[39]

BROMELAIN ENZYMES

475

TABLE II ESTERASE ACTIVITY OF STAPHYLOCOCCAL PROTEASE ISOLATED FROM VARIOUS STRAINS ON ~r-CBZ-GLUTAMYL-~-PHENYL ESTER Strain

Km 104 M

kcat sec -1

V8 A B C D

0.78 5.10 1.17 0.97 1.03

230 44 219 167 175

kcat/Km 104 M -1 sec -1 295 8.6 187 172 170

enzyme from another strain has a molecular weight of about 24,000. This enzyme does not cross-react immunologically with any of the lowmolecular-weight proteases. Recently, the purification of a protease from another strain of S. aureus having the same substrate specificity has been reported? 3 The molecular weight of this enzyme was estimated to be 29,000. It would appear that extensive autodigestion can occur with this class of enzymes without significantly affecting their activities. Table II shows some kinetic parameters for the protease isolated from different S. aureus strains? 4 ~ A. C. Ryden, L. Ryden, and L. Philipson, Eur. J. Biochem. 44, 105 (1974). ~4j. Houmard and G. R. Drapeau, in preparation.

[39] Bromelain Enzymes B y TAKASHI MURACHI N o m e n c l a t u r e . Bromelain enzymes are found in tissues of the plant family Bromeliaceae of which pineapple, A n a n a s comosus (L), is the best known. The proteolytic enzyme found in the juice of pineapple stem is called stem bromelain and the enzyme in the fruit was first described under the name of bromelin 1 and is now called fruit bromelain. 2 Systematic number EC 3.4.22.4 is given to stem bromelain, and EC 3.4.22.5 to fruit bromelain. The purpose of the present chapter is to review only recent information about these two enzymes and to focus on data accumulated since publication of Chapter 18 in Vol. 19 of this series? Thus it is suggested that the reader consult that reference first. R. H. Chittenden, Trans. Conn. Acad. Sci. 8, 281 (1892). R. M. Heinicke and W. A. Gortner, Econ. Bot. 11,225 (1957). T. Murachi, Vol. 19, this series [18].

476

ENDOPEPTIDASES

[39]

Stem lBromelain Purification General Comments Regarding Purification. The crude stem bromelain preparation, usually obtained commercially (Dole Company, Honolulu, Hawaii), contains a number of proteolytic enzymes and nonproteolytic enzymes, including phosphatase, 2 peroxidase, -° cellulase, ~ and other glycosidases. 5 Purification of a carboxypeptidase from commercial bromelain has recently been described. 6 The existence of at least two major components in stem bromelain having similar proteolytic activities and molecular properties except for electrophoretic mobility was first demonstrated by Murachi and Neurath. r E1-Gharbawi and Whitaker s separated five proteolytically active components from a crude stem bromelain preparation by chromatography on Bio-Rex 70 resin at pH 6.1 or by zone electrophoresis in Sephadex G-75 gel. Feinstein and Whitaker 9 compared the physical and chemical properties of these five components. Further characterization of multiple components has also been reported by Silverstein and K6zdy. 1° Using SE-Sephadex C-50, Scocca and Lee ll were able to separate two proteolytically active components that had identical carbohydrate composition. These previous reports together may imply (1) that the crude starting material contains several proteolytically active components, (2) that among these components some differ significantly in molecular size and also in electric charge, so that they can be separated by conventional chromatography or electrophoresis, and (3) that some other components, however, resemble one another so closely t h a t they usually behave as a single component and may be fractionated only under special conditions. Therefore, the purification procedures must be such that one proteolytie enzyme could be separated from another and also from nonenzyme proteins. Purification Procedure of Murachi et al? 2 The method consists of six steps, which have been given in detail in Vol. 19. 3 A few additional points should be noted, however. Sephadex G-50 can be used in place

