The Sulfhydryl Proteases - Advances in Chemistry (ACS Publications)

8 The Sulfhydryl Proteases IRVIN E . L I E N E R

Downloaded by CORNELL UNIV on October 21, 2016 | http://pubs.acs.org Publication Date: October 1, 1974 | doi: 10.1021/ba-1974-0136.ch008

Department of Biochemistry, University of Minnesota, St. Paul, Minn. 55101

The sulfhydryl proteases—papain, ficin, and bromelain— all have a sulfhydryl group at their active site and are thus readily inactivated by reagents or conditions which modify this functional group. These enzymes are used extensively in the food industry for the tenderization of meat and chillproofing of beer, have application in the tanning and textile industries, and are used medicinally as digestive aids and debriding agents. The enzymatic and physicochemical properties and structural features of these enzymes are compared with particular emphasis on the relationship of structure to mechanism of action.

Among the proteolytic enzymes, the plant proteases are the most widely used in the food industry. Most of the plant proteases which have been studied are characterized by a free sulfhydryl group which is essential for their activity. The most important of these so-called sulfhydryl or thiol proteases are papain, ficin, and bromelain. Since the literature on these enzymes has been the subject of several recent reviews (J, 2, 3, 4), major emphasis is placed in this presentation on the use of these enzymes in the food industry. Some of the more recent developments relating to the structure and function of the sulfhydryl proteases are discussed. Commercial Preparations Papain is the main protein constituent of latex of the green fruit, leaves, and trunk of Carica papaya, a small softwood tree which is native to most tropical countries. Although the protein-digesting property of papain was first recorded in 1873 (5), the native custom of tenderizing meats by wrapping them in leaves of the papaya tree prior to cooking dates back centuries. Commercial papain is collected from full-grown but still unripe fruits by making shallow, longitudinal scratches in the fruit and allowing the collected drippings to coagulate. The coagulated 202 Whitaker; Food Related Enzymes Advances in Chemistry; American Chemical Society: Washington, DC, 1974.


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latex is dried in the sun or i n heated chambers to reduce the moisture content to 5 - 8 % . Since F D A regulations do not permit the use of crude papain in food products, most commercial preparations have been further purified by precipitation with an organic solvent such as acetone. A n excellent monograph on the commercial preparation of papain was pub lished in 1963 by the Central Food Technological Research Institute, Mysore, India (6). Proteolytic activity in the juice of the pineapple plant (Ananas comosus) was first reported in 1879 (7). More recently, the juice from the stem of the pineapple plant was shown to be a rich source of stem bromelain. This name is used to distinguish the enzyme from another which is derived from the fruit (8, 9). Mature pineapple stems are col lected by special harvesting machines. The juice is pressed by special mills and then filtered. Most commercial preparations of bromelain have been precipitated from the stem juice by acetone. Although ficin has been commercially available for many years, it has not attained the importance of papain and bromelain. F i c i n is i m ported mainly from South America where it is obtained from the latex of tropical fig trees of the genus Ficus. There are over 2000 species of this genus, and the amount of activity varies considerably from one species to another (10). Most of the commercial preparations of ficin are derived from F . glabrata. The latex is collected from the trees by tapping, and the juice separated after the latex has coagulated. The juice is then spray-dried, or the protein may be precipitated with acetone. Food, Industrial, and Medicinal Uses Since several excellent books and monographs are available on this subject (11, 12, 13, 14, 15), only a brief survey is presented here. The principal commercial uses of the sulfhydryl proteases are summarized i n Table I. Although papain is used mainly for these purposes, ficin and bromelain also have been used with essentially the same results. Chill-Proofing Beverages. Papain is used mainly in the beverage industry for stabilizing and chill-proofing beer. W h e n beer is made, some of the protein which is soluble at room temperature is apt to pre cipitate on chilling, causing a cloudy product. Because papain digests proteins in a slightly acid environment ( p H 4.5), it prevents the separation of the protein in the cold beer. About 1 gram of commercial papain is added per barrel (8 ppm) prior to pasteurization at 60°C. This tempera ture is not severe enough to denature the enzyme to any appreciable extent. Meat Tenderizing. This is the second largest use of papain. About one-third of all the papain sold in this country is used by the housewife

Whitaker; Food Related Enzymes Advances in Chemistry; American Chemical Society: Washington, DC, 1974.

