptides only, has become somewhat uncertain. The ability to hydrolyze dipeptides has frequently been found to be associated with an activity toward polypeptides. Thus, crystallized carboxypeptidase splits the dipeptide 2-tyrosyl-2-tyrosine (7). Similarly, an aminopeptidase con- tained in beef spleen extracts was shown to hydrolyze 2-leucylglycine as well as 2-leucineamide (8).

Using the intestinal mucosa of higher animals, the classical source for “dipeptidase,” a rather indicative experiment may be performed (9). Crude aqueous extracts of swine intestinal mucosa are highly active toward glycylglycine, alanylglycine, leucylglycine, leucylglycylglycine, leucineamide and many other peptides. If such mucosa extract is precipitated with acetone and the precipitate fractionated with ammonium sulfate, an enzyme preparation may be obtained which no longer has any activity toward the dipeptides glycylglycine and alanylglycine, but still possesses high activity toward the dipeptide leucylglycine, the tripeptide leucylglycylglycine and leucineamide. * This demonstrates that the enzyme that hydrolyzes the dipeptide leucylglycine does not at- tack the other previously mentioned dipeptides but is active toward other leucine deriva- tives.

Apparently, the specificity of the hitherto known peptidases is not adapted to the structural differences between dipeptides and polypeptides, but is adapted to the nature of the so-called side groups, i. e., the atomic groups that represent the differences between the various a-amino acids. Consequently, one must expect to find in the future not only one but sev- eral aminopeptidases which should differ in their side group specificity and, similarly, to encounter several carboxypeptidases of distinct side group specificity.

Many of the enzymes that previously had been regarded as dipeptidases simply because they were found to hydrolyze leucylglycine or similar di- peptides will in the future be recognized as aminopeptidases or carboxy-

8 This enzyme might belong to the group of leucylpeptidwes studied by Linderstr6m- Lang (25) and by Berger and Johnson (18).

CLASSIFICATION OF PROTEOLYTIC ENZYMES 53

peptidases. I t cannot bc predicted how many enzymes, if any, will re- main to he classifird as dipeptidases. The information that is available at the present time neither proves nor disproves the existence of an enzyme or a class of cnzymrs that is specifically adapted to the exclusive hydrolysis of dipeptidrs.

The sidc group spwificity of the protrinasrs was first demonstrated for the gastro-intestinal enzymes trypsin (lo), chymotrypsin (11) and pepsin (12) and more recently also for the intracelIular enzymes or cathepsins (8). For each of these proteinases there could be found several low mo- lecular substrates containing the requisite side groups. Many of these low molecular substrates are hydrolyzed by proteinases with a speed of the same order of magnitude as that observed in the enzymatic hydrolysis of genuine proteins (13). Thus, it must be concluded that the specificity of the proteinases cannot be restricted or adapted to high molecular sub- strates.

On inspection of the revised classification of proteolytic enzymes, as presented in Table 111, it will be noticed that i t is based on two characteristics of the proteolytic specificity, namely, that each proteolytic enzyme requires the presence of certain atomic groupings within the so- called backbone and within the side chain of the substrate molecule. In order to determine for each enzyme the nature of these requisite groups, it was hitherto necessary to study the action of the enzyme upon a con- siderable number of synthetic substrates, the structures of which were systematically modified. The indispensable groups within the backbone of the substrate are italicized in Column 2 of Table 111. The requisite side groups of the substrates for each enzymatic specificity are present in a precisely defined structural relation to the sensitive peptide bond. The location of the side groups is marked by the letter R in the formulas of Column 2, and their detailed structural formulas are given in Column 3.

It is indicated in Table I11 that the substrates for the aminopeptidases contain as requisite groups in the backbone of the substrate an amino group in addition to the carbonyl of the sensitive peptide bond and in close proximity to it. The carboxypeptidases, on the other hand, require in their substrates a carboxyl group in addition to the imido group of the attacked or synthesized peptide bond. The hydrolytic action of both aminopeptidases and carboxypeptidases upon their substrates invariably leads to the production of free amino acids.

When proteinases hydrolyze or synthesize a peptide bond, they require, in proximity to their point of action, another peptide bond. Thus, sub- strates hydrolyzed by pepsin, trypsin or chymotrypsin contain a second

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56 MAX BERQMA"

peptide bond in direct proximity to the carbonyl group of the hydrolyzed peptide bond.