4H. Suzuki, S. Imai, K. Nisizawa, and T. Murachi, Bot. Mag. Tokyo 84, 389 (1971). ~Y. T. Li and Y. C. Lee, J. Biol. Chem. 247, 3677 (1972). ST. Doi, C. Ohtsuru, and T. Matoba, J. Biochem. (Tokyo) 75, 1063 (1974). 7T. Murachi and H. Neurath, J. Biol. Chem. 235, 99 (I960). s M. ENGharbawi and J. R. Whitaker, Biochemistry 2, 476 (1963). 9G. Feinstein and J. R. Whitaker, Biochemistry 3, 1050 (1964). lo R. M. Silverstein and F. J. K6zdy, Fed. Proc., Fed. Am. Soc. Exp. Biol. 29, 929 (abstr.) (1970). 11j. Scocca and Y. C. Lee, Y. Biol. Chem. 244, 4852 (1969). 12T. Murachi, M. Yasui, and Y. Yasuda, Biochemistry 3, 48 (1964).

[39]

BROMELAIN ENZYMES

477

of Sephadex G-100.13 In order to minimize the hazard of autodigestion during the purification procedure, it is recommended to use sodium tetrathionate, which may reversibly block the catalytic sulfhydryl group of the enzyme protein. The concentration of sodium tetrathionate in the buffers used is 0.1 mM. la F u r t h e r F r a c t i o n a t i o n on S P - S e p h a d e x . 13 The step 6 preparation can be further fractionated into two very similar components by means of chromatography on SP-Sephadex. An aqueous solution of tetrathionateblocked fraction 6 protein (2-4 g in 100 ml) is dialyzed against 5 liters of the starting buffer, 0.05 M Tris-HC1, pH 8.0, at 4 ° overnight. The dialyzed solution is centrifuged and applied to a 2.5 X 100 cm column of SP-Sephadex C-50 (2.3 m E q / g ) , which has been equilibrated with the starting buffer containing 0.1 m M sodium tetrathionate and 0.1 m M EDTA, The column is washed with 500 ml of the starting buffer, then adsorbed enzyme proteins are eluted from the column with 2.5 liters of 0.2 M Tris-HC1, pH 8.0, containing 0.1 m M sodium tetrathionate and 0.1 m M E D T A , at a flow rate of 15 ml/hr, at 4 ° . This gives rise to one major and one minor component on the chromatogram with almost equal specific activity values toward casein. Yields after rechromatography of the major and minor components are 15-25% and 7.5-15% of the fraction 6 preparation on a weight basis. A l t e r n a t i v e Purification Procedure2 ~ Five grams of commercial bromelain preparation are dissolved in 30 ml of 0.02 M sodium citrate buffer, pH 5.5, saturated with phenylmcrcnric acetate (less than 0.5 mM). After insoluble material has been removed by centrifugation at 7000 g for 20 rain, the supernatant solution is applied to a 5 X 85 cm column of Sephadex G-75 (medium particle size), which has been equilibrated at room temperature with 0.02 M sodium citrate buffer, pH 5.5, saturated with phenylmercuric acetate. The column is washed with the same buffer at a flow rate of 40 ml/hr at room temperature. The major proteolytically active fractions are eluted between 600 and 1060 ml. The p H of the pooled fractions is adjusted to 7.2 with 0.2 M NaOH, and the solution is then applied to a 5 X 20 cm column of DEAE-cellulose (0.96 m E q / g ) , which has been equilibrated with 0.02 M sodium phosphate buffer, pH 7.2, containing 0.5 m M phenyhnercuric acetate. The colunm is washed with 1 liter of the same buffer at a flow rate of 40 m l / h r and at room temperature. Most of the proteolytic activity comes off the column. The pH of the combined effluent solution is ~'~N. Takahashi, Y. Yasuda, K. Goto, T. Miyake, and T. Murachi, J. Biochem. (Tokyo) 74, 355 (1973). ~ S. Ota, K. Horie, F. Hagino, C. Hashimoto, and H. Date, J. Biocher~. (Tokyo) 71, 817 (1972).

