9
THE JOUHNAL OF Bmmo~cnr. CHEMISTRY Vol. 244, No. 21, Issue of November 10, PD. 59285935, 1969 Printed in U.S.A. The Activation of Papain and the Inhibition of the Active Enzyme by Carbonyl Reagents* (Received for publication, May 29, 1969) IRA B. KLEIN AND J. F. KIRSCH From the Department of Biochemistry, University of California, Berkeley, California 94720 SUMMARY The activation of papain with four different activators is not accompanied by the binding of any of them to the protein. These experiments, taken together with previously reported results, show that the inactive form of papain prepared by the method of Kimmel and Smith (J. Biol. Chem., 207, 5’75 (1954)) is a mixed disulfide formed between the active site sulfhydryl group of the protein and free cysteine. The known inhibition of the activated enzyme by reagents having affinity for carbonyl groups has been investigated in order to determine whether an aldehyde residue which is intimately connected with the activation process is present on the enzyme, or if the observed inactivation of the protein by this class of reagents can be accounted for in some other manner. The following relevant observations were made. (a) Phenylhydrazine inactivates and binds to cyanide-acti- vated papain but neither inactivates nor binds to cysteine- or borohydride-activated papain in the presence of excess ac- tivator. (b) The cysteine-activated enzyme is also inhibited by phenylhydrazine when the cysteine to papain ratio is low. (c) This inhibition in the presence of cyanide or low con- centrations of cysteine is readily reversible with excess cysteine which also releases the bound phenylhydrazine from the protein. (d) Treatment of the enzyme with either phenylhydrazine or hydrogen peroxide in the presence of WN- results in the binding of one cyanide group per active site on the enzyme. Carboxamidomethylation of the sulfhy- dry1 group at the active site prevents this reaction. (e) Semi- carbazide and hydroxylamine, other reagents having high affinity for the carbonyl group, are much less effective in- hibitors of papain. (f) Reactivation of cyanide-activated, peroxide-inactivated papain by cysteine yields Z-iminothia- zolidine-4-carboxylic acid. Model studies show that phenyl- hydrazine, but not semicarbazide or hydroxylamine, is capable of oxidizing cysteine to cystine during 1 hour of incubation with 30 mM reagent. Phenylhydrazine does not bind to cyanate-inactivated papain. Activator-free papain is irreversibly inhibited by phenylhydrazine, without con- comitant binding of the reagent. These and other experi- ments show that the inhibition of cyanide-activated papain by carbonyl reagents is not due to the affinity of these com- pounds for a carbonyl group on the enzyme, but rather to an oxidative coupling of cyanide to the enzyme resulting in the * This research was supported by National Institutes of Health Grant GM12278 and National Science Foundation Grant GB4606. conversion of the cysteine residue at the active site to @- thiocyanatoalanine. Reactivation can be effected by excess cysteine, whereby the cyanide moiety is transferred from the enzyme to the free sulfhydryl group of the cysteine residue. Papain, when isolated by the method of Kimmel and Smith (l), is generally inactive and must be treated with either mer- captans (l-5), cyanide (2-4), or other reducing agents (4) in order to release the free active thiol and the concurrent prote- olytic or esterase activity. The structure of the major fraction of inactive papain has recently been demonstrated to be a mixed disulfide of papain and cysteine (6, 7). The evidence in support of this model is as follows. Activation of papain with K14CN results in the release of thiol equivalent to the amount of cyclized fl-thiocyanatoalanine released from the protein (6). This compound is the expected product of the reaction of cyanide upon the nonprotein sulfur atom of the mixed disulfide. Cys- teine can be oxidatively bound to active papain, and the kinetics of reactivation of this species are identical with those observed for the activation of native papain (7). The activation is apparently accomplished without a major conformational change as determined by fluorescence, ultraviolet difference spec- troscopy, and circular dichroism (8). These recent findings are difficult to reconcile with other phenomena associated with activation which have been de- scribed in the literature. It has been reported for example that phosphorothioate ion (PO&?) becomes reversibly bound to papain during the course of activation (5). It has also been ar- gued, from the sensitivity of papain to carbonyl reagents (%12), that an aldehyde moiety is present on the protein and that the inactive form is an hemithioacetal formed between the active site thiol residue and this aldehyde (9, 12). The experiments reported herein were designed to determine the total extent of binding of activators during the course of activation, and to elucidate the mechanism of the inactivation of papain by carbonyl reagents. EXPERIMENTAL PROCEDURE Materials Radiochemicals-85S-Thiophenol, 26.5 mCi per mmole, pur- chased from the Radiochemical Centre (Amersham, England), by guest on June 4, 2015 http://www.jbc.org/ Downloaded from

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The activation of papain and inhibitors

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  • THE JOUHNAL OF Bmmo~cnr. CHEMISTRY Vol. 244, No. 21, Issue of November 10, PD. 59285935, 1969

    Printed in U.S.A.

    The Activation of Papain and the Inhibition of the Active Enzyme by Carbonyl Reagents*

    (Received for publication, May 29, 1969)

    IRA B. KLEIN AND J. F. KIRSCH From the Department of Biochemistry, University of California, Berkeley, California 94720

    SUMMARY

    The activation of papain with four different activators is not accompanied by the binding of any of them to the protein. These experiments, taken together with previously reported results, show that the inactive form of papain prepared by the method of Kimmel and Smith (J. Biol. Chem., 207, 575 (1954)) is a mixed disulfide formed between the active site sulfhydryl group of the protein and free cysteine.