From what has been said before, it is obvious that both classes of en- zymes, peptidases and proteinases are concerned with the hydrolysis and synthesis of peptide bonds and that both classes of enzymes are capable of acting upon low molecular peptides. However, the class comprising the aminopeptidases and carboxypeptidases acts upon terminal peptide bonds only and, therefore, can be termed ezopeptidases (14). The other class comprising enzymes such as pepsin, trypsin, chymotrypsin and others is capable of attacking centrally located peptide bonds as well as terminal peptide bonds and has therefore been designated as endopeptidases. The terms exopeptidases and endopeptidases are in good agreement with the specific requirements of the two classes of enzymes, while the terms pep- tidases and proteinases have their origin in a specificity concept that is now recognized as erroneous.

With regard to the side group specificity, i t may be stated that pepsin and chymotrypsin require the presence in their substrates of a tyrosine or phenyl- alanine residue, i. e., a p-hydroxylbenzyl or benzyl side chain, while trypsin requires the side groups characteristic for arginine or lysine residues. In the case of trypsin and chymotrypsin, the characteristic side group is located between the requisite backbone groups; in the case of pepsin, it is located outside the requisite groups of the backbone.

Of special interest with respect to the problem of protein synthesis are the intracellular proteolytic enzymes. Therefore, in the author's laboratory there was recently begun a study of the specificities of the proteolytic en- zymes of animal tissues such as spleen and kidney. These organs were found to contain a surprisingly large number of proteolytic enzymes. An extract of beef spleen, for instance, was found to contain a t least three pro- teinases, an aminopeptidase and a carboxypeptidase. As yet, none of these enzymes has been isolated in pure form, However, the various individual enzymes contained in a mixture such as a tissue extract can be distinguished from one another and the specificity of each enzyme can be characterized by a method developed recently (15). This method involves the study of the action of the enzyme mixture upon a number of synthetic substrates, each of which is hydrolyzed by the enzyme preparation with the scission of only one peptide linkage per substrate molecule. In proteolytic experi- ments performed with such substrates, first order reaction kinetics are fre- quently obtained and the numerical values of the reaction rates are found to be a direct measure of the enzyme concentration. It is frequently ob- served that the reaction rates for the hydrolyses of two substrates are

CLASSIFICATION O F PROTEOLYTIC ENZYMES 57

affected to a different degree when the enzyme preparation under study i s subjected to purification procedures. If such is the case, it may be con- cluded that the observed hydrolyses of the two substrates are effected by two or more enzymes. By such a systematic quantitative study of the ac- tion of beef spleen extracts upon a number of synthetic substrates, it has become possible to differentiate the previously mentioned five spleen en- zymes.

One of theAe spleen enzymes-designated as beef spleen cathepsin I or beef spleen pepsinase-hydrolyzes the typical pepsin substrates. More- over, the spleen pepsinase requires the same groups in the backbone of the substrate and has the same side group specificity as pepsin. However, de- spite these similarities, the spleen pepsinase is by no means identical with pepsin. That pepsin and spleen pepsinase are two different individual en- zymes is convincingly demonstrated by the fact that even rather impure preparations of spleen pepsinase have a much greater proteolytic activity per milligram of protein nitrogen than that of crystalline pepsin. The pH optima of the two enzymes toward identical substrates are different- 5.4 and 4.0, respectively, for the hydrolysis of carbobenzoxy glutamyl ty- rosine by the spleen pepsinase and by pepsin. Beef kidney also yields a cathepsin I, i. e., a pepsinase possessing a specificity similar to that of pep- sin and of beef spleen pepsinase. We are therefore confronted with the remarkable fact that various animal organs adapted to such different physi- ological functions as are the fourth stomach, spleen and kidney of cattle, produce enzymes which exhibit an identical specificity type (16).

Other examples of enzymes possessing similar specificities are beef spleen cathepsin 11, beef kidney cathepsin I1 and swine kidney cathepsin 11. All three enzymes exhibit the same type of specificity and therefore split the same synthetic substrates as does trypsin. These enzymes may therefore be designated as beef spleen trypsinase, beef kidney trypsinase and swine kidney trypsinase. We have, at present, no method at our dis- posal to ascertain whether or not the three catheptic trypsinases are iden- tical enzymes. There can be no doubt, however, that the catheptic trypsin- ases, on the one hand, and trypsin, on the other, are different chemical entities. The spleen and kidney trypsinases, in contrast to trypsin, act upon their substrates only in the presence of an activator. Moreover, the pH optimum of the catheptic trypsinases is 4.9; that of trypsin is 7.8.