478

ENDOPEPTIDASES

[39]

adjusted to 6.0 with 0.1 M acetic acid, and the solution is then applied to a 5 }( 35 cm column of SE-Sephadex C-25 (medium particle size, 2.0 mEq/g), which has been equilibrated with 0.03 M sodium citrate buffer, pH 6.0, containing 0.5 mM phenylmercuric acetate. The column is washed at a flow rate of 40 ml/hr and at room temperature first with 1.5 liters of the starting buffer and then with 1.5 liters of 0.3 M sodium citrate buffer, pH 6.0, containing 0.5 mM phenylmereuric acetate. The fractions that have high proteolytic activity are pooled, concentrated, and rechromatographed twice on a 5 }( 35 cm column of SE-Sephadex under the identical conditions as above. The final preparation of the main enzyme component amounts to 10.4% of the crude enzyme. Several minor proteolytically active components can be separated during the chromatographic procedures described above with yields ranging from 0.03% to 2.2% of the starting material. TM Isolation of stem bromelain from an acetone powder by a single passage through a column of e-aminocaproyl-D-tryptophan methyl ester coupled to Sepharose 4B has been described. 15 P u r i t y . The enzyme preparation purified up to step 612 contains at least two very closely similar components, which can be resolved only by chromatography on SP-Sephadex under specified conditions. 13 In this respect, the step 6 preparation is not pure enough. Nevertheless, since these two components are almost indistinguishable from each other and from the step 6 preparation on any other chemical, physical, and enzymic criteria, 13 the step 6 preparation can be employed as a practically pure material for most purposes. The homogeneity of the two components obtained by SP-Sephadex chromatography is verified by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) and immunoelectrophoresis23 Enzyme preparations purified by alternative procedures are also found to be chromatographically homogeneous~,~6,~7 and migrate as a single band upon electrophoresis on cellulose acetate ~ or on polyacrylamide gel. l~,~s The two components obtained by SP-Sephadex chromatography have each single amino-terminal residue valine. 13 The main enzyme purified by Ota et al. ~4 and the purified preparation obtained by Wharton TM always contain significant amounts of extraneous amino end groups, particularly alanine, in addition to valine. The implication of such persistent heterogeneity of these preparations with respect to the amino end group has not been fully understood. 15D. Bobb, Prep. Biochem. 2, 347 (1972). leC. W. Wharton, Biochem. J. 143, 575 (1974). 17S. Ota, S. Moore, and W. H. Stein, Biochemistry 3, 180 (1964). 18L. P. Chao and I. E. Liener, Biochem. Biophys. Res. Commun. 27, 100 (1967).

[39]

BROMELAIN ENZYMES

479

Properties Physical Properties. Physical data for stem bromelain were listed in Table II of Ref. 3. The results showed the enzyme to be a more basic and larger protein than papain. The molecular weight of the step 6 preparation obtained by the method of Muraehi et al. was calculated to be approximately 33,000 from experimentally determined sedimentation and diffusion coefficients. 1-~ Using a further purified preparation, reexamination of the molecular weight has been made by polyaerylamide gel eleetrophoresis in the presence of SDS and also by sedimentation equilibrium nltraeentrifugation. 13 Measured values range from 25,600 to 28,100, and in practice it is recommended to adopt a tentative value of 28,000.13 Chemical Properties. Amino acid compositions of stem bromelain as reported by different investigators, 9,13,14 are given in the table. Characteristically, stem bromelain has only one cysteinyl and histidyl residue per molecule whereas papain has two histidyl residues; also bromelain contains methionyl residues that papain does not. have. The aminoterminal residue is valine, 13 and the principal carboxyl terminal is glyeine. 14 The enzyme is a glyeoprotein having one oligosaeeharide moiety per moleeule, which is eovalently linked to the peptide chain. 11,19 The proposed structure of the earbohydrate moiety of stem bromelain follows.2O,21 L-Fuc (~1~6 (al~2) D-Man

(al~2 D-Man

o r 6) D-Man

D-Xyl o r 2) (fl)

(;3) D-GlcNAc

I

(~1~3

or 4)

D-GIcNAc I (~1--~3 NH2--N)

I

- Ala- A r g - V a l - P r o - A r g - A s n - A s n - G l u - S e r - S e r - MetT y r - Ala - V a l - S e r - L y s - G l n - P r o - Ile -

The amino acid sequence of the peptide fragment that attaches the sugar residues has been determined. 2°,2-~ The B-configuration of the most proximal D-mannosyl residue has been demonstrated by the use of fl-D-mannosidase isolated from a crude stem bromelain preparation. 2~ 19T. Muraehi, A. Suzuki, and N. Takahashi, Biochemistry 6, 3730 (1967). 20y. Yasuda, N. Takahashi, and T. Murachi, Biochemistry 9, 25 (1970). 21y. C. Lee and J. R. Scocca, J. Biol. Chem. 247, 5753 (1972). ~ K. Goto, T. Murachi, and N. Takahashi, FEBS Lett. 62, 93 (1976).