    The known inhibition of the activated enzyme by reagents having affinity for carbonyl groups has been investigated in order to determine whether an aldehyde residue which is intimately connected with the activation process is present on the enzyme, or if the observed inactivation of the protein by this class of reagents can be accounted for in some other manner. The following relevant observations were made. (a) Phenylhydrazine inactivates and binds to cyanide-acti- vated papain but neither inactivates nor binds to cysteine- or borohydride-activated papain in the presence of excess ac- tivator. (b) The cysteine-activated enzyme is also inhibited by phenylhydrazine when the cysteine to papain ratio is low. (c) This inhibition in the presence of cyanide or low con- centrations of cysteine is readily reversible with excess cysteine which also releases the bound phenylhydrazine from the protein. (d) Treatment of the enzyme with either phenylhydrazine or hydrogen peroxide in the presence of WN- results in the binding of one cyanide group per active site on the enzyme. Carboxamidomethylation of the sulfhy- dry1 group at the active site prevents this reaction. (e) Semi- carbazide and hydroxylamine, other reagents having high affinity for the carbonyl group, are much less effective in- hibitors of papain. (f) Reactivation of cyanide-activated, peroxide-inactivated papain by cysteine yields Z-iminothia- zolidine-4-carboxylic acid. Model studies show that phenyl- hydrazine, but not semicarbazide or hydroxylamine, is capable of oxidizing cysteine to cystine during 1 hour of incubation with 30 mM reagent. Phenylhydrazine does not bind to cyanate-inactivated papain. Activator-free papain is irreversibly inhibited by phenylhydrazine, without con- comitant binding of the reagent. These and other experi- ments show that the inhibition of cyanide-activated papain by carbonyl reagents is not due to the affinity of these com- pounds for a carbonyl group on the enzyme, but rather to an oxidative coupling of cyanide to the enzyme resulting in the

    * This research was supported by National Institutes of Health Grant GM12278 and National Science Foundation Grant GB4606.

    conversion of the cysteine residue at the active site to @- thiocyanatoalanine. Reactivation can be effected by excess cysteine, whereby the cyanide moiety is transferred from the enzyme to the free sulfhydryl group of the cysteine residue.

    Papain, when isolated by the method of Kimmel and Smith (l), is generally inactive and must be treated with either mer- captans (l-5), cyanide (2-4), or other reducing agents (4) in order to release the free active thiol and the concurrent prote- olytic or esterase activity. The structure of the major fraction of inactive papain has recently been demonstrated to be a mixed disulfide of papain and cysteine (6, 7). The evidence in support of this model is as follows. Activation of papain with K14CN results in the release of thiol equivalent to the amount of cyclized fl-thiocyanatoalanine released from the protein (6). This compound is the expected product of the reaction of cyanide upon the nonprotein sulfur atom of the mixed disulfide. Cys- teine can be oxidatively bound to active papain, and the kinetics of reactivation of this species are identical with those observed for the activation of native papain (7). The activation is apparently accomplished without a major conformational change as determined by fluorescence, ultraviolet difference spec- troscopy, and circular dichroism (8).

    These recent findings are difficult to reconcile with other phenomena associated with activation which have been de- scribed in the literature. It has been reported for example that phosphorothioate ion (PO&?) becomes reversibly bound to papain during the course of activation (5). It has also been ar- gued, from the sensitivity of papain to carbonyl reagents (%12), that an aldehyde moiety is present on the protein and that the inactive form is an hemithioacetal formed between the active site thiol residue and this aldehyde (9, 12).

    The experiments reported herein were designed to determine the total extent of binding of activators during the course of activation, and to elucidate the mechanism of the inactivation of papain by carbonyl reagents.

    EXPERIMENTAL PROCEDURE

    Materials

    Radiochemicals-85S-Thiophenol, 26.5 mCi per mmole, pur- chased from the Radiochemical Centre (Amersham, England),

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  • Issue of November 10, 1969 I. B. Klein and J. F. Kirsch 5929

    was diluted with redistilled unlabeled compound obtained from Matheson, Coleman and Bell, East Rutherford, New Jersey, to a specific activity of 173 cpm per mpmole. K14CN, about 6 mCi per mmole, was obtained from New England Nuclear Corpora- tion and diluted to a specific activity of 100 to 500 cpm per mpmole with Mallinckrodt unlabeled KCN. Volk 35S-~- cysteine, 43.1 mCi per mmole, was diluted with Calbiochem nn-cysteine HCl monohydrate to 243 cpm per mpmole. Ac- tivation with unlabeled cysteine was performed using either Nutritional Biochemical Corporation L-cysteine or the DL- cysteine HCl monohydrate from Calbiochem. 14C-Phenylhy- drazine HCl, 126 mCi per mmole from Tracerlab, Richmond, California, was diluted with unlabeled compound obtained from Eastman (White Label) to a specific activity of from 50 to 100 cpm per mFmole. Labeled P-thioatel was synthesized from 32PSC13 (Volk Radiochemical Company, Irvine, California) by the method of ikerfeldt (13) to a final specific activity of 167 cpm per mpmole. This preparation was used within a week of synthesis, and the specific radioactivity was redetermined daily. 14C-Semicarbazide, 7.5 mCi per mmole, was purchased from Volk Radiochemical Company, and diluted with unlabeled compound (Eastman White Label) to 50 cpm per mpmole.

    Other Reagents-Iodoacetamide was obtained from Calbio- them and recrystallized from 1007, ethanol. Hydroxylamine HCl was purchased from Fisher Scientific Company, Pittsburgh, Pennsylvania; hydrogen peroxide as a 30% solution, Su- peroxol, from Merck; potassium cyanate from Matheson, Coleman and Bell; 5,5-dithiobis(2-nitrobenzoic acid) from Aldrich and benzaldehyde (White Label) from Eastman. NaBH4 was obtained from Metal Hydrides, Inc., Beverly, Massachusetts. Bio-Gel P-2 was purchased from BioRad Laboratories, and Sephadex G-10, G-25, and G-75 from Phar- macia Fine Chemicals. Z-glycine p-NP was prepared by the method of Bodanzky and du Vigneaud (14), m.p., 127.5128, literature 128.