The observation that several enzymes may resemble one another with regard to side group and backbone specificity is not restricted to protein- ases. Analogous similarities were observed for the specificities of the pan- creatic carboxypeptidase and the catheptic carboxypeptidases (i. e. ,

58 MAX BERGMANN

cathepsins IV) of beef spleen, beef kidney and swine kidney; moreover, for t.he specificities of the catheptic leucine-aminopeptidases (i. e. , cathepsins 111) of spleen and kidney and of the leucine-aminopeptidase from intestinal niucosa. Thus, enzymes of undoubtedly different chemical individuality may correspond to a similar type of backbone and side group specificity and may, therefore, occupy identical places in the scheme of classification presented in Table 111. One might speak of the members of such a group of enzymes as “homospecific” enzymes in contrast to “heterospecific” enzymes, i. e., enzymes exhibiting differences of the backbone or side group specificity. For example, trypsin and beef spleen trypsinase are homo- specific, while trypsin is heterospecific when compared with chymotrypsin.

The phenomenon of homospecificity has been placed on a quantitative basis by comparing the reaction kinetics of the action of homospecific and of heterospecific enzymes upon several synthetic substrates (16). In Table IV each of the five homospecific enzymes-trypsin, beef spleen trypsinase,

Enzyme

Beef spleen trypsinase Beef kidney trypsinase Swine kidney trypsinase Trypsin Papain trypsinase

TABLE IV

HOMOSPECIFIC ENZYMES HYDROLYSIS OF BENZOYL-GARQININEAMIDE AND BENZOYLZ-LYSINEAMIDE BY SEVERAL

Tempera- ture, ’ C.

40 40 40 25 40

4.7 4 .7 4.7 7 . 4 5 . 0

Activator

8 . 3 3 . 8 8 . 7 3 . 7

27 11 42 20

167 78

Cysteine Cysteine Cysteine None Cysteine

I ~ c-x 108

Benroyl- Benzoyl- pH arginine- lysine- 1 amide I amide

C B A A CBLA

2 . 2 2 . 3 2 . 5 2 .1 2 . 1

-

The symbols used in describing the proteolytic coefficient C and the proteolytic quo- tient for the substrates benzoylarginineamide and benzoyllysineamide-CBAA/CBLA- have been defined in (15).

beef kidney trypsinase, swine kidney trypsinase and the trypsinase con- tained in papain-were tested with the two substrates benzoyl-bargi- nineamide and benzoyl-blysineamide. The experimental conditions were such that good first order reaction constants were obtained with both substrates and with each of the enzymes. These reaction constants for en- zyme concentrations of 1 mg. of protein N per ccm. of test solution are the so-called proteolytic coefficients C, reported in Table IV for the action of the five enzymes upon benzoyl-l-argineamide ( C B A A ) and upon benzoyl-dly- sineamide ( C B L ~ ) . The quotient C B A A I C B L A , as given in the last vertical

CLASSIFICATION OF PROTEOLYTIC ENZYMES 59

columri of Tahle I V , coniparw the reaction rat-es of the cnzymatic Iiydroly- sis of tlio 1,wo substrates or, in other words, states the change of the nu- niericd \ d u e of t,lw react.ion r:tt,r produced wlieri tlie argiiiirie residue of the first substrat,(. is replawd hy the lysiiie residuct i n t,he sccond sulistrate. I t will be noted that this quotient was found to bc 2.1 for trypsin, also 2.1 for papain trypsinase, and that almost identical valucs were obtained for the spleen and kidney trypsinases.

In Table V the hornospecific carboxypeptidases from beef pancreas, beef spleen, beef kidney and swine kidney are compared with respect to their action upon the substrates carbobenxoxyglycyl-Z-phenylalanine and car- bobenzoxyglycyl-Z-tyrosine. In all instances the proteolytic quotient CCGP/CCGT is found to be 1.6 to 1.8.