480

[39]

ENDOPEPTIDASES AMINO ACID COMPOSITION OF STEM AND F R U I T BROMELAINS a

Stem bromelain Components

Amino acids Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan

A m m o n i a (amide) Glucosamine C a r b o h y d r a t e (%)

Fruit bromelain

1

2

3

4

5

6

14 1 8 23 l0 21 19 11 27 30 9 16 3 17 7 14 8 7 245

17 1 8 23 10 21 17 13 24 26 9 16 3 17 8 16 8 7 244

17 1 9 23 11 22 18 11 26 28 7 18 4 18 8 16 7 6 250

12 1 6 16 8 16 12 8 19 20 5 12 2 12 5 11 5 5 179

5 1 5 17 8 18 13 7 18 14 6 11 3 9 6 13 4 3 161

8 1 9 32 14 30 25 10 35 25 9 20 5 18 10 16 8 7 282

2 2.1

21 2b 2.1

28 2 0.9

19 4 2.0

24 < 0.2 3.2

0 0

a Sources of values are as follows. Column 1: N. Takahashi, Y. Yasuda, K. Goto, T. Miyake, a n d T. Murachi, J. Biochem. (Tokyo) 74, 355 (1973). Nearest integral n u m b e r of residues per mole of M W 28,000 for component SB1. Column 2: N u m ber of residues per mole of M W 33,000 reported earlier for step 6 preparation [T. Murachi, Biochemistry 3, 932 (1964)] has been recalculated on the basis of an M W of 28,000. Column 3: S. Ota, K. Horie, F. Hagino, C. Hashimoto, a n d H. Date, J. Biochem. (Tokyo) 71, 817 (1972). N u m b e r of residues per mole of M W 28,000 for component I-1. Column 4: G. Feinstein a n d J. R. Whitaker, Biochemistry 3, 1050 (1964). For c o m p o n e n t II, t a k e n methionine as two residues per molecule. Column 5: S. Ota, K. Horie, F. Hagino, C. Hashimoto, and H. Date, J. Biochem. (Tokyo) 71,817 (1972). Nearest integral n u m b e r of residues per mole of M W 18,000 for c o m p o n e n t A. Column 6: F. Yamada, N. Takahashi, a n d T. Murachi, J. Biochem. (Tokyo) 79 in press. Nearest integral n u m b e r of residues per mole of M W 31,000 for c o m p o n e n t FA2. b N. Takahashi, Y. Yasuda, M. Kuzuya, and T. Murachi, J. Biochem. (Tokyo) 66, 659 (1969).

[39]

BROMELAIN ENZYMES

481

Oxidative degradation of the carbohydrate residues by sodium periodate does not cause much alteration in enzymic activity toward casin as well as synthetic substrates38 Stem bromelain has one reactive sulfhydryl group per molecule as determined by titration with p-chloromercuribenzoate. The sulfhydryl group is essential for catalytic activity 24 and reacts stoichiometrically with 5,5'-dithiobis(2-nitrobenzoic acid) 13 or with 2,2'-dipyridyl disulfide. ~5 This cysteinyl residue ( C y s ) is located at position 25 from the amino-terminus32 1