    Four papain preparations were used in these experiments: Worthington Biochemicals, twice recrystallized enzyme, lot numbers 5629, 7DB, and 8CA, and enzyme purified in this laboratory from dry papaya latex (Wallerstein Company, New York, New York) by the method of Kimmel and Smith (l), as modified by Masuda (15). This is referred to as IK(-cys). These Worthington Biochemical preparations had similar maxi- mal velocities when assayed with Z-glycine p-NP, while IK(-cys) was 60% as active. In the absence of added cysteine, lot 5629 had an activity of 40% of the maximum obt.ainable (16), while IK(-cys) had 25yo of its maximum rate that was obtained in the presence of cysteine.

    Methods

    Enzyme concentrations were calculated from absorbance measurements at 280 rnp using an extinction coefficient of 5.1 x lo4 M- cm+ (17) except in the experiments with thio- phenol, in which the method of Lowry et al. (18) was used.

    A Packard Tri-Carb model 3003 liquid scintillation spec- trometer was used to measure radioactivity. Samples were counted in 10 ml of a solution containing 5 g of 2,5-diphenyl- oxazole and 2 g of 1,4-bis[2-(5-phenyloxazolyl)]benzene per liter of toluene-ethanol mixture (19). In the ?S-thiophenol

    1 The abbreviations used are: P-thioate, trisodium phosphoro- thioate; Z-glycine p-NP, benzyloxycarbonylglycine p-nitrophenyl ester.

    experiment, 0.5-ml samples were counted; in all other experi- ments, 0.2-ml samples were counted.

    The enzyme was assayed essentially by the method previously described (20). A catalytic rate was determined as follows. Regardless of previous treatment, the enzyme was assayed in a solution containing 20 mM phosphate buffer, pH 6.8, 1.0 mM EDTA, and a final papain concentration of 0.3 to 0.7 PM. The reaction was started by adding 0.2 ml of a 1.5 mM solution of Z-glycine p-NP in redistilled acetonitrile to 2.8 ml of the above solution in a cuvette maintained at 25 with a thermostat in the cell compartment of a Zeiss PM& II spectrophotometer. The esterase activity was calculated by dividing the initial change in optical density per min at 400 rnp by the optical density at the end of the reaction. This number was then divided by the protein concentration in micrograms per ml and multiplied by 10 to give the reported activity. A rate of 1.0 mini (micro- grams of enzyme per ml)- was generally the highest attainable and was taken as 1OO70 or optimal activity. For maximal recoverable activity after inactivation or for the determination of maximal activity for a particular batch of papain, a final concentration of 0.3 mM cysteine was used in the standard assay solution as described above and allowed to react for periods of up to 3 hours with the enzyme.

    Activation of Papain Using Low Activator Concentrations-In one activation experiment, the method of Soejima and Shimura (21) was used with the following modifications. %-Thiophenol was used as an activator instead of p-thiocresol, and the papain was not subjected to prior inactivation with KI and Iz. The activation was performed on Worthington lot 5629 in 40 mM citrate buffer, pH 5.0, at 25. After gently shaking 2.5 ml of the 250 pM solution of papain with 2.5 ml of toluene containing 5 mM 35S-thiophenol, a 0.5-ml aliquot of the aqueous phase was filtered through fine sintered glass and then chromatographed on a column (1 x 8 cm) of Sephadex G-25 using 0.1 M phosphate buffer, pH 6.8, containing 1 mM EDTA at 25 under nitrogen. The flow rate was about 1 ml per min. Fractions of 1.5 ml were collected in serum capped tubes, assayed, and counted as de- scribed above.

    Papain was activated with K14CN as described previously (6) Papain, which had been carboxamidomethylated or diluted due to passage through a gel column, was at about 80 pM and was treated with labeled 1.0 mM KCN. This cyanide treatment was generally carried out for 4 hours. All experiments were done at pH 6.8 and 25 unless otherwise stated. In certain experiments, papain was made activator-free after activation by elution through a Bio-Gel P-2 column (1 x 10 cm) in 0.01 M phosphate buffer. When activated by 3SS-cysteine, the papain was at 240 PM, and the labeled thiol at 2 InM; the activation mixture also contained 35 InM EDTA and 35 mM phosphate buffer. Reaction time was 2 hours. The 3zP-P-thioate used to activate papain was 0.17 mM as was the enzyme. Activation proceeded for 1 hour in a 30 mM EDTA, 30 InM phosphate buffer, and then the solution was chromatographed as described in Table I. NaBH4 activa- tion was done with 180 mM NaBH4 in 0.67 mM EDTA and 25 mM phosphate buffer at pH 7 and 0 for 45 min, similar to the procedure described by Glazer and Smith (4). Thiol titer was determined by the method of Ellman (22).

    Inactivation Conditions-Inactivation reactions were usually performed for 1 hour at pH 6.8 and 25 in the dark at concentra- tions of papain ranging between 80 and 300 pM in the presence of 6 mM carbonyl reagents unless otherwise stated. Potassium cy-

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  • 5930 Activation and Inhibition of Papain Vol. 244, No. 21

    TABLE I

    Extent of binding of activators to various forms of papain4

    Papain was activated and then eluted through Sephadex G-10 or Bio-Gel P-2 as described under Methods.

    K14CN treatment of Inactive papain (7DB). . . . Carboxamidomethyl papain

    (7DB). Cysteine activated papain (ac-

    tivator free) (7DB).

    32P-P-thioate treatment of Inactive papain (7DB).

    35S-Cysteine treatment of Papain (IK-(cys)) .

    Amount of activator bound per

    Protein Thiol reIessedb

    ?nolc/mole

    0.04

    0.06

    0.09

    0.01

    0.07 I -

    0.14

    0.18

    0.04

    0.19

    D Lot 7DB was purchased from Worthington, while IK(-cys) was isolated by the method of Kimmel and Smith (1) as modified by Masuda (15).

    b Based on thiol determination by the method of Ellman (22). The thiol released per mole of protein in each experiment is given by dividing the figure in Column 2 by that in Column 3.

    anate and HzOz were used at concentrations of 2 mM with 30-min reaction times.

