TE JOURNAL OF BIOLOGICAL CHEMISTRYVol. 241, No. 16, Issue of August 25, pp. 3811-3817, 1966

Printed in U.S.A.

Transesterification Reactions Catalyzed by Papain*(Received for publication, March 24, 1966)

A. N. GLAZER

From the Department of Biological Chemistry, School of Medicine, University of California, Los Angeles, Cal-ifornia 90024

SUMMARY

The hydrolysis of a series of esters of benzoyl-L-arginineand glycylglycine by papain has been examined in mixturesof water with various alcohols in the pH range of 5 to 6. Thepresence of straight chain alcohols resulted in an apparentinhibition of acid release. It was shown that this inhibitioncould, in part, be accounted for by papain-catalyzed trans-esterification reactions between the various substrates andthe alcohols studied. The products of transesterification,transamidation, and hydrolysis resulting on incubation ofpapain with esters of glycylglycine were separated andidentified, and the kinetics of their formation were examined.Ficin was found to behave similarly to papain. The presentstudy, in conjunction with earlier reports, suggests that theability to catalyze alcoholytic reactions may be a property ofmany hydrolytic enzymes.

It is known from the work of a number of investigators thatmany proteolytic enzymes are inhibited by relatively low con-centrations of normal aliphatic alcohols. In 1956, McDonaldand Balls (1) showed that the liberation of titratable acid fromL-tyrosine ethyl ester by a-chymotrypsin was inhibited by aseries of primary and secondary alcohols, and were able to showthat the observable inhibition was due, at least in part, to chymo-trypsin-catalyzed transesterification reactions. Chymotrypsin-catalyzed transesterification reactions have since been reportedby a number of other investigators (2-4). Glazer showed thattrypsin (5) and subtilisins (6) from the Carlsberg, Novo, andBPN' strains of Bacillus subtilis also readily catalyzed transesteri-fication reactions.

Alcoholytic reactions are not restricted to the proteolytic en-zymes. Indeed, the first instance recorded of such a reactionwas the transformation of chlorophyll to ethyl chlorophyllide inthe presence of the enzyme chlorophyllase noted by Tswett in1908 (7) as explaining a result obtained by Borodin in 1882 (8).Among more recent examples, the following may be cited: themethanolic cleavage of adenosine triphosphate by myosin (9),the alcoholytic cleavage of cytidine 2',3'-phosphate by bovinepancreatic ribonuclease (10), and the transesterification reactions

*This investigation has been aided by Grant GM 11061 fromthe National Institutes of Health, United States Public HealthService.

between acetylcholine and various alcohols catalyzed by a choli-nesterase from Pseudomonas fluorescens (11).

These observations suggested that many other hydrolytic en-zymes may be capable of catalyzing alcoholytic reactions andthat inhibition by normal aliphatic alcohols might be regardedas a suggestive indication in this connection.

The plant proteases, papain and ficin, are known to be in-hibited by alcohols (12, 13). Since no transesterification reactioninvolving a sulfhydryl proteolytic enzyme has as yet been re-ported, it was of interest to examine these two enzymes with re-spect to their ability to catalyze such reactions.

It was found that both papain and ficin are capable of catalyz-ing transesterification reactions between ester substrates and avariety of straight chain aliphatic alcohols.

EXPERIMENTAL PROCEDURE

Materials-Papain was prepared by the method of Kimmel andSmith (14) and recrystallized three times. The protein wasstored at 4° as a crystalline suspension in saturated sodium chlo-ride. Stock solutions were prepared either by dissolving a suit-able aliquot of the suspension in distilled water, or, if a concen-trated solution of the enzyme was required, by dialysis againstdistilled water. All of the enzyme solutions were kept in an icebath and discarded after 24 hours. Less than 5 % loss of activitywas observed over this time period. Papain concentrations weredetermined spectrophotometrically by using Elm of 25.0 at 278m,> (15). When assayed in the presence of 0.004 M 2,3-dimer-capto-l-propanol, with 0.05 M benzoyl-L-arginine ethyl ester assubstrate at pH 5.5 and 37 °, the papain preparation had a C1 of1.9 0.1 (mean of 10 determinations).

A suspension of twice-crystallized ficin in 0.001 M ethylene-diaminetetraacetic acid, lot 660, was obtained from Worthington.Stock ficin solutions were obtained by the addition of suitablealiquots of the suspension to 0.05 M acetate buffer at pH 5.0.Traces of insoluble material were removed from the resultingsolutions by high speed centrifugation and the solutions werestored in an ice bath. Ficin concentrations were determinedspectrophotometrically on suitably diluted aliquots by using therelation A2so X 0.44 = milligrams of protein per ml (16).