204

FOOD R E L A T E D

Table I.

ENZYMES

Commercial Uses of Sulfhydryl Proteases

In Food Industry 1. Chill-proofing beverages 2. Meat tenderizing 3. Other: Milk-clotting agent, dough ingredient, protein hydrolysates


Industrial Uses 1. Leather industry 2. Textile industry 3. Cleaning agent Medicinal Uses 1. Digestive aid 2. Prevention of post-operative adhesions 3. Debridement for tenderizing small cuts of meat. The product used for this purpose contains in addition to papain ( 2 % ) , varying amounts of salt, glucose, monosodium glutamate, flavoring agents, and spices. The powder is applied to meat before it is cooked and is forced into the tissues by stabbing with a fork. The meat is then cooked in the usual way. Muscle protein and connective tissue, primarily collagen and elastin, are digested to the point where a definite softening of the flesh is observed. Papain is relatively resistant to heat, and most of the proteolysis takes place during the early stages of cooking, the greatest breakdown of tissue occurring at 70 °C. When the enzyme concentration is too high or the period of treatment too long, overtenderization and mushiness w i l l result. Unlike the housewife, the meat packer is faced with the problem of applying the enzyme uniformly throughout very large pieces of meat. One effective way of doing this is to inject the enzyme into the animal prior to slaughter. It w i l l be carried by the vascular system into all parts of the body and exert its tenderizing action after the animal is dead. The recommended dosage for antemortem injection is approximately 5 ppm of commercial papain based on the live weight of the animal. A n enzyme solution may also be injected into freshly slaughtered carcasses, placing the needles so that there is intramuscular distribution of the enzyme. Other techniques for tenderizing meat with papain which have been patented since 1960 are described by Wieland (13). The extent to which ficin and bromelain are used in place of papain is determined largely by their availability and other economic factors. Other Food Uses. Papain has also been used to hydrolyze Tenderers meat scraps to make a product which can be used for feeding farm animals. Because of their ability to coagulate casein in much the same fashion as rennet, papain and ficin have been used as milk-clotting agents in place of rennet in the production of cheese. Papain may be substituted



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for fungal enzymes as a dough ingredient. It serves to improve quality by lowering the viscosity of the dough. This effect results from its ability to degrade and depolymerize the gluten of the flour. In actual commer cial practice, however, the thiol proteases are little used for bread making or milk clotting, at least in the United States. Industrial Uses. Papain is used in the leather industry to pre pare the sides for tanning. Its proteolytic action removes some of the undesirable proteins which adhere to the hide and thus facilitates the subsequent tanning process. In the textile industry, the treatment of wool fibers with papain has been found to reduce the shrinkage from launder ing. This appears to be caused by the ability of the enzyme to destroy the elastic properties of wool protein. Because of its digestive action on protein, papain is used as a spot remover in the laundry and dry cleaning business. Medicinal Uses. One of the earliest medicinal applications of the thiol proteases was the use of fig latex as an anthelmintic agent (16, 17). In more recent times papain has been used as a digestive aid i n the treatment of dyspepsia and gastric distress. Its usefulness in this respect derives from the fact that papain remains active under acid conditions and is resistant to attack by pepsin. The intraperitoneal injection of sterile solutions of papain has proved to be effective in preventing post-operative adhesions. Papain is sometimes referred to as a "bio logical scalpel" because of its specific proteolytic action on dead tissue without affecting live tissue. For this reason it serves as a very useful debridement in the treatment of burns and the removal of scar tissue. When used in combination with antibiotics, it has proved to be effective for topical treatment of ulcerating and infected lesions. Other Uses. The thiol proteases have proved to be extremely useful tools in studying protein structure (18). Their utility in this respect has been considerably enhanced by the preparation of insolubilized derivatives (see, for example, 19, 20, 21). Purification Papain. Papain was first isolated in crystalline form from fresh latex by Balls et al. (22), but it is more conveniently isolated from commer cially available dried latex by the procedure of K i m m e l and Smith (23). Papain prepared by this procedure, however, is not fully active (see "Activation and Inhibition" below) and usually contains only 0.5 mole S H per mole of protein. Fully active papain containing 1 mole S H per mole of protein may be prepared by affinity chromatography on a column of an inhibitor, G l y - G l y - T y r ( B z l ) - A r g , covalently linked to agarose (24). Two other techniques which have been used to purify papain involve