TABLE V

COMPARISON OF SEVERAL HOMOSPECIFIC CARBOXYPEPTIDASES

Beef spleen carboxy-

Beef kidney carboxy-

Swine kidney carlmxy-

Beef pancreas car-

peptidase

peptidase

peptidase

boxypeptidase

40

40

40

25

Cysteine

Cysteine

Cysteine

None

Beef spleen pepsinase Beef kidney pepsinase Swine kidney pepsinase

5 . 0

5 . 1

5 . 0

7 . 7

25 25 25

c x 103 2arbobensoxy-

glyryl-l- pheiiylalaninc

2 . 5

6 . 3

34

6570

Zarbobenzoxy- glycyl-l- tyrosine

1 .5

4 . 0

19

3620

‘CQP

c,,,

1 . 7

1 . 6

1 .8

1.8

In Table V1 there is reported a comparison of the action of the three pepsinases from spleen and kidney as tested with the aid of the two sub- strates carbobenzoxy-Z-glutamyl-Z-phenylalanine and carbobenxoxy-l-glu- tamyl-Z-tyrosine. The proteolytic quotients CCCP/CCGT were found to be 0.48, 0.42 and 0.50.

TABLE VI

COMPARISON OF SEVERAL PEPSINASES

I I

None Kone None

5 . 4 5 . 4 5 . 4 -

c x 103 Carbobenzoxy- Carbobenzoxy

I-glutamyl- I-glutamyl- I-phenylttlanine I-tyrosine 1 3 . 3 1 . 6

0.84 2 . 0 1 .6 3 . 2

0.48 0.42 0.50

60 MAXBERQMANN

The carboxypeptidases of Table V and the pepsinases of Table V I have the same side group specificity in that both require the side groups of a phenylalanine or a tyrosine residue. On the other hand, the two substrates discussed in Table V differ from one another only with respect to these side groups-the phenylalanine residue of one substrate is replaced by a tyro- sine residue in the other substrate and the same difference exists between the two substrates discussed in Table VI. However, while this modifica- tion of the substrates effects a retardation of the carboxypeptidme action, it accelerates the action of the pepsinases. One arrives, then, at the follow- ing conclusion : Homospecific enzymes respond similarly, heterospecific enzymes respond differently when the characteristic side group of the sub- strate is altered. Thus, the proposed classification of the proteolytic en- zymes by their specificity requirements finds support in a quantitative comparison of the reaction kinetics of the enzymes belonging to the various subgroups. Conversely, it will become possible in the future to assign to a proteolytic enzyme of unknown specificity its proper place within the system by studying the kinetics of its action upon a few well-chosen synthetic substrates and by thus determining whether this enzyme is homo- specific or heterospecific with respect to other enzymes of known specificity.

In the previously discussed examples, the homospecificity of a group of enzymes was concluded from proteolytic quotients which were in every case determined with the aid of two substrates that differed only with respect to the nature of their characteristic side groups. Thus, the two substrates benzoylarginineamide and benzoyllysineamide had been employed for the determination of proteolytic quotients of the trypsinases. The question might be asked whether a fundamentally different value of the proteolytic quotient would have resulted if the two substrates benzoylglycyl-Z-argi- nineamide and benzoylglycyl-Z-lysineamide, which also contain arginine and lysine residues as characteristic side groups, had been employed. It will be noted from Fig. 1 that the rate of trypsin action upon benzoylgly- cylarginineamide is ten times greater than that of trypsin action upon benzoylarginineamide, C B G A A / C B A A being equal to 10.0. However, a similar ratio results for the action of trypsin upon benzoylglycyllysine- amide and benzoyllysineamide, C B C L A / C B L A being 11 .O. As a consequence, the proteolytic quotient C B G A A / C B c L A is almost identical with the quo- tient C B A A / C B L A .