Chromatographically pure a-N-benzoyl-L-arginine methylester HC1, lot N-1365, and a-N-benzoyl-L-arginine, lot G2336,were obtained from Mann and a-N-benzoyl-L-arginine ethylester.HCl, lot 611282, was from Calbiochem. Glycylglycineethyl ester HCI, lot R-4093, was obtained from Cyclo ChemicalCorporation. Glycylglycine methyl ester HC1, lot P2006, wasobtained from Mann. This preparation was contaminated with

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Transesterification by Papain

TABLE I

Chromatographic and electrophoretic mobilities of glycylglycine,tetraglycine, and certain of their esters

Distance of migration

CompoundChromatog- Electrophoresis

b

raphya

cm

Glycylglycine ................. : ....... 7.7 6.8Tetraglycine .......................... 5.2 6.7Glycylglycine methyl ester ............ 15.4 31.5Glycylglycine ethyl ester ............. 20.7 30.2Glycylglycine n-propyl ester .......... 26.1 28.1Glycylglycine n-butyl ester ........... 30.2 26.8Glycylglycine n-amyl ester ............ 33.0 25.5Tetraglycine methyl ester ............. 10.7 24.1Tetraglycine n-amyl ester ............. 24.9 20.7

a Chromatography on Whatman No. 3MM paper in n-butylalcohol-acetic acid-water (200:30:75, v/v) for 15½ hours.

b Electrophoresis on Whatman No. 3MM paper in pyridine-acetate buffer, pH 4.6, for 35 min at 60 volts per cm.

approximately 1.5% glycylglycine and was used without furtherpurification. Glycylglycine, triglycine, and tetraglycine wereobtained from Nutritional Biochemicals. Hexaglycine was ob-tained from Cyclo Chemical Corporation.

Benzoyl-L-arginine butyl ester hydrochloride was synthesizedby the general procedure for the preparation of esters of benzoyl-L-arginine described by Bergmann, Fruton, and Pollok (17).The crystalline hydrochloride melted at 142 to 142.5° .

C17H27N403C1

Calculated: C 55.05, H 7.35Found: C 55.07, H 7.37

Synthesis of n-Propyl, n-Butyl, and n-Amyl Esters of Glycyl-glycine-Glycylglycine (10 g) was suspended in 180 ml of theappropriate normal alcohol and the suspension was chilled in anice bath. Dry HC1 gas, 12.5 g, was slowly bubbled in and themixture stirred at 500 for 90 min (or longer if the glycylglycinehad not gone completely into solution at the end of this time).The reaction mixture was then chilled in a Dry Ice-alcohol bathand the product collected on a sintered glass filter. The productwas washed with the appropriate ice-cold alcohol followed byanhydrous ether. It was then stored in a vacuum over P205.Care was taken to exclude moisture from the reaction mixturethroughout the preparation.

Analysis of glycylglycine propyl ester hydrochloride, m.p.156-157 ° is as follows.

C7Hs1 N203C1

Calculated: C 39.93, H 7.20Found: C 39.79, H 7.17

Analysis of glycylglycine butyl ester hydrochloride, m.p.153-154 °, is as follows.

CsH17N20 3C1

Calculated: C 42.76, H 7.63Found: C 42.86, H 7.80

Analysis of glycylglycine amyl ester hydrochloride, m.p. 126-127, is as follows.

CgH,gN 203C1

Calculated: C 45.30, H 8.04Found: C 45.39, H 7.90

The n-propyl, n-butyl, and n-amyl esters of glycylglycinsynthesized by the above procedure were found to be pure ajudged by chromatography in n-butyl alcohol-acetic acid-wate(200:30:75, v/v), n-butyl alcohol-pyridine-acetic acid-wate(15:10:3:12, v/v), and by high voltage electrophoresis at plI4.6.

Tetraglycine methyl ester and tetraglycine amyl ester weresynthesized by the procedure described above for the preparatiof glycylglycine esters. The products were purified by chromatog.raphy in n-butyl alcohol-acetic acid-water (200:30:75, v/v),These derivatives were used solely for the comparison of theirchromatographic and electrophoretic mobilities with those of theproducts of enzymatic transamidation reactions.