206

FOOD R E L A T E D E N Z Y M E S

affinity chromatography on columns of agarose to which p-aminophenylmercuriacetate (25) or glutathione-2-pyridyl disulfide (26) have been attached. The reactions involved i n these techniques are depicted in Figure 1. I. INHIBITOR

COLUMN

( Blumberg et al, 1970 )

Λ

ρΗ3

Υ

1

^j-(Gly) -Tyr-Arg + Ρ - S H

•

2

"Ά

A

Η 0

V

2

%-(Gly) -Tyr-Arg— 2

P - S H ^

(Gly) -Tyr-Arg + P-SH 2


Z. MERCURIAL COLUMN ( Sluyterman and Wijdenes , 1970 )

-NH-^

W

\ " H g + P-SH

—U-m-f~\-HQ-SP

+

3. DISULFIDE-CONTAINING

COLUMN

||-G-S-S-/~~\ + P-SH

Figure 1.

>

HgClp

NH-^

>HgCI + P-S-Hg-CI

( Brocklehurst et al, 1973) ii-G-S-S-P-^*

i "

É~

6

_

S

H

+

R

- -Ss

R

+

P

"

S

H

Purification of papain by affinity chromatography

In addition to papain, at least two other proteolytic components have been shown to be present in crude extracts of commercial papaya latex, namely, chymopapain (1,27) and papaya peptidase A (28). Since these isolated enzymes are not used commercially, they w i l l not be con sidered further. Ficin. This enzyme was first isolated i n crystalline form by W a l t i (29) although subsequent studies have shown this preparation to be heterogeneous (30). Since several active components are generally ob served when crude preparations of ficin are chromatographed (31-36), the designation of any one active component as "ficin" has been quite arbitrary. The possibility has been excluded that these multiple forms of ficin could have arisen by autodigestion from a common precursor or as artifacts of the purification procedure (32). A representative result from an attempt to purify ficin is shown in Figure 2. Despite the differ ences i n techniques which were used i n these experiments (see Table I of Ref. 3 ) , it is evident that there are at least three major proteolytic components present i n most preparations generally referred to as "ficin." Bromelain. Although several investigators have reported the prepa ration of chromatographically pure stem bromelain (37, 38), there is some


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uncertainty as to whether these preparations are really homogeneous since other workers have observed several proteolytically active com ponents to be present (39,40, 41, 42). It is still not clear how much of this heterogeneity is caused by autodigestion (43). Bobb (44) recently de scribed the purification of stem bromelain by affinity chromatography on c-aminocaproyl-D-tryptophan methyl ester coupled to Sepharose. A n apparently homogeneous preparation of fruit bromelain can be prepared by acetone precipitation of the protein from the juice of the green or ripe fresh fruit followed by chromatography on D E A E - c e l l u l o s e (38).

IOOO

2000

Elution volume (ml)

Figure 2. Chromatography of preparations of ficin on CM-cellulose as performed in several laboratories. A: (31); B: (30); C. (55). Solid line denotes protein as measured by absorbance at 280 nm. Dotted line denotes activity on benzoyl-Oiu-arginine-^-nitroanilide in A , casein in B, and radioactivity of C-14 carboxymethyl group in C.