5

10

H-Val-Pro-GIn -$er - Ile - Asp-Trp-Arg-Asp-Tyr-Gly 16

20

15 -Ala-Val-Thr-Ser-

25

Val- Lys-Asn-Gln -Ash- Pro- Cys-Gly -Ala -Cys - T r p -

J

-

Gly - Tyr- Cys- Lys-

A nonapeptide sequence, which corresponds to positions 18 to 26 in the sequence illustrated, except for Asp in place of Asn at position 20, was first reported by Husain and Lowe 26 but a neutral amino acid residue Asn at this position has been confirmed. TM The unique histidine residue, involved in the intramolecular crosslinking of the enzyme by 1,3-dibromoacetone, has an apparent pK~ value of 6.4 as determined directly by the photooxidation reaction in the presence of methylene blueY ,2s This value is within the n o r m a l range for an imidazole group. The following is the amino acid sequence of carboxyl-terminal 33 residues~-2: -Arg-Trp-Gly-Glu-Ala-Gly-Tyr-Ile-Arg- Met-Ala-Arg-Asp-Val-SerSer-Ser-Ser-Gly-Ile-Cys-Gly-Ile-Ala-Ile-Asp-Pro-Leu-Tyr-ProThr-Glu-Gly-OH Evidence for the structural homology among stem bromelain and other thiol proteinases, including fruit bromelain, papain, and ficin, are now being accumulated by elucidating the primary structure of the fragments ~2 and by studying the immunological cross-reactions between thiol proteinases? 9-31 2~y. Yasuda, N. Takahashi, and T. Murachi, Biochemistry 10, 2624 (1971). T. Murachi and M. Yasui, Biochemistry 4, 2275 (1965). :: C. W. Wharton, E. M. Crook, and K. Blocklehurst, Eur. J. Biochem. 6, 565 (1968). :~S. S. Husain and G. Lowe, Chem. Commun., p. 1387 (1968). 27T. Murachi and K. Okumura, FEBS Lett. 40, 127 (1974). 3'ST. Murachi, T. Tsudzuki, and K. Okumura, Biochemistry 14, 249 (1975). :~S. Iida, M. Sasaki, and S. Ota, J. Biochem. (Tokyo) 73, 377 (1973). 30M. Sasaki, T. Kato, and S. Iida, J. Biochem. (Tokyo) 74, 635 (1973). ~IT. Kato and M. Sasaki, J. Biochem. (Tokyo) 76, 1021 (1974).

482

ENDOPEPTIDASES

[39]

Activators, Inhibitors, and Chemical Modifications. In addition to the reactions already reviewed in Vol. 19, 3 the following should be considered. The tyrosyl residues in stem bromelain, which are in "exposed" state, and hence readily accessible to the solvent, can be acetylated with N-acetylimidazole at p H 7.5 or nitrated with tetranitromethane at pH 8,0 without change in catalytic activities22 Photosensitized oxidation of stem bromelain in the presence of methylene blue results in partial loss of the enzymic activity even when the essential sulfhydryl group is protected from the oxidation. The photooxidation involves histidyl, methionyl and t r y p t o p h a n y l residues. 27,2s R a b b i t anti-stem bromelain antibodies inhibit the catalytic activity of stem bromelain. The inhibition occurs more strongly when casein is used as the substrate than with N~-benzoyl-L-arginine ethyl ester (BAEE).33 Stem bromelain cross-reacts with rabbit anti-fruit bromelain, anti-papain, and anti-ficin antibodies to varying degrees resulting in partial loss of its enzymic actvity. ~9-31 H u m a n plasma inhibits the hydrolysis of casein by stem bromelain24 a~-Macroglobulin shows inhibitory effects on various proteolytic enzymes including stem bromelain, but al-antitrypsin does not inhibit bromelain. 35 The occurrence of several inhibitor proteins, which bind stem bromelain, in acetone powder of pineapple juice has recently been described26 One of these inhibitors has a molecular weight of 5600 and is composed of two peptide chains with an interchain disulfide. The amino acid sequences of these two chains have been determined. 37 The preparation and some properties of stem bromelain covalently attached to O-carboxymethyl cellulose have been described. ~5 Specificity, Kinetic Propertie?, and E n z y m i c Mechanism. Substrate specificity and kinetic data for stem bromelain were summarized in Table IV of Chapter 18 in Vol. 19, 3 concerning the hydrolysis of amino acid esters and amides. A further significant finding is the fact that the enzyme seems to be able to interact with two or more sequential amino acids in a peptide substrate is demonstrated by using N-benzyloxycarbonyl-L-phenylalanyl-L-serine methyl ester, 16 with a K,n(~pp~ of 0.53 m M and/~c~t of 3.4 sec -~. 33K. Goto, N. Takahashi, and T. Murachi, J. Biochem. (Tokyo) 70, 157 (1971). 33M. Sasaki, S. Iida, and T. Murachi, J. Biochem. (Tokyo) 73, 367 (1973). 34M. Sasaki, H. Yamamoto, S. Iida, and S. Kimura, Nagoya Med. J. 17, 49 (1972). 3~M. Sasaki, H. Yamamoto, H. Yamamoto, and S. Iida, J. Biochem. (Tokyo) 75, 171 (1974). S. H. Perlstein and F. J. K6zdy, J. 8upramol. 8truct. 1, 249 (1972). 3TM. N. Reddy, P. S. Keim, R. L. Heinrikson, and F. J. K6zdy, J. Biol. Chem. 250, 1741 (1975).