Analytical reagent grade alcohols were used throughout andwere obtained from the following sources: methyl alcohol, J. TBaker; ethyl alcohol, United States Industrial Chemical Company; n-amyl alcohol, Mallinckrodt; and the other alcohols usedMatheson, Coleman, and Bell.

Determination of Esterase Activity-The rates of substrate hydrolysis were determined with the aid of a Radiometer pH-statmodel TTTlc, equipped with a model SBR2 recorder and a thermostated reaction vessel. The volume of the reaction mixturewas 5 ml, and the titrations were performed at 370 with either 0.or 0.25 N NaOH as titrating agent. The details of the compoitions of the various reaction mixtures used are given in the appropriate places in the text.

Chromatographic and Electrophoretic Separations-The variousesters of glycylglycine could all be separated by one-dimensionaldescending chromatography in n-butyl alcohol-acetic acid-wate(200:30:75, v/v) on Whatman No. 3MM paper for 15½ hours orby high voltage electrophoresis at pH 4.6. The chromatographyand electrophoretic properties of the various compounds ex,amined are listed in Table I.

Separation of a-N-benzoyl-L-arginine and its methyl and butyesters can be readily achieved by electrophoresis at pH 4.6 oWhatman No. 3MM paper at 60 volts per cm for 60 min. Underthese conditions, a-N-benzoyl-L-arginine moved 9.1 cm, thbbutyl ester, 27.3 cm, and the methyl ester, 30.5 cm. The methyand ethyl esters of a-N-benzoyl-L-arginine did not separate comrpletely in this system.

Kinetics of Transesterification and Transamidation-The Idnetics of these reactions was followed by allowing the enzymaticreaction to proceed for various suitable time intervals and thestopping it by the addition of sufficient 1 N HC1 to bring the lilto 2.4. The acidified mixtures were immediately frozen. Underthese conditions, no detectable spontaneous hydrolysis of benzoyl-L-arginine esters occurred over a period of at least 4 hours 0of glycylglycine esters over a period of at least 24 hours. At tbend of the experiment, appropriate aliquots of the acidified miitures were subjected to either electrophoretic or chromatograpbseparation. Several standards of different concentrations of thappropriate compounds were included on each chromatogramelectrophoretogram. At the conclusion of the separation prcedure, the papers were dried for 30 min at 60°. In the casethe glycylglycine compounds, the papers were then dippedcadmium-ninhydrin reagent (18). They were then immediateplaced in a drying oven at 800 for 30 min; following this the eol

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A. N. GlazerIssue of August 25, 1966

development was allowed to go to completion by leaving the chro-matograms in an ammonia-free atmosphere for 48 hours. Thered bands were then cut out, the color was eluted with absolutemethyl alcohol, and the amounts of the various components inthe reaction mixture were determined from the absorbance at500 m/. In this procedure, the color yields given by the variousglycylglycine esters and by glycylglycine were equal within ex-perimental error. The color yield of tetraglycine esters wasassumed to be equal to that of tetraglycine. In the case ofseparations involving a-N-benzoyl-L-arginine and its esters, thepapers were dipped in a modified Sakaguchi reagent (19) and theamounts of the various components determined qualitatively bycomparison of color intensities with those given by knownamounts of standards.

RESULTS

Hydrolysis of oc-N-Benzoyl-L-arginine Esters by Papain inWater-Alcohol Mixtures-On incubation of the butyl and methylesters of benzoyl-L-arginine with papain in water-alcohol mix-tures, products of transesterification could be readily detected.The proportion of transesterification product to hydrolysis prod-uct observed under various conditions is given in Table II.The size of the alcohol used appeared to be an important factorin determining the extent of transesterification. Thus, from aconsideration of solely the Km and Vmax values for the benzoyl-L-arginine esters (Table III), one would expect that the greatestaccumulation of transesterification product would be found onincubation of the butyl ester with papain in presence of ethylalcohol since the Km for benzoyl-L-arginine ethyl ester is consider-ably higher than that for the butyl ester and the V..m values areapproximately equal; whereas, in actual fact, it is found thatmethyl alcohol is considerably more effective than ethyl alcohol inthe transesterification reaction. No accumulation of the butylester was detected on papain-catalyzed hydrolysis of benzoyl-L-arginine ethyl ester in presence of 0.55 M n-butyl alcohol (TableII). This result is consistent with the low Km value of the butylester as compared to that of the ethyl ester and.the large size ofn-butyl alcohol. The relatively low solubility of n-butyl alcoholin water precluded studies in solutions of considerably higheralcohol content.