Vhysicochemical Properties A comparison of the major physicochemical properties of the sulf hydryl proteases is shown i n Table II. Generally speaking there do not appear to be any major differences in the size and charge distribution among the various enzymes. The data pertaining to bromelain and ficin, however, must be accepted with the reservation that the preparations used for obtaining these data may not have been homogeneous and that differences may exist with respect to the particular components which various investigators may have used. Nevertheless, despite their distinguishability by ion-exchange chromatography, it has proved difficult to differentiate among the various active components of ficin and brome-


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Table II.

Papain

S ,w D ,w Molecular weight sedimentation amino acid composition N o . amino acid residues Carbohydrate, % Isoelectric point, p H Absorbancy A ^ at 280 nm

2.66 10.23

20

l

lcm

Unless specified otherwise, most of the and Smith (1). rt


Comparison of the Physicochemical

Property 20

ENZYMES

23,700 23,000 212 0 8.75 25.0 e

Ficin 2.66 25,500 23,800 248 3.4 9.0 21.0

c

6

Λ

data shown here are taken from Glazer and

(27). (45). (46)

b b

d

lain on the basis of amino acid composition, amino terminal groups, sub strate specificity, kinetic parameters (31-36, 39, 40), and active site sequences (48). One of the most characteristic differences between papain and the other sulfhydryl proteases, ficin and bromelain, is the absence of carbo hydrate in papain. Glycopeptides have been isolated from ficin ( 48 ) and bromelain (49, 50, 51, 52). In the case of bromelain the carbohydrate moiety composed of mannose, xylose, fucose, and N-acetylglucosamine is covalently linked to an asparagine residue of the peptide chain through a glucosamine component of the sugar moiety (51, 52). Activation and Inhibition Activation. Papain prepared in the presence of cysteine according to the method of Kimmel and Smith (23) is composed of three species of the enzyme: (1) active papain with a free thiol group (about 50% of the protein), (2) inactive papain in which the thiol group has formed a disulfide bond with cysteine, and (3) inactive papain i n which the thiol group has been oxidized to a sulfonic acid group. Thiol proteases are activated by mild reducing agents, low molecular weight thiol com pounds, and cyanide because of the formation of active papain (species 1) from disulfide-linked papain (species 2). Species 3 has undergone irreversible oxidation and cannot be activated under these conditions. Brocklehurst and Kierstan (53), however, found that when papain was isolated from dried latex in the absence of cysteine, very little active papain was obtained despite the fact that this preparation contained about 0.4 mole SH/mole protein. Papain prepared in this manner could be activated with cysteine and was indistinguishable from activated papain


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Properties of the Sulfhydryl Proteases

0

Stem bromelain Fruit bromelain

Chymopapain B

b

2.73 7.77 20,000-33,200 35,730' 179-285* 1.46-2.1* 9.55 19.0 Downloaded by CORNELL UNIV on October 21, 2016 | http://pubs.acs.org Publication Date: October 1, 1974 | doi: 10.1021/ba-1974-0136.ch008

e

2.82 18,000

d

34,500

3.2*

318 10.4 18.4

Based on revised sequence reported by Drenth et al. (47).

f (38).

isolated in the presence of cysteine. O n the basis of this and other evi dence the authors postulate that papain exists i n dried latex as a zymogen (propapain) i n which the active S H group (cys-25) forms a disulfide bond with a neighboring cysteine residue, but upon treatment with a reducing agent or cyanide it undergoes intramolecular thiol-disulfide interchange to yield the active isomer (see Figure 3 ) . Whether a similar mechanism of activation applies to the other thiol proteases has yet to be determined. The thiol—disulfide mechanism described here could con ceivably account for the differences i n amino sequence around the S H group of ficin reported by Metrione et al. (54) and Friedenson and Liener (55). Inhibition. Since papain, ficin, and bromelain are all enzymes whose activity depends on a free S H group, it is to be expected that all thiol reagents act as inhibitors. Thus, α-halogen acids or amides and N-ethylmaleimide irreversibly inhibit the thiol proteases. Heavy metal ions and organic mercurial salts inhibit i n a fashion that can be reversed by low molecular weight thiols, particularly i n the presence of E D T A which

PROPAPAIN (INACTIVE)

PAPAIN (ACTIVE) Nature New Biology

Figure 3.