    RESULTS

    On Binding oj Activators to Papain-The extent of binding of a%-cysteine, K%N, and a2P-P-thioate to papain is shown in Table I. After the incubation with the activator as described under Methods, the mixtures were chromatographed on Sepha- dex G-10 or Bio-Gel P-2 to remove nonprotein-bound activator. It can be seen that neither of the two thiol activators nor cyanide was bound in quantities comparable with the enzyme thiol formed upon activation. The binding of a%-thiophenol was also investigated on a sample of papain that had some activity in the absence of activator (lot 5629), and the thiol was observed to bind to an extent of less than 0.1 mole per mole of protein thiol formed while fully activating the enzyme.

    It has been shown before (6), and here again, that only about 0.1 mole of C-cyanide was bound during cyanide-mediated ac- tivation of papain per mole of protein thiol released. This could be due to a nonspecific reaction of cyanide with one of the three disulfide bridges on the papain molecule (23, 24) or to a wrong side attack at the active site which would release free cysteine from the inactive form and leave /3-thiocyanatoalanine at position 25 in the amino acid sequence as shown in Equation 1. This de- rivative would presumably be inactive.

    r PHI+ r -+ SCN + CYSTEINE L (1) coo-

    The fact that cyanide also binds to a similar extent to car- boxamidomethyl- and cysteine-activated papain suggests that the former explanation is more probable. The cyanide bound to

    1 A

    0 0.2 0.4 0.6 0.8

    [ Thiol I/[ Protein ]

    FIG. 1. Enzymatic activity as a function of the number of titratable -SH groups per mole of protein. All the experiments were done on papain lot 7DB activated by either of three different activators and at different ratios of activator to papain; v, 0.18 mM papain-O.18 mM P-thioate; A, 0.16 mM papain-O.8 mM P- thioate; l , 0.3 mM papain-O.5 mM cysteine; n , 0.3 mM papain-3.0 mre CN-; 0, no activator. The enzyme activity was measured as described under Methods and the thiol titer was determined by the method of Ellman (22).

    cyanide-activated Worthington papain was stable to rechro- matography, probably indicating that it is in covalent linkage with the protein. The extent of binding of thiol activators to the enzyme was likewise small compared with the amount of protein- bound thiol released. Similar results using P-thioate and KCN were obtained with another preparation of papain (lot 8CA).

    The binding ratio is presented in two ways in the table to re- illustrate a characteristic of papain which has been noted before (3, 4, 6, 7, 25); that is, that the usual preparations of fully acti- vated papain contain considerably less than 1 mole of thiol per mole of protein, presumably indicating the presence of some ir- reversibly inactivated papain. As shown in Fig. 1, the activity of the enzyme is directly proportional to the titrable thiol on the protein irrespective of the mode of activation. This result is similar to the one obtained previously by Sanner and Pihl with no added activator (2). At the maximum rate of the Z-glycine p-NP assay of 1.0 mine1 (micrograms of enzyme per ml)+, this particular batch of papain had 0.54 mole of SH per mole of protein based on ~280 = 51,000 (17). This rate of esterase ac- tivity is the highest that could be obtained on several batches of Worthington papain, and on papain isolated by the method of Kimmel and Smith (1) as modified by Masuda (15) under many different activating conditions.

    In some of the experiments it was necessary to use low ratios of activator to protein so that all of the potential protein-bound thiol was not released. Since the amount of nonspecific binding of the activators was fairly constant, a small thiol titer resulted in a higher molar ratio of bound activator to thiol released as is seen by the difference between the amount of activator bound per mole of protein and per mole of thiol (Table I).

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  • Issue of November 10, 1969 I. B. Klein and J. F. Kirsch 5931

    Volume Eluted (mls)

    FIG. 2. Bio-Gel P-2 chromatography in 10 mM phosphate buffer, pH 6.8, at 25. A, 2 mM 3%-cysteine-activated 0.24 mM papain in 35 mM phosphate, 35 mM EDTA, pH 6.8, for 2 hours. B, 0.170 rnM 32P-thioate activation of 0.170 mM papain in 30 mM phosphate buffer, 30 mM EDTA for 1 hour. 0, protein was de- termined using the optical density at 280 rn#; 0, thiol by the method of Ellman (22) ; and A, radioactivity of s2P and 3% as de- scribed under Methods.

    The chromatography on Bio-Gel P-Z of 36S-cysteine- and 32P-P- thioate-activated papain is shown in Fig. 2. The elution profiles show that a small amount of radioactivity comes out with the void volumes of the columns and was taken to be protein bound. These elution profiles are similar to the one previously shown for W-cyanide-activated papain (6), except that Bio-Gel P-2 was used here instead of Sephadex G-10. Fig. 2B illustrates how small an amount of P-thioate is bound to papain, but, more interestingly, shows that a new 32P-containing compound is formed as a result of the reaction of P-thioate with inactive papain. The small thiol peak on the right of the graph corre- sponds to the elution of P-thioate when chromatographed alone. By assuming an analogous mode of activation to that by cyanide, and because oxidized P-thioate, ostensibly the disulfide (03PSSP03) comes out at a different point than either the large azP-containing peak, the protein peak, or the thiol peak, this new product is considered to be a mixed disulfide of cysteine and P-thioate. The small amount of P-thioate that remains unre- acted further illustrates that the reaction of P-thioate with pa- pain is a very efficient one at this dilute concentration of acti- vator, as has been reported previously by Neumann, Shinitzky, and Smith (5).

    From the fact that cyanide-activated papain is particularly sensitive to inhibition by carbonyl reagents (10, ll), Morihara et al. (9,12) have postulated that the inactive form of the enzyme has a structure in which the active thiol is bound as an internal hemithioacetal. The reactions of papain with this type of re- agent were, therefore, investigated in order to attempt to find a satisfactory explanation for this phenomenon.