Hydrolysis of Glycylglycine Esters by Papain in Aqueous Solu-tion-In earlier work with trypsin' (5), it was found that the rateof transesterification observed, at a given alcohol concentration,with "good" substrates, such as benzoyl-L-arginine ethyl ester,was lower than that observed with "poor" substrates, such as L-

lysine methyl ester. This observation prompted a search for"poor" ester substrates of papain. The glycylglycine esters werechosen for further study for a number of reasons. First, they arehydrolyzed by papain at rates considerably lower than the ben-zoyl-L-arginine esters. Second, the products of papain actioncould be completely and readily separated by suitable chromato-graphic and electrophoretic techniques. Last, the amounts ofthe various components of the reaction mixture could be accu-rately determined, following separation, by quantitative reactionwith ninhydrin on paper.

Papain is known to be an efficient catalyst of transamidationreactions at alkaline pH values where a significant proportion ofthe a-amino group of peptides is in the uncharged form (21-24).To minimize these reactions, all of the studies with the glycyl-

1 Unpublished observations.

TABLE II

Hydrolysis of a-N-benzoyl-L-arginine esters by papainin water-alcohol mixtures

The reaction mixture contained, initially, 0.02 M ester sub-strate in 5 ml of 0.1 M KCI-alcohol solution containing the per-centage of alcohol indicated below, 0.004 M 2,3-dimercapto-1-pro-panol, and a papain concentration of 0.04 mg per ml (introducedin 0.02 ml of water). Titration was performed at pH 5.5 and 37 °

with 0.1 N NaOH in the pH-stat. The reaction was stopped byaddition of 1 N HC1 to pH 2.4 when the base uptake indicated35% hydrolysis to a-N-benzoyl-L-arginine. The reaction mixturewas then frozen. All of the experiments were performed in dupli-cate. For the details of separation and quantitation of thecomponents of the reaction mixture see "Experimental Proce-dure."

Ratio oftrans-

Substrate Percentage of alcohol, catior-by volume product to

hydrolysisproduct

Benzoyl-L-arginine butyl ester 20% methyl alcohol -0.2Benzoyl-L-arginine butyl ester 20% ethyl alcohol 0.01Benzoyl-L-arginine methyl ester 5% n-butyl alcohol -0.01Benzoyl-L-arginine ethyl ester 5% n-butyl alcohol 0

TABLE III

Kinetic constants of papain-catalyzed hydrolysis ofca-N-benzoyl-L-arginine esters

The reaction mixture contained, initially, 0.0037 to 0.1 M sub-strate in 5 ml of 0.3 M KCI, 0.004 M 2,3-dimercapto-l-propanol,and 1.6 to 3.8 X 10-7 M papain. The rate of reaction was propor-tional to the enzyme concentration over this range. Titrationwas performed at pH 5.5 and 37° with 0.1 N NaOH in a pH-stat.Initial velocities were determined from the slopes of the zeroorder curves obtained for the initial 8 to 10%o hydrolysis as calcu-lated from the base uptake. All of the experiments were per-formed in triplicate. A molecular weight for papain of 22,000was used in the calculations.

Substrate K, Vmax

M sec-1

a-N-Benzoyl-L-argininemethyl ester ......... 0. 0066 - 0.0003 22.9 1.1

a-N-Benzoyl-L-arginineethyl ester .................. 0.022 - 0.003a 18.7 1.9

c -N-Benzoyl-L-argininen-butyl ester ............... 0.0039 - 0.0005 23.6 - 3.0

a A K, of 0.018 was reported for a-N-benzoyl-L-arginine ethylester by Smith and Parker (20) under the same conditions asabove.

glycine esters were performed at pH 5.0. At this pH, the a-amino group of glycylglycine ethyl ester, pK' (NH3+) = 7.75(25), and, of glycylglycine, pK2 = 8.25 (25), is 99.82% and99.94% in the NH3

+ form, respectively; nevertheless, papain-catalyzed transamidation reactions were found to proceed at asignificant rate at this pH. Inasmuch as the over-all qualitativepattern of results was the same for the methyl, ethyl, n-propyl,n-butyl, and n-amyl esters of glycylglycine, the discussion willdeal mainly with the results obtained with glycylglycine n-amylester.