[39]

BROMELAIN ENZYMES

483

With B A E E or N"-benzoyl-L-arginine amide (BAA) as a substrate, the p H profile of Km(a,,)/kcat shows a wide plateau in the range from pH 5 to pH 8. 3s This is in general agreement with the pH-profile of kinetic parameters for papain :'9,'~° and ficin. 4~,~-~ The p H optimum for denatured hemoglobin is around pH 5. 43 Evidence is given for an S-acyl-enzyme intermediate by observing an absorption band having a maximal intensity at 316 nm after admixture of bromelain and methyl thionohippurate. 4~ In the acylation step, a controversial, i.e., either a proton-donating or a proton-withdrawing, function is assigned to the imidazole group of the unique histidyl residue that is spatially very close to the sulfhydryl group. 4~ F r o m the results of the photooxidation experiments, however, an entirely different mechanism has recently been proposed in which the histidyl residue m a y not have intimate electronic interaction with the substrate molecule during catalysis. ~ Fruit Bromelain Methods of assay and preparation of the acetone powder of this enzyme from the fruit juice have been described in detail in Vol. 19. 3 Here, only additional steps of purification and recently available information concerning the properties of the purified enzyme will be given. Purification F r a c t i o n a t i o n Procedure. TM Five grams of the acetone powder is dissolved in 30 ml of 0.02 M sodium phosphate buffer, p H 7.2, containing 0.5 m M phenylmercuric acetate. After centrifugation, the supernatant solution is applied to a 5 X 20 cm column of DEAE-cellulose (0.96 m E q / g ) , which has been equilibrated with 0.02 M sodium phosphate buffer, p H 7.2, containing 0.5 m M phenylmercuric acetate. The column is washed with 1 liter of the same buffer. The adsorbed enzyme is eluted from the column using two-convex-gradient system, 0.06 M 0.3 M-0.5 M sodium phosphate buffer, p H 7.2, containing 0.5 m M

.~3T. Inagami and T. Murachi, Biochemistry 2, 1439 (1963). 39E. L. Smith and M. J. Parker, J. Biol. Chem. 233, 1387 (1958). s0j. R. Whitaker and M. L. Bender, J. Am. Chem. ~oc. 87, 2728 (1965). 41L. A. AE. Sluyterman, Biochim. Biophys. Acta 85, 305 (1964). 42B. R. Hammond and H. Gutfreund, Biochem. J. 63, 61 (1956). 43F. Yamada, N. Takahashi, and T. Murachi, J. Biochem. (Tokyo) 79, in press. 44K. Brocklehurst, E. M. Crook, and C. W. Wharton, Chem. Commun. p. 1185 (1967). '~ D. M. Blow and T. A. Steitz, Annu Rev. Biochem. 39, 63 (1970).

484

ENDOPEPTIDASES

[39]

phenylmercuric acetate. The fractions that represent the major portion of the proteolytic activity applied are pooled, concentrated, and rechromatographed on DEAE-cellulose at pH 7.2. The yield of the final product is 6.4%. Besides the main component, two minor proteolytically active components may be isolated during the course of the fractionation with yields of 0.3 and 0.5%. Alternative Purification Procedure. 43 After the first DEAE-cellulose chromatography at pH 7.2, fruit bromelain may be further chromatographed on ECTEOLA-cellulose (0.45 mEq/g) at pH 7.2. Rcchromatography on ECTEOLA-cellulose yields 260-300 mg of the purified material from 10 g of acetone powder of the juice of pineapple fruit. The product gives a single "precipitation arc to anticrude fruit bromelain antibody upon immunoelectrophoresis at pH 8.6.