    The Reaction of Papain with Carbonyl Reagents-Of the reagents

    TABLE II

    Reaction of phenylhydrazine with papain

    Papain concentrations between 80 and 300 pM were activated with 4- to lo-fold molar excesses of cysteine or cyanide, at 25, or with a 450 molar excess of NaBH4 at pH 6.8 to 7.0 at 0. A 30- to loo-fold excess of phenylhydrazine was employed to inactivate the activated papain at 25 for 1 hour at pH 6.8. Where desig- nated as activator-free, the activated papain was eluted through a Bio-Gel P-2 column (1 X 15 cm), 0.01 M phosphate buffer, pH 6.8, before treatment with phenylhydrazine.

    Activator

    None, native, inactive papain

    None, carboxamido- methyl derivative of papain

    KCN

    Cysteine

    Sodium borohydride

    .ctiva- tor

    resent when henyl.

    hy- razine i adde

    - ! I-

    1

    b a

    --

    I

    I

    I

    I

    I

    I

    I

    -

    Esterase activity

    After initial rctiva. tion

    0.97 0.91

    0.80 D.89 Cl.75

    0.97 D.83

    1 1

    ._

    -

    After phenyl-

    hydrazine :reatment

    0.066 0.049

    0.61 0.061 0.043*

    0.84 0.0476

    -

    - After reacti- vation with cys-

    tein@

    0.61 0.07:

    0.79 0.17 0.57

    0.57

    - I

    1

    1

    1

    1

    -

    Phenyl- hydrazine mund,per pa:tn

    treatment

    soze/moze 0.12

    0.01

    0.64 0.076

    0.08 0.22 0.46

    0.01 0.42

    (1 After inactivation, an aliquot of the mixture was diluted into 12 ml of the standard assay solution to a final enzyme concentra- tion of 0.3 to 0.7 PM. Two portions of this diluted solution were assayed immediately, and the remaining two portions were treated with 0.3 mM cysteine for 3 hours and then assayed to determine recoverable activity as described under Methods.

    * A lo-fold molar excess of cyanide over protein was added with the phenylhydrazine.

    having affinity for carbonyl groups which have been investi- gated, phenylhydrazine has been shown to be the most potent inhibitor of papain. A particularly puzzling aspect of the in- activation of papain by phenylhydrazine is that the inhibition is more pronounced when papain is activated by cyanide than when it is activated by cysteine (9, 12). The experiments reported in Table II confirm this observation, and provide a plausible ex- planation for it. Papain activated either by KCN, cysteine, or NaBH4 was inactivated after the activator had been removed by gel filtration before treatment with phenylhydrazine. Under these conditions, very little radioactive phenylhydrazine was bound to the protein as a result of the inactivation process. When cyanide was used as the activator and was not removed prior to the addition of phenylhydrazine, papain was also in- activated by the phenylhydrazine. In this case about 1 mole of phenylhydrazine was bound per mole of active enzyme.2 If either cysteine or NaBH4 was present during the phenylhydrazine treatment, there was no appreciable inactivation, nor did phenyl- hydrazine become bound to the protein. The inactivation medi-

    2 According to Fig. 1, there is about 0.5 mole of free thiol, and, according to Table III, about 0.5 mole of phenylhydrazine bound.

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  • 5932 Activation and Inhibition of Papain Vol. 244, No. 2I

    ated in the presence of cyanide was partly reversed by the addi- tion of excess cysteine, and this reactivation was accompanied by the displacement of phenylhydrazine from the enzyme (Fig. 3). The inactivation caused by phenylhydrazine in the absence of activator cannot be reversed by cysteine. The requirement for cyanide for binding of phenylhydrazine and reversible inactiva- tion was explored further by the experiments shown in Table III. The results demonstrate that, in addition to the presence of cyanide being required for the binding of r4C-phenylhydrazine to papain (Table II), either phenylhydrazine or hydrogen peroxide will couple 14C-cyanide to the enzyme. These experiments sug- gest that the mechanism of inactivation of papain in the presence of cyanide by these reagents is not due to a reaction of a carbonyl group on the enzyme, but rather to an oxidative coupling of cya- nide to the active sulfhydryl group of the enzyme converting the essential cysteine residue at position 25 in the primary sequence

    0.0 w 0 20 40 60 80 100

    % Phenylhydrazine Removed

    FIG. 3. Reversal of phenylhydrazine inactivation by cysteine. Phenylhydrazine-inhibited cyanide-activated papain was pre- pared as described in Table II. The inactivated papain was chromatographed on Bio-Gel P-2. Two fractions containing 40 and 30 PM papain were treated with 0.3 mM cysteine for 15 hours, and 30 mM cysteine for 24 hours, respectively, at pH 6.8 and 25 in 10 mM phosphate buffer. Rechromatography on Bio-Gel P-2 was done to determine activity recovered and the remaining bound phenylhydrazine as described under Methods.

    L- -SH CN- CeH,NHNH, > or H,Q

    SCN + C,HaHNH,

    - CeH,N& + NH3

    I$ (2) r NH* / -s-c \N-NH-C H 6 5 TABLE III

    Oxidative coupling of WY-cyanide to activated papain

    The reactions were carried out at pH 6.8 and 25 in 10 or 30 mM phosphate buffer containing 1 mM or 10 mM EDTA. Papain concentrations varied from 80 to 400 PM. Phenylhydrazine was about 6 mM, cyanide, 1.6 mM, and peroxide, 2 mu. Cyanide activation was done for 4 hours; cysteine activation, 45 min; and hydrogen peroxide and phenylhydrazine inactivation 30 and 60 min, respectively. -

    I I Esterase activity CN bound per nole of protein

    before reactivation

    After phenyl-

    hydrazine treatment

    After reactiva-

    tion After

    :tivation a,

    --

    -

    Carboxamidomethyl papain with W- cyanide and phen- ylhydrazine.