Activation of papain according to Brocklehurst and Kierstan (53)



210


chelates such metals. The chloromethyl ketone derivatives of lysine ( T L C K ) and phenylalanine ( T P C K ) , which are known to react with the active site histidine residue of the serine proteases, also inhibit the thiol proteases. In this instance, the S H group is the site of reaction (56, 57, 58, 59). Diisopropylphosphofluoridate ( D F P ) has also been reported by some investigators (60, 61, 62, 63) to inhibit the thiol proteases, but this inactivation was subsequently found to be caused by the presence of an unidentified impurity present i n some lots of D F P (64). Westrik and Wolfenden (65) have recently reported that aldehydes, with side chains similar to those comprising the acyl portion of substrates which papain effectively hydrolyzes, were very potent inhibitors of this enzyme. Umezawa and his associates (66) have also recently reported that certain microorganisms ( actinomyces ) produced an aldehyde, acetylL-leucyl-L-leucyl-L-argininal (leupeptin), which inhibits papain. The structures of some of the more effective aldehyde inhibitors of papain are shown in Figure 4. I.

SYNTHETIC ALDEHYDES (Westerik and Wolfenden,1972)

C-NH-CH -C-H

Ki =0.025

2

benzoylaminoacetaldehyde

\

>-CH -0-C-NH-CH -C-H 2

2

Ki

=0.0072

Cbz - aminoacetaldehyde Ac CH -CH-C-NH-CH -C-H 2

2

acetyl-L-phenylalanyl 2.

NATURAL ALDEHYDE

Ki = 0.000046

aminoacetaldehyde

( Umezawa , 1973 )

Acetyl - L-Leu-L- Leu-L- Argininal " leupeptin "

Figure 4.

Aldehyde inhibitors of papain

Substrate Specificity In general the thiol proteases catalyze the hydrolysis of a variety of peptide, ester, and amide bonds of synthetic substrates. Employing the general formula R ' — N H — C H R — C O — X , cleavage of the — C O — X — bond has been demonstrated when R represents the side chain of glycine, threonine, methionine, lysine, arginine, citrulline, leucine, and tyrosine.


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—h Lys — ΔΙα — COOH

ρ;—

Ρ' —

Ρ'

1 Biochemical and Biophysical Research Communications


Figure 5. Specificity of papain in re lation to its active site (67) The X component of the substrate may be derived not only from amino acids but also from alcohols, ammonia, mercaptans, aniline, and p-nitrophenol. A benzoyl or carbobenzoxy group has generally been employed as the R ' substituent of the synthetic substrate. Examination of the pep tides produced by cleavage of various proteins has confirmed the rather indiscriminate specificity of this group of enzymes. The variety of peptide bonds cleaved by thiol proteases may be superficially interpreted as indicating a low degree of specificity. Schech ter and Berger (67), however, have shown that the active site of papain can accommodate a peptide sequence of up to seven amino acid residues. One can visualize (Figure 5) the active site of papain as consisting of seven subsites where C denotes the catalytic site and the point of cleavage of the substrate. F r o m the examination of a large number of peptides it was found that if P , the amino acid residue which specifically interacts with subsite S , is L-phenylalanine or L-tyrosine, then the cleavage of the peptide bond one amino acid residue away was considerably en hanced. These results show that the specificity of papain is determined not so much by the side chain of the amino acid containing the susceptible bond but rather by the nature of the amino acids adjacent to it. 2

2

Structure/Function Relationship As shown in Figure 6, the similar properties that exist among the plant proteases can also be extended to the amino acid sequences which surround the active thiol group of these enzymes (68-73). Evidence that a histidine residue is also involved at the active site of the thiol proteases is largely inferential and rests mainly on chemical (73, 74, 75), kinetic (76, 77), and crystallographic data (see below). For example, dibromoacetone has been used to demonstrate that a histidine residue is close enough (within 5 A ) to the S H group to form a covalent bridge between these two residues (see Figure 7). The use of active-sitedirected reagents such as T L C K and T P C K , which have proved so useful in identifying a histidine residue at the active site of the serine proteases,


212


Papain:

Pro- Val-Lys{Asn-Gln-Gly4Ser- •Cys-Gly- Ser- Cys -Trp

Ficin:

Pro

Tie- Arg-GlnfGln-Gly Gin Cys-Gly-Ser-Cys-Trp Αεη-Gln· Asp- Pro4Cys-Gly Ala Cys-Trp

Bromelain:

t Active S H group

Amino acid sequences around the active SH group of thiol proteases


Figure 6.

to

Br-CH

1

L-N — C H I

2

C=0 I Br-CH 2

Figure 7.