The above data demonstrate that both the action of a proteolytic en- zyme upon various substrates and the action of various homospecific en- zymes upon identical substrates are sometimes interrelated by quanti- tative rules of a surprisingly simple nature. In such cases it becomes pos-

CLASSIFICATION OF PROTEOLYTIC ENZYMES 61

Fig. 1.-Proteolytic Coefficients and Quotients fnr the Action of Trypsin upon Several Substrates

1-argininearnide t -- Z-arginineamide

(CBAA = 0.040) CBAA

Benzoyl- Benzoylgly cyl-

(CBOAA = 0.40) !?,,,A = 10.0

I I Benzoy I- Benzoylglyc yl-

Glysineamide 4 + I-lysineamide

(CBLA = 0.020) CBLA (CBGLA = 0.22) C B o L A = 11.0

sible to calculate the approximate rates of certain enzymatic reactions before they have been studied experimentally. If, for instance, the pro- teolytic coefficient for the hydrolysis of bcnzoylarginineamide by a prepara- tion of beef spleen trypsinase is known, then one may calculate the ap- proximate rates a t which this enzyme preparation will hydrolyze the suh- strates benzoylglycylarginineamide and benzoylglycyllysineamide. If, on the other hand, the unknown substrate benzoyl-Z-alanyl-Z-arginineamidc were synthesized and its hydrolysis by pancreatic trypsin were studied, then one could predict, on this basis, the reaction rate with which trypsin would hydrolyze the also unknown substrate benzoyl-2-alanyl-Z-lysinc- amide.

111. The Activation of Proteolytic Enzymes

The classification of the proteinases and peptidases, as suggested in Table 111, is based on the specificity of the enzymes as expressed in struc- tural requisites to be found in the substrates. It should be our goal to proceed from these symptoms of the enzymatic specificity to its origin, namely, the constitution of the various proteolytic enzymw. For example, one would like to learn which constitutional properties of the various pro- teolytic enzymes are responsible for their specificities and, in particular, for their differences in specificity, and whether there are certain constitu-

62 MAXBERGMANN

tional properties common to a group of homospecific enzymes that might account for the similarity of their specificity requirements. A t present such questions cannot be answered since our knowledge of the constitu- tional details of proteolytic enzymes is almost nil. There is only one con- stitutional property that we know to be common to many proteolytic enzymes and that has frequcntly been discussed in connection with their classification. It consists in the aforementioned fact that many pro- teolytic enzymes are active only after they have been activated by HCN or sulfhydryl compounds; others are active only when activated by metals. The activation by HCN and sulfhydryl compounds is frequently-and, in the author's opinion, erroneously-regarded as a reduction of the general type described schematically in Table VII.

TABLE VII

THE ACTIVATION OF INTRACELLULAR ENZYMES Oxidation-Reduction Theory

Oxidation

Reduction 2(Enz*SH) -- Enz s--S Ene

(Proteolytically active) (Proteolytically inactive)

On the other hand, a group of enzyme chemists (17) have advanced the hypothesis that all proteolytic enzymes have a dualistic nature in that they consist of a colloidal protein, acting as carrier, and a specific active part of unknown chemical nature. On the basis of recent experiments, it ap- pears that both theories-the activation theory and the dualistic theory- when properly modified, might be combined to include all the activatable proteolytic enzymes that are known at the present time.

It is known from the experiments of Johnson (18) and of Maschmann (19) that the activity of the intestinal enzyme that hydrolyzes Z-leucylgly- cine increases considerably on the addition of manganese or magnesium salts. This enzyme is also known to be inhibited by HCN or HgS (20). These properties may be taken as indications that the active enzyme is a dissociable metal-protein compound. Indeed, it ha,q recently been ob- served (21) that on dialysis of the active enzyme a prcparation is obtained which is completely inactive toward Z-leucylglycinc but regains a high ac- tivity on addition of manganese or magnesium salts. This reactivation is a time reaction and the degree of the final activity depends upon the con- centration of the metal ions added. Several other proteolytic enzymes of

CLASSIFICATION O F PROTEOLYTIC ENZYMES 63

the intestines and many bacterial enzymes investigated by Maschmann (19) behave similarly.

The activation of t IN. intracc~llrilar cnzyincls of plants and animals by HCN and sulfhydryl compounds is complicated by the fart that these en- zymes are almost always accompanicd by sulfur-containing compounds which are sonictimcs referred to ah natural activators. Recently papain and cathepsin preparations have been obtained that contain no natural activators. Such a purified papain preparation was found to be com- pletely inactive toward the substrate benzoylarginineamide and to remain inactive after HCN had been added (Table VIII). This is somewhat sur- prising since ordinary inactive papain preparations contain an enzyme- i t was called papain trypsinase in Table IV-which can be activated by HCN and then will hydrolyze benzoylarginineamide, benzoyllysineamide and similar substrates. The indifference of the purified papain trypsinase toward HCN is the more surprising since the HCN activation of papain is generally regarded as typical for this whole group of activation phenomena. The purified papain trypsinase which is inactive and not activated by HCN and which, for the sake of convenience, may be called papain a-trypsinase, may be activated by HzS. When the H2S is subsequently removed in vmuo, a second inactive form-papain P-trypsinase-is obtained. In con- trast to a-trypsinase, /3-trypsinase can be activated by HCN. Thus, pa- pain trypsinase exists in two inactive forms: one that cannot be activated by HCN, and one that can.