    Cysteine-activated, cysteine-free pa- paina with W-cy- anide and phenyl- hydrazine

    i4C-Cyanide activation of papain followed

    by Phenylhydrazine.. . . Hydrogen peroxide. .

    0.06

    ._1 30 35 40 -15 20 25

    0.76

    0.69 0.69

    0.070

    0.11 0.05 0.69

    0.42 Volume (mls)

    FIG. 4. The oxidative binding of cyanide to the active site of papain. Sephadex G-75 chromatography in 10 mu phosphate buffer, pH 6.8, of cyanide-activated papain (see Methods). A, half the activation mixture was inactivated with 20 mM phenyl- hydrazine. B, the other half was treated with 50 mM iodoaceta- mide which completely abolished the activity, and then followed by 20 mM phenylhydrazine. Protein (0) was determined using the optical density at 280 rnp, and radioactivity (A) as described under Methods.

    0.46 0.88

    a Cysteine was separated from papain using a Bio-Gel P-2 column (1 X 15 cm), 0.01 M phosphate buffer, pH 6.8, at 25.

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  • Issue of November 10, 1969 I. B. Klein and J. F. Kirsch 5933

    TABLE IV

    Hydroxylamine and semicarbazide reactions with cyanide-activated papain

    Papain (0.4 mM) was activated with 1.6 mM cyanide for 4 hours at 25 in 0.01 M phosphate buffer, and then incubated with 6 mM carbonyl reagent or 3 mM Hz02. The hydrogen peroxide inacti- vation was completed after 30 min. Semicarbazide and hydroxyl- amine were incubated with the enzyme for 2 hours unless other- wise indicated. All reactions were in 30 mM phosphate-30 mM EDTA, pH 6.8, at 25.

    Esterase activity

    After activation

    Azeert tre&

    carbonyl reagent

    W bound per mole of

    protein --

    -

    1%~Semicarbazide reacted with cyanide-activated papain

    2 hours.. 12 hours..

    W-Semicarbazide reacted with peroxide-inactivated cyanide-activated papain.

    Hydroxylamine reacted with W-cyanide-activated pa- pain.....................

    0.93 o.e3 0.17 0.71a 0.61 0.19

    0.87

    0.81

    0.03

    0.54

    0.24

    0.26

    3x10+ 3x10+ 3x10-2

    Concentration of Carbonyl Reagent (M)

    FIG. 5. The oxidation of cysteine by carbonyl reagents. Cys- teine (24 PM) was treated with semicarbazide (A), phenylhydra- zine (O), or hydroxylamine (0) for 1 hour at 25, pH 7.0 (10 mM phosphate, 1 mM EDTA). Thiol was determined by the method of Ellman (22). Cysteine which was not treated with carbonyl reagents showed less than 2% loss of thiol under these conditions. -

    a This rate was determined 12 hours after the enzyme was initially activated, but without semicarbazide treatment. The original rate was 0.762.

    TABLE VI

    Cysteine protection of papain against phenylhydrazine inhibition Papain was reacted with phenylhydrazine in the presence of

    increasing amounts of cysteine. The initial enzyme concentra- tion was 0.3 rnl\b in 2.5 mM phosphate buffer, 2.5 mM EDTA at pH 6.8. The protein was incubated 25 min at 25 with cysteine and then diluted to 24 ,UM before treatment with phenylhydrazine.

    TABLE V

    Reaction of papain with carbonyl reagents and aldehyde protection Papain (0.3 mM) was incubated with the reagents indicated in

    0.01 M phosphate buffer, pH 6.8, for the times shown, after being activated with 1.6 mM cyanide for 4 hours.

    -

    -

    Esterase activity

    Cysteine concentration Treatment -

    Recoverable with more activatora Concentra- tions of

    reagents

    DUI% tion 0 treat- ment

    After treatment

    0.82

    0.05

    0.12 0.87 1.03

    Esterase activity Reagents added

    None

    Phenylhydrazine Phenylhydrazine

    + Benzaldehyde (added together)

    Phenylhydrazine +

    Benzaldehyde (added sequentially)

    Semicarbazide

    Semicarbazide

    + Benzaldehyde (added together)

    Benzaldehyde

    None

    Phenylhydrazine 3 mM for 1 hour in dark

    m.M

    3.75 3.75

    25.0

    3.75

    25.0

    3.75

    3.75

    25.0

    25.0

    hrs

    0.85

    1 0.04

    1 0.31

    1

    1t 0.08

    1 0.91

    1 0.59

    1 0.58

    hour before the 25 a Phenylhydrazine (3.75 mM) was added mM benzaldehyde.

    0.90

    0.07

    0.84 1.01 0.93

    - o Inactivation mixture (0.2 ml) was added to 12.17 ml of the

    standard assay solution described under Methods which con- tained a final concentration of 35 mM phosphate, pH 6.8, 1 rnM EDTA, and 0.5 pM papain. One-half of this was assayed, and the remainder was made 0.3 mM in cysteine, incubated at 25 for 3 hours, and then assayed to determine the recoverable activity.

    (23, 24) to P-thiocyanatoalanine. These reactions a.re sum- marized in Equation 2.

    The proposed structure of the adduct produced by the addition of phenylhydrazine to the thiocyanate group is based on the stoichiometrv of the binding of atmroximatelv 1 mole of cvanide u II * I

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  • 5934 Activation and Inhibition of Papain Vol. 244, No. 21

    per mole of phenylhydrazine, and is supported by analogous model reactions (see Discussion). The location of the bound cyanide at the active site cysteine is strongly suggested by the fact that alkylation at this position completely prevents the bind- ing of cyanide caused by phenylhydrazine (Fig. 4).