5Â

2

i-ln. -o s-s- - % 3

S03

u

i-S-CH

^ _

s

.

s

.

S

O

j

3

ACTIVATABLE

Br-CH -C-CH 2

É-Im-CH -C-CH 2

3

3

I ·

I •?

^ - S-CH2-C-CH

2

^-S-S-SOj

U-S-CH3

ACTIVATABLE Br-CH -C-CH 2

•I-CH -C-CH 2

^-s-s-so %hs-CH

3

S-CH -C-CH

CH

,.

^-S-CH

3

ACTIVE

^

I - CH

Reaction of thiol and histidine residues of thiol protease with dibromoacetone

-Im

#

^ L o

2

c=o

3

3

3

3

3

NON-ACTIVATABLE

3

Figure 8. Reactions involved in directing the speciINACTIVE ficity of bromoacetone to the alkylation of a histidine residue in ficin is not applicable to the thiol proteases since the thiol group seems to react preferentially with such reagents (56, 57, 58, 59). Evidence of the partici pation of a histidine residue at the active site of ficin was recently obtained in our laboratory by the sequence of reactions shown in Figure 8 (78). B y reversible protection of the active thiol group with sodium tetrathionate and oxidation of a vulnerable methionine residue with metaperiodate, it was possible to direct the specificity of bromoacetone to the alkylation of a single histidine residue. The modification of histidine in this manner


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Sulfhydryl Proteases Enzyme

Reagent

Sequence

Papain

Dibromacetone

Bromelain

Di bromoacetone

•His -Ala--Val -Thr- -Ala-

Ficin

Dibromoacetone

-Asp-His -Ala -Val -Ala -Ala-

Ficin

Bromoacetone

- Asp-His--Ala- Val -Δ1α· -Leu-

-

His -Ala -Val -Ala

Active histidine residue


Figure 9. Amino acid sequences around the active histidine residue of thiol proteases was accompanied by a complete loss of enzymatic activity. As shown in Figure 9, the amino acid surrounding the modified histidine residue was found to be virtually identical to the sequence surrounding the histidine residue which can be crosslinked to the active S H groups of papain, ficin, and bromelain with dibromoacetone (73, 74, 75). The complete amino acid sequence of papain is shown in Figure 10. The structure is based on the combined efforts of several groups of inves tigators (69, 72, 79). Cysteine-25 has been identified as the active thiol

Biochemical Journal

Figure 10.

Amino acid sequence of papain (72)


214



group (64). The secondary and tertiary structure of papain based on the x-ray crystallographic studies of Drenth and associates (47, 80) is shown in Figure 11. The molecule has a deep cleft running diagonally down the center so that it appears to be almost two molecules. Only three segments of the chain cross the cleft, and the essential cysteine residue-25 lies to one side of the cleft. Histidine-159 lies on the other side of this cleft. Because its close proximity to cysteine-25 (about 4.5 A ) , it is be lieved to constitute the active-site histidine residue already referred to. The substrate is stereospecifically bound to groups i n the cleft so that cysteine-25 and histidine-159 are properly oriented to participate i n the catalytic mechanism described below.

Figure 11.

Main chain conformation of papain based on x-ray crystallographic data (47, 80)

Mechanism of Action The overall reaction pathway for the catalytic activity of the thiol proteases is best described by the scheme shown in Figure 12. This mechanism shows the formation of an enzyme-substrate complex which results i n the acylation of the enzyme (to form a thiol ester) and its sub sequent deacylation, the overall reaction leading to a regeneration of the enzyme, and the elimination of the products of hydrolysis. Evidence for a thiol ester intermediate is provided by spectrophotometric data which show the formation of N-frans-cinnamoylpapain (81)


8.