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3814 Transesterificat

TABLE IV

Papain-catalyzed release of titratable acid from glycylglycineesters in aqueous solution at pH 5.0

The reaction mixture contained, initially, ester substrate (atthe concentration indicated) in 5 ml of 0.3 M KCI solution con-taining 0.004 M 2,3-dimercapto-1-propanol and a papain concen-tration of 0.2 to 0.3 mg per ml. Titration was performed at pH5.0 and 37° with 0.1 N NaOH in the pH-stat. The initial velocityvalues given in this table were determined from the apparent zeroorder curves obtained for the first 10% hydrolysis as calculatedfrom the base uptake. All measurements were performed induplicate.

Bas upaewth olwnggyyyieetr

Substrateconcentration

M

0.1220.0980.0490.0370.02450.0122

Base uptake with the following glycylglycine estersused as substrate:

Methyl Ethyl I n-Propyl n-Butyl I n-Amyl

meq/min/ml/mg protein/ml

0.83 1.03 4.070.68 0.81 3.430.41 0.49 2.030.34 0.390.26 0.29 1.16

5.86r4 -

0.760.670.34

0.22

TABLE V

Inhibition of papain-catalyzed release of titratable acidfrom glycylglycine methyl ester by alcohols

The reaction mixture contained, initially, glycylglycine methylester (0.125 M), alcohol (0.55 ), 2,3-dimercapto-1-propanol(0.004 M), and papain (0.22 mg per ml) in a total volume of 5.0 mlof 0.1 M KC1. Titration was performed at pH 6.0 and 37 ° with0.1 N NaOH. The percentage of inhibition was determined fromthe initial velocities of base uptake. Each value is a mean ofthree determinations.

Alcohol Activity

None ............................ 100Methyl alcohol .................... 94 1Ethyl alcohol ..................... 80 i 4n-Propyl alcohol ................. 60 l 1n Butyl alcohol .................. 34 = 1

Glycylglycine n-amyl ester was incubated with papain at pH5.0 in aqueous solution, and the reaction mixture was analyzed atvarious times during the first 35 % of substrate hydrolysis. Thereaction mixture was found to contain, in addition to thesubstrate, tetraglycine n-amyl ester, glycylglycine, tetragly-cine, and traces of hexaglycine. All of these products wereidentified by direct comparison with the authentic syntheticcompounds in both chromatographic and electrophoreticsystems. On further incubation ( 50% hydrolysis), tracesof higher glycine polymers appeared. When glycylglycine n-amyl ester (0.06 M) in 0.14 M acetate buffer, pH 5.0, and 0.004 M

2,3-dimercapto-l-propanol was incubated with papain (0.5 mgper ml) for 20 hours at 39°, the composition of the reaction mix-ture at the end of this time was found to be 90% glycylglycine,8% tetraglycine, and 2% hexaglycine. In the control mixture,from which only the enzyme was omitted, no measurable break-down of glycylglycine n-amyl ester was observed. In a separate

ion by Papain Vol. 241, No. Iro

experiment, it was found that tetraglycine was split to glycylglycine extremely slowly by papain under the above conditions

The results presented above can be readily accounted for by th5following papain-catalyzed transamidation and hydrolytic reactions.

Glycylglycine n-amyl ester + papain

[glycylglycyl-papain] + n-amyl alcohol (1}

[Glycylglycyl-papain] + H20 = glycylglycine + papain (2a)

[Glycylglycyl-papain] + glycylglycine n-amyl ester =

tetraglycine n-amyl ester + papain (2b)

Tetraglycine n-amyl ester + papain

[tetraglycyl-papain] + n-amyl alcohol (3)

[Tetraglycyl-papain] + H2 0 = tetraglycine + papain (4)

a. 4" During the initial stages of hydrolysis, Reactions 2a and 2b3.13

were found to proceed at comparable rates. The transamidation1.71 reaction (2b) is analogous to those described for papain a number0.96 of years ago by Fruton et al. (21-24), e.g.

Carbobenzoxyglycinamide + L-Leu-Gly =

carbobenzoxy-Gly-L-Leu-Gly + NI,

The reaction

papainGlycylglycine n-amyl ester + glycylglycine

tetraglycine n-amyl ester

does not appear to take place at a measurable rate alpH 5.0 even when glycylglycine is present in equimolar con-centration with the ester substrate. Since Reaction 2b,above, does proceed readily, it is clear that the ester substratecompetes successfully with glycylglycine as a replacement agentunder these conditions. This observation complements that olMycek and Fruton (24), who showed that in an equimolar mix-ture of carbobenzoxyglycinamide, Gly-Gly, and L-Leu-Gly at7.3, L-Leu-Gly inhibited the reaction leading to carbobenzoxytriglycine by 75% while the reaction leading to carbobenzoxyGly-L-Leu-Gly proceeded at the same rate as in the absence 1Gly-Gly.