Properties Unlike stem bromelain, the main fruit enzyme is an acidic proteinY Its isoelectric point is pH 4.6 as determined by isoelectric focusing technique. 4~ The amino-terminal residue is alanine, 1~ with sequence43: Ala-Val-Pro-Gln-Ser-Ile-Asp-Trp-Arg-Asp-Tyr-Gly-Ala The principal carboxyl-terminal residue is glycine.1. The reported amino acid compositions of fruit bromelain preparations are shown in the table. One preparation is reported to contain carbohydrate that cannot be removed by purification procedures used thus far, 14 and another preparation obtained by different investigators has neither hexosamine nor neutral sugars. 43 The molecular weight of fruit bromelain is still a subject to controversy: 18,000 as determined by Sephadex G-75 gel filtration14 and 31,000 by polyacrylamide gel electrophoresis in the presence of SDS and by Sephadex G-75 gel filtration. 43 The fruit enzyme is more active against BAA and BAEE than the stem enzyme. The following kinetic parameters are reported: for BAA ~4 Km(.~,p~ = 4.0 mM and k c a t = 0.033 sec -~ at pH 6.0 and 25 °, and for BAEE ~3 K,n(.~pp) = 0.043 M at pH 6.0 and 25 °. The pH optima for casein and denatured hemoglobin are pH 8.3 and pH 8.0, respectively..3 Fruit bromelain catalyzes synthesis of acylamino acid anilides. 46 The enzyme is inhibited by mercurials, and activity is restored by cysteine or mercaptoeth~nol. The amino acid sequence around the reactive cysteinyl residue (Cys) is Asn-Glx-AsnPro-Cys-Gly-Ala-Cys. 43 Fruit bromelain cleaves bradykinin at either 4~S. Ota, T. Fu, and R. Hirohata, J. Biochem. (Tokyo) 49, 532 (1961).

[40[

PEPTIDOGLUTAMINASE

485

Gly(4)-Phe(5) or Phe(5)-Ser(6) bond at comparable rates, whereas it splits angiotensin II almost exclusively at Tyr(4)-Ile(5) bond. 43 Rabbit antifruit bromelain antibodies inhibit the catalytic activity of fruit bromelain, and they also cross-react with stem bromelain. 29,31

[40] Peptidoglutaminase (Bacillus circulans) 1 By

MAMORU KIKUCHI a n d K E N J I SAKAGUCHI

Peptidoglutaminase catalyzes the deamidation of the 7-carboxyamide of peptide-bound glutamine. -~ There are two peptidoglutaminases in B. circulans cells. The two enzymes, designated peptidoglutaminase I and peptidoglutaminase II, can be separated by DEAE-Sephadex chromatography2 The former catalyzes reaction (1) and the latter reaction (2) preferentially. 4 CONH 2

COOH

i

(CH~)2 ' R--NH--CH--COOH

I

+ H20

~"

CONH 2

(1)

COOH

r

I

(CH2)2 R--NH--CH--COR

(CH~)2 I R--NH--CH--COOH + NH3

(CH2)~ +

H20

*~

R--NH-- CH--COR

+ NHs

(2)

Assay Method Principle. Enzyme activity is measured by determination of ammonia formed during hydrolysis at the ~/-carboxyamide of carbobenzoxy (CBZ)L-glutamine for peptidoglutaminase I or tert-amyloxycarbonyl (t-AOC)L-glutaminyl-L-proliue for peptidoglutaminase II. The assay procedure described below is based on the direct nesslerization of ammonia. Reagents Sodium phosphate buffer, 0.05 M, pH 7.5 CBZ-L-glutamine or t-AOC-L-glutaminyl-L-proline (Peptide Insti1EC class 3.5.1. M. Kikuchi, H. Hayashida, E. Nakano, and K. Sakaguchi, Biochemistry 10, 1222 (1971). 3 M. Kikuchi and K. Sakaguchi, Agric. Biol. Chem 37, 827 (1973). 4 M. Kikuchi and K. Sakaguchi, Agric. Biol. Chem 37, 1813 (1973).