Activator, rnhl per PC.

( a ) None (b) HCN (0.02) (c) H2S (0.003)

( e ) Cysteine (0.00004) cf) (el + HCN (0.02) (a) cf) evacuated

(d ) (c) evacuated

Hydrolysis 111 substrate K (first order)

0.0000 0.0000 0.0019 0.0003 0.0000 0.0020 0.0000

Reduction H2S a-Papain t-- /%Papain - -- H2S-@-Papain trypsinase Oxidation trypsinase Evacuation trypsinase (inactive) (inactive) (active)

A

TABLE VIII

REVERSIBLE ACTIVATION O F PAPAIN TRYPSINASE Transformation of a- to 8-Trypsinase by Truces of SH-Compounds

Substrate: Benzoyl-l-itrginineamide

64 MAX BERQMANN

We assume that the above-mentioned action of HzS upon papain trypsin- ase consists of two steps, namely, first the transformation of a-trypsinasr into p-trypsinase and, second, the activation of P-trypsinase. Both steps are effected by HzS. The second step, the activation of b-trypsinase by HzS, may be reversed by evacuation. By the same procedure, the activa- tion of 8-trypsinase by HCN may be reversed. The reversibility of these activations shows that they consist in the formation of the two dissociable compounds, HCN-b-trypsinase and HzS-p-trypsinase. HCN and HzS play for papain /3-trypsinase the same role as do manganese and magnesium in the activation of many intestinal and bacterial enzymes. With respect t o the a- and /3- forms of papain trypsinase, i t seems probable that 8-trypsin- ase is the reduced form of a-trypsinase and may be reversed to a-trypsin- ase by oxidation. However, it should be understood that the reduction of a-papain to &papain is not an activation, since both forms are enzymatic- ally inactive toward benzoylarginineamide.

The activable spleen trypsinase was also found to exist in two inactive forms and to be activated through the formation of dissociable activator- @-trypsinase compounds. This appears to indicate that the HzS-activable intracellular enzymes of the higher animals follow an activation pattern similar to that discussed for the plant enzyme, papain trypsinase.

Hence, two mechanisms of activation of proteolytic enzymes are known: the activation by metals and the activation by sulfhydryl compounds or HCN. Both these activation processes have the common characteristic that the active enzyme is composed of an inactive apoenzyme and an acti- vator.

Thus far, the structure of the activated enzymes agrees with the theory of the dualistic nature of proteolytic enzymes. However, specificity studies do not support the claim that the protein part of the dualistic enzyme is merely a colloidal carrier for another and supposedly active and specific part of the enzyme. On the contrary, the protein part of the activated enzymes exclusively determines the character of the specificity of the acti- vated enzyme.

IV. Coupled Reactions as Induced by Proteolytic Enzymes

The classification of proteolytic enzymes by their specificities has its basis in experiments in which the enzymes act upon simple substrates of known structure. The fact that a substrate is attacked by an enzyme has always been taken as an unequivocal indication that the structure of the substrate falls within the specificity range of the enzyme. However, recent

CLASSIFICATION O F PROTEOLYTIC ENZYMES 65

experiments have demonstrated that such reasoning does not always con- form to the truth. Peptides which are perfectly stable in contact with a certain proteolytic enzyme are sometimes attacked by the same enzyme after a second peptide has been added to the mixture. To cite the best known example of this type of enzymatic action, glycyl-Z-leucine is not hydrolyzed by cystine-activated papain, but is hydrolyzed when acetyl- phenylalanylglycine is simultaneously present in the test solution (22). It could be shown that under these conditions the enzymatic hydrolysis of glycylleucine takes an indirect course and represents the result of three distinct enzymatic reaction steps. The first step consists in a combination of glycyl-kleucine and acetylphenylalanylglycine resulting in the formation of the acetylated tetrapeptide, acetylphenylalanyldiglycyl-l-leucine. From this tetrapeptide, first leucine and subsequently one glycine residue are split off , thus regenerating acetylphenylalanylglycine.