No measurable amounts of free glycine or triglycine were detested in the reaction mixtures. Clearly, the ester linkage shydrolyzed very much faster than the peptide linkages. Papadid not release glycine or triglycine from tetraglycine or glycylglycine. This is in accord with the known specificity of papaon peptide substrates. Papain cleaves the peptide bond adjacento a free a-carboxyl group extremely slowly (26).

The relative affinity of papain for the various glycylglycinesters under the conditions used for this study was examined bdetermining the initial rate of production of titratable acid froeach of the Bsters over a wide range of substrate concentrationThe observed base uptake was a measure of the sum of Reaction2a and 4, i.e. of the rate of production of glycylglycine and tetrglycine. From the values given in Table IV, it may be seen thlthe relative rate of hydrolysis of the glycylglycine esters was in tbstrict order of chain length: n-amyl > n-butyl > n-propylethyl > methyl. Obviously, it is impossible to calculate frothese data alone actual Km and Vmax values for these substrat

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Transesterification by Papain

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0

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o

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I-

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0

Q

a-

TIME (IN MINUTES)

Fio. 2. Kinetics of papain-catalyzed hydrolysis of glycylglycine esters. A, glycylglycine n-amyl ester (0.1 M), KC1 (0.1 M), metha-nol (10% (v/v)), 2,3-dimercapto-l-propanol (0.004 M), and papain (0.2 mg per ml) at pH 5.0 and 37°. B, glycylglycine n-butyl ester(0.1 M) and papain (0.26 mg per ml). All other conditions were as in A. For details of determination of the composition of the re-action mixture at different time intervals see "Experimental Procedure." All determinations were performed in duplicate.

of the papain-catalyzed hydrolysis of the n-amyl and n-butylester of glycylglycine in water-methyl alcohol solutions werestudied in detail. The results are presented in Fig. 2. Fromthese results, it, may be seen that the transesterification reaction(Reaction 5) proceeds at a rate comparable to that of the hydro-lytic reaction (Reaction 2a) and to that of the transamidation re-action (Reaction 2b).

Ficin-catalyzed Transestrification and Transamidation Reac-tions-A brief survey was carried out of the action of ficin on thevarious glycylglycine esters in both aqueous and alcoholic solu-tions. The considerable similarity, which has long been recog-nized to exist between the mechanisms of action of these two en-zymes (see Reference 26 for a review), was clearly evident in thepresent study. Ficin catalyzed all of the transamidation andtransesterification reactions described above for papain (see Fig.1, for example), and showed, qualitatively, the same pattern ofinhibition by straight chain aliphatic alcohols.

DISCUSSION

In earlier studies (1-6), it has been shown that the "serine"enzymes (chymotrypsin, trypsin, and the subtilisins) are all effi-cient catalysts of transesterification reactions. The present in-vestigation has shown that the "sulfhydryl" enzymes (papain andficin) are also able to catalyze such reactions. This finding,taken in conjunction with reports of studies on a variety of hy-drolytic enzymes (7-11), strongly suggests that many morehydrolytic enzymes will be found capable of using alcohols inplace of water, and that the possibility of transesterificationshould be examined carefully whenever studies are performed inwater-alcohol media.

The transesterification reactions appear to account for onlyabout 20% of the observed inhibition by alcohols of papain-cat-alyzed release of titratable acid from esters of glycylglycine. Theeffect of the alcohols on the dielectric constant of the mediumundoubtedly plays a major role in the inhibition as shown inearlier studies on papain by Stockell and Smith (12) and Smith,Finkle, and Stockell (27). The esters of benzoyl-L-arginine havea lower K, and higher Vmax values than the amide (28). Thefailure to observe alcoholytic reactions in earlier studies withpapain (5, 12) in which benzoyl-L-arginine amide was used assubstrate was undoubtedly in part due to the failure of the prod-uct of the alcoholytic reaction to accumulate under the partic-ular conditions used. This emphasizes the importance of in,vestigating a variety of combinations of substrates, alcohols, andpH in order to rule out alcoholytic reactions whenever alcoholsare used in studies of the influence of dielectric constant on thecatalytic process.

Acknowledgments-I would like to thank Mrs. M. Desser forexpert technical assistance. I am also grateful to Dr. Emil DSmith for his interest in this work and many helpful suggestion

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A. N. GlazerTransesterification Reactions Catalyzed by Papain

1966, 241:3811-3817.J. Biol. Chem. 

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