    The oxidation hypothesis was further tested by the experi- ments reported in Table IV, wherein it is shown that neither one of two other well known carbonyl reagents, semicarbazide or hydroxylamine, significantly inhibit cyanide-activated papain under these conditions, nor is 14C-semicarbazide bound to the enzyme to any appreciable extent. It is shown below that these compounds are poor oxidizing agents as well. Hydroxylamine, moreover, does not cause as much 14C-cyanide to bind to papain as does phenylhydrazine or hydrogen peroxide.

    Benzaldehyde has been reported to provide partial protection against the phenylhydrazine-induced inactivation of the enzyme (11). This result is confirmed by some of the experiments shown in Table V, where it is observed in addition, that benzaldehyde itself causes some inactivation of the enzyme. The protection by benzaldehyde need not, however, reflect a competition be- tween this aldehyde and a similar group on the enzyme, but only the complexation of the phenylhydrazine presumably through the formation of the benzaldehyde phenylhydrazone. The inacti- vation caused by the benzaldehyde itself was not further in- vestigated, but may be due to the formation of a hemithioacetal between the active site thiol group and the added aldehyde. It should be further noted that benzaldehyde was only able to moderate the phenylhydrazine-induced inhibition when it was added with the phenylhydrazine; benzaldehyde added after treatment with phenylhydrazine was totally ineffective in re- versing the inhibition of the enzyme.

    The oxidative inactivation hypothesis was further tested by the model experiments shown in Fig. 5 where phenylhydrazine, but neither semicarbazide nor hydroxylamine, is shown to com- pletely oxidize cysteine at a concentration of 0.03 M. This observation, along with the experiments reported in Table VI, provides a plausible explanation for the fact that phenylhydra- zine does not inactivate cysteine-activated papain in the pres- ence of excess cysteine; i.e. sufficient cysteine remains after treatment with phenylhydrazine to either prevent oxidation of the enzyme or to reactivate the oxidized papain. Papain itself is oxidized in 1 hour at a phenylhydrazine concentration of one- tenth the amount necessary to oxidize cysteine.

    Sluyterman (26) has shown that cyanate ion inactivates papain by carbamylation at the active site thiol. The possibility that the inactivation of the enzyme promoted by oxidizing agents in the presence of cyanide is due to carbamylation by cyanate formed by the oxidation of cyanide was excluded by the following two observations. (a) The extent of r4C-phenylhydrazine bind- ing to carbamylated papain prepared from activator-free papain, which was inactivated by 20-fold molar excess of potassium cyanate was only 0.15 mole per mole of protein as opposed to an average of 0.65 mole per mole of protein when cyanide itself was present; and (b) incubation of the enzyme with cysteine after it was inactivated with hydrogen peroxide in the presence of r4C- cyanide resulted in the formation of 2.iminothiazolidine-4- carboxylic acid, presumably by the mechanism shown in Equa- tion 3.

    \ coo-

    r NH3+ SH + N=X!%H~--C.i; (3) L \ coo- \

    2-Iminothiazolidine- 4-carboxylic acid

    This process is the reverse of the mechanism of activation of papain described previously (6). This product could not have arisen from X-carbamylated papain, and its appearance proves that the cysteine used in the reactivation experiments must attack the thiocyanato moiety at the carbon rather than the sul. fur atom, since 2-iminothiazolidine-4-carboxylic acid, rather than CN-, was released.

    DISCUSSION

    The radioactively labeled mercaptans, YS-thiophenol, 3%- cysteine, and 3P-P-thioate, along with Kr4CN did not bind to the protein in sufficient quantity to account for the amount of active thiol released on activation of the enzyme by these re- agents. These results do not support models that require that the activation process consists of a noncatalytic cleavage of a simple intramolecular bond of the active thiol of papain.

    Although the evidence discussed above for an inactive form of papain in which there is a mixed disulfide formed between a sulfhydryl group on the enzyme and cysteine (6, 7) is convinc- ing, there are experiments suggesting other inactive forms of papain that must be explained. Some of these have led to proposals in which the sulfhydryl group which, after activation, becomes the catalytic nucleophile of the enzyme, is actually bound intramolecularly to some group on the polypeptide chain (5, 9, 12). It has been reported in one instance that the binding of the activator, P-thioate, was proportional to the increase in the catalytic activity of the enzyme; the conclusion being based upon observed changes in fluorescence of the protein upon activation, and on the binding of the activator to the enzyme as shown by Sephadex chromatography in 0.1 M acetic acid. In contrast to the former result, Bare1 and Glazer (8) have concluded that the activation of papain has little effect on the ultraviolet circular dichroism or the extent of perturbation of the enzymes aromatic chromophores. The experiments we have just presented show that no activator, including P-thioate, binds to papain under the activation and assay conditions described in this paper.

    We have no definite explanation for the differences between our results and those in the literature (5) with respect to the apparent binding of P-thioate. This reagent is known to attack dithio bridges in proteins (27) and such a reaction might have occurred in acetic acid with denatured protein. There is no doubt how- ever that papain activity is not dependent upon the continued presence of the activator (Table I) (2, 6, 8, 21).

    Although the inhibition of papain by carbonyl reagents has been interpreted in terms of a reaction of this reagent with a critical aldehyde moiety on the enzyme, the experiments de- scribed above show that the mode of inhibition of papain by phenylhydrazine is due to the ability of this compound to oxidize

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  • Issue of November 10, 1969 I. B. Klein and J. F. Kirsch 5935

    nanain rather than to the formation of an enzyme-bound phenyl- 4. GLAZER, A. N., AND SMITH, E. L., J. Biol. Chem., MO, 201

    hydrazone. This conclusion is further supported by the obser- vation that carbonyl reagents such as semicarbazide and hy- droxylamine, which do not have the ability to oxidize cysteine at low concentrations (Fig. 5), are not effective inhibitors of the enzyme (Table IV).