Acetylphenylalanylglycine + Glycylleucine

I TI J . 1 I

Acetylphenylalanylgly c ylgl ycylleucine

J.4 I AcetylphenylalanylglycylgIycine + Leucine

In this indirect hydrolysis of glycylleucine an enzyme, or rather a mix- ture of enzymes (papain, as obtained from the latex of carica papaya con- tains a considerable number of proteolytic enzymes4) attacks a substrate which is clearly outside the specificity limits of all the enzymes present. This transgression of the specificity limits is made possible through the presence of a so-called cosubstrate, that is, a second peptide (acetylphenyl- alanylglycine) that does not appear in the over-all equation of the reac- tion. The over-all reaction, the hydrolysis of glycylleucine, is the sum of three reaction steps which are coupled to one another.

Coupled reactions of the type just described are not rare exceptions but take place rather frequently when two or more peptides are in contact with proteolytic enzymes. It has repeatedly been observed (22) that peptides

Two crystalline enzyme preparations obtained from the latex of carica papaya have heen designated papain and chymopapain (23).

66 MAX BERQMANN

that are resistant to certain proteolytic enzymes are hydrolyzed by the same enzymes after hemoglobin, or casein, or blood serum has been added to the peptide-enzyme mixture.

When proteolytic enzymes have at their disposal a number of peptides, as will he the case in the course of an enzymatic hydrolysis of a protein, the possibility will frequently arise for the occurrence of a sequence of coupled reactions-synthetic as well as hydrolytic reactions-the over-all result of which might give a misleading picture of the specificity of the enzymes involved. All attempts in the past to study the specificity of proteolytic enzymes by using proteins or mixtures of protein split products as substrates have unwittingly neglected this possibility.

The course of a sequence of coupled proteolytic reactions must depend upon the specificities of the enzymes involved and upon the constitution of the peptides present. Consequently, if a given proteolytic system con- sisting of several enzymes and peptides is augmented by the addition of a foreign peptide or protein, the fate of the proteolytic system will, in all probability, be changed by the establishment of a sequence of coupled re- actions in which the foreign peptide participates.

The sequence of coupled proteolytic reactions in the various tissues of an organism can follow its normal course only when it is protected against interferences by intruding foreign peptides or proteins. The presence within the gastro-intestinal tract of a multitude of proteolytic enzymes each of which exhibits a highly developed specificity may be explained as a mechanism providing such a protection against foreign peptides containing undesirable combinations of amino acids.

It is well known that the intrusion or introduction of a foreign protein into an organism by ways other than the oral route results in a number of characteristic reactions which interest us here only in so far as they are accompanied by the production of abnormal proteins. The question arises whether some of these abnormal proteins are produced by an interaction between the foreign protein and the normal proteolytic system of the or- ganism in a manner which is comparable to the aforementioned cosub- strate action. One might consider, for instance, the possibility that the infection of a host by a virus protein might be explained as an alteration of the normal course of the coupled protcolytic and proteosynthetic reactions of the host tissues resulting, in the final outcome, in the synthetic produc- tion of more virus protein.

As a result of the latter part, of this discussion, we recognize that pro- teolytic enzymes may act in two different manners. One type of reaction is represented by the specific action of a single enzyme upon a single pep-

CLASSIFICATION O F PROTEOLYTIC ENZYMES 67

tide bond, while the other type of reaction consists in a sequence of coupled reactions involving several peptide bonds and several enzymes. If the assumption that the biological synthesis of proteins is effected by pro- teolytic enzymes6 is correct, then the second type of reartion must play a paramount role in protein synthesis. The proteins genrratcxd by living cells under normal or pathogenic conditions inu.;t bc the product of coupled reactions in which many proteolytic erizyincs and many substrates pnrtici- pate The synergy of proteolytic enzymes iii a sequence of coupled reac- tions requires a certain harmony between the specificitiw of the synergetic enzymes. A close study of the specificity requirements of the coupled reactions produced by proteolytic enzymes may help, in the future, to ap- proach a better understanding of thc biological synthesis of thc many pep- tide bonds of a genuine protein.

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68 MAX BERGMANN

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