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LIENER

E-SH + R-C-X ~ ± E S H R - C - X — ^ E S - C - R

k HgO 3

+

ΗΧ(Ρ,)

Ο

ESH + R-C-OH(P ) 2

Figure 12. Reaction pathway depicting the mechanism of action of papain where ESH and Ο

II

R—C—X are enzyme and substrate respectively and P and P are products formed from the substrate Downloaded by CORNELL UNIV on October 21, 2016 | http://pubs.acs.org Publication Date: October 1, 1974 | doi: 10.1021/ba-1974-0136.ch008

t

2

and thionohippurylpapain (82). From a study on the effect of p H on various kinetic parameters of papain (83,84, 85), it may be concluded that a group having a p K of 8.5 is involved in the acylation step. This is pre sumably the active thiol group which reacts i n its unionized form i n the catalytically active enzyme. Kinetic evidence also implicates a group having a p K of about 4 as being involved in the acylation and deacylation steps. The precise identification of the responsible amino acid residue is a matter of controversy. Stockell and Smith (86) originally proposed that the group responsible for ρ Κ near 4 was a carboxyl group which from the x-ray data would most likely be aspartic-158. The chemical and x-ray evidence already referred to, however, suggest that a more likely candidate for such a group would be histidine-159. Sluyterman and Wolthers (87) believe that this residue should really have a p K of 9.5-10 rather than 4 because of hydrogen bonding to asparagine-175 and electroa

a

α

a

ACYLATION R SH

R

0 + R-C-X

+

HX

DEACYLATION

Figure 13. A possible mechanism of action for papain catalyzed hydrolysis


216


static interaction with aspartic acid-158. They have accordingly proposed a mechanism i n which histidine-159 participates i n the catalytic process as a conjugate acid. The pH-dependence of the acylation and deacylation rate constants on an apparent p K near 4 was considered to be caused by a carboxyl group which i n its undissociated state led to an inactive conformation of the enzyme. O n the other hand, Allen and L o w e (88) argue that the abnormally low p K of histidine-159 can be attributed to its enclosure by a hemisphere of hydrophobic residues, particularly tryptophan-177. A mechanism which portrays histidine as playing a key role i n catalysis is presented i n Figure 13, although other mechanisms are not necessarily precluded (87, 89). a


a

PAPAIN ACTING ON A SUBSTRATE

(ACYLATION):

Γ

OH ES-C-R —• E S - C - R + HX I X tetrahedral adduct acylenzyme

Ε SH + R-C-X

ACTION OF ALDEHYDE INHIBITOR : Ο ESH + R-C-H

OH ;

*

ES-C-R

—X—•

ι

Η

stable thiohemiacetai

Figure 14. Analogy of formation of thiol ad duct with aldehyde inhibitors to formation of tetrahedral intermediate in papain catalysis One of the key intermediates shown i n this reaction scheme is the formation of a tetrahedral adduct during acylation and deacylation (84). Additional support for the formation of a tetrahedral intermedite comes from the observation already referred to—that aldehydes may act as potent inhibitors of papain. Westerik and Wolfenden ( 65 ) attribute the inhibitory effect of aldehydes to the formation of a stable thiol adduct (thiohemiacetai) analogous to the tetrahedral intermediate produced when papain acts on a substrate. This relationship is depicted i n Figure 14. When the complete picture for the mechanism of catalysis b y the thiol proteases finally emerges, it w i l l no doubt be similar to the mecha nism of action of the serine proteinases.

Literature Cited 1. Glazer, A. N., Smith, E. L., "Papain and Other Plant Sulfhydryl Proteolytic Enzymes" in "The Enzymes," P. D. Boyer, Ed., Vol. III, p. 501-546, Academic, New York, 1971. 2. Arnon, R., "Papain," Methods Enzymol. (1970) 19, p. 226-244.



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