    5. (1965)

    NEUMANN, H., SHINITZKY, M., AND SMITH, R. A., Biochemistry, 6, 1421 (1967).

    6.

    7.

    The proposed structure of the phenylhydrazine-thiocyanato papain adduct shown in Equation 2 is, to a certain extent, speculative, but it is supported by the fact that phenylhydrazine will not bind to the protein unless cyanide is also present, and that these two compounds are bound in approximately equal stoichiometry (Tables II and III). The structure is, moreover, chemically reasonable since the thiocyanato carbon atom is known to form adducts under mild conditions with nitrogen nucleophiles as occurs in the cyclizations of P-thiocyanatoalanine to form 2-iminothiazolidine-4-carboxylic acid (28) and o-amino- thiocyanatobenzene to form 2-aminobenaothiazole (29). The fact that there is more cyanide bound when peroxide rather than phenylhydrazine inactivates CN--activated papain may be be- cause a similar mechanism is operating in which a second mole of cyanide attacks the carbon atom of the initially formed thio- cyanato group; a reaction which is, to a certain extent, analogous with the dimerization of HCN (30).

    KLEIN, I. B., AND KIRSCH, J. F., Biochem. Biophys. Res. Com- mun., 34, 575 (1969).

    SLUYTERMAN, L. A. AL, Biochem. Biophys. Acta, 139, 430 (1967).

    8. BAREL, A. O., AND GLAZER, A. N., J. Biol. Chem., 244, 268 (1969).

    9. M~RIH~RA, K., J. Biochem. (Tokyo), 62, 250 (1967). 10. BERGMANN. M.. AND Ross. W. F.. J. Biol. Chem.. 111. 659

    11. (1935).

    ,

    BERGMANN, M., AND Ross, W. F., J. Biol. Chem., 114, 717 (1936).

    12. MORIHARA, K., MONNA, K., AND AKABORI, S., Proc. Jap.

    13. 14.

    Acad., 41, 828 (1965). ARERFELDT. S.. Acta Chem. Stand.. 14. 1980 (1960). BODANZKY, M.; AND DU VIGNEAUD V.,.Biochem. Prep., 9, 110

    (1962). 15. MASUDA, T., J. Biochem. (Tokyo), 46, 1489 (1959). 16. HENRY, A. C., AND KIRSCH, J. F., Biochemistry, 6, 3536 (1967). 17. GLAZER, A. N., AND SMITH, E. L., J. Biol. Chem., 236, 2948

    (1961). 18. LOWRY. 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL,

    The treatment of activator free papain with oxidizing agents leads to an inactive form of the enzyme that cannot be reacti- vated with cysteine. The nature of this species is unknown, but it presumably arises through the conversion of the active site cysteine residue to a sulfinic or a sulfonic acid. In the presence of activating nucleophiles, the intermediate papain sulfenium ion is trapped (Equation 2), preventing further irreversible oxi- dation.

    19. 20. 21.

    R. J.; J. Biol. Chem., 193; 265 (1951). HALL. T. C.. AND COCKING. E. C.. Biochem. J.. 96. 626 (1965). KIRS~H, J. f., AND IGELSTR~M, M:, Biochemist&, 5; 783 (1966). SOEJIMA, M., AND SHIMURA, K., J. Biochem. (Tokyo), 49, 260

    (1961). 22. 23.

    24.

    25.

    Acknowledgments-We are grateful to Dr. W. Dixon Riley for the gift of Tracerlab 14C-phenylhydrazine, and we wish to thank Mrs. Patricia Hinkle for preparing the carboxamidomethylated derivative of papain.

    REFERENCES

    26.

    27.

    28.

    ELLMAN, G. L., Arch. Biochem. Biophys., 82, 70 (1959). LIGHT, A., FRATER, R., KIMMEL, J. R., AND SMITH, E. L., Proc.

    Nat. Acad. Sci. U. S. A., 62, 1276 (1964). DRENTH, J., JANSONIUS, J. N., KOEKOEK, R., SWEN, H. M.,

    AND WOLTHERS, B. G., Nature, 218, 929 (1968). BENDER, M. L., BEGUE-CANTON, M. L., BLAKELEY, R. L.,

    BRUBACHER, L. J., FEDER, J., GUNTER, C. R., KEZDY, F. J., KILLWEFFER, J. V., MARSHALL, T. H., MILLER, C. G., ROESKE. R. W.. AND STOOPS. J. K.. J. Amer. Chem. Sot., 88. 5890 (1966).

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    SLUYTERMAN, L. A. AX., Biochem. Biophys. Acta, 136, 439 (1967).

    NEUMANN, H., GOLDBERGER, R. F., AND SELA, M., J. Biol. Chem., 239, 1536 (1964).

    SCHOBERL, A., KAWOHL, M., AND HAMM, R., Chem. Ber., 84, 1. KIMMEL, J. R., AND SMITH, E. L., J. Biol. Chem., 207, 515 571 (1951).

    (1954). 29. SPRAGUE, J. M., AND LAND, A. H., in R. C. ELDERFIELD (Edi- 2. SANNER, T., AND PIHL, A., J. Biol. Chem., 238, 165 (1963). tor), Heterocyclic compounds, Vol. 6, John Wiley and Sons, 3. SMITH, E. L., AND KIMMEL, J. R., in P. D. BOYER, H. LARDY, New York, 1957, p. 585.

    AND K. MYRBACK, (Editors), The enzymes, Vol. 4, Ed. 2, 30. WADSTEN, T., AND ANDERSSON, S., Acta Chem. Stand., 13,1069 Academic Press, New York, 1960, p. 133. (1959).

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  • Ira B. Klein and J. F. Kirsch

    Reagentsof the Active Enzyme by Carbonyl The Activation of Papain and the InhibitionMACROMOLECULES:CHEMISTRY AND METABOLISM OF

    1969, 244:5928-5935.J. Biol. Chem.

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