Cbdbrmational Changes in Papain During Catalysis and
~~~r~~rn:Concentrationsof2-buturolaslowasl xlO-4~ were found to cause a timo-deptndent, essentially imversible inactivation of papain. me inactivationappears to be specific for derivativesof &butane (such as 2-butylamine, 2-butanonc, I-butanof). The kinetics of the imctivation reaction were firjt o r b in 2-butanol and first order in active enzyme. The #ieudo-fkst-ordcr rate constant for’hctivation at 25’ and pH 5.2 is 24.3 f 2.3 kf-1 mh-1, and the energy of activation is 10.1 f 1.0 kcaI/mOl. From the effect of substrate on the inactivation proctss it is concluded that papain undergoes a conformational change during the catalyzed hydrolysis of ester suhstratcs. inactivation proass is dependent on a
p p a i n is a sulfhydryl protease which catalyzes the hydrolysis of N-acyldno acid esters accordhg to Scheme I, SC”S1 Rs
k
kr
E+SeES--+EAdE+Pa
+
R
where ES = Michaelis complex and EA = acyl-enzyrne (Glazer and Smith, 1971). One of the distinguishing features of papain is its unusual stability toward denaturing teagents, e.g., urea (Sluyterman, 1967) or high concentfations of organic solvents (Drenth et al., 1968; Barel and Glazer, 1969). This characteristic may be due to its compact structure (Drenth et al., 1968). Several previous studies have shown that the presence of aIcohoIs during papain-catalyzed hydrolyses results in transesterification(My& and Fruton, 1957; Glazer, 1966; Brubacher and Bender, 1965; Fink and Bender, 1969). During an investigation of the effects of small liwnds on papainatalyzed reactions we wished to determine whether asymmetric synthesis would occur during the transesterification reaction with 2-butanol (A. L. Fink, In preparation). m e observed results, however, were rather unusual and unexpected. Rather than acting as a nucleophile to compete with water in partitioning the acyl-cnzyme in the transesterification reac$ion (Scheme II), the 2-butanol caused the inactivation of SCHEME I1
E+S
A ES
E+P2 EAhROH .).h E+PJ PI
papain. Since the effect is observed with concentrations of 2-butanol as low as 1 X 10-4 M it seems unlikely to be the usual kind of solvent-induced inactivation (denaturation) of proteins in the presence of organic solvents. A few reports ~
t Prom the Division of Natural Sciences, University of California,
Santa Cruz, California 95064. Receloed Ocrobrr 10,1972. This research was supported by Faculty Rnearch Furldr granted by the University of California at SInta Cruz.
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13, NO. 6, 1 9 7 4
group with pKaUu= 4.0 which could be the same group which is involved in the catalytic reaction. Sulfhydryl group titratim indicates that the inactivation is associated with a loss of the free thiol. The dt‘ects of Zbutanol on the uv abb)otpdon, circular dichroic, and fluorescent &on spectra of papain are consistent with a small conformational change being assoCiated with the inactivation. The following mechanimfor the inactivation process k most consistent with the cxperimental observations. 2-Butanol binds bo papain in a rapid, reversible step to form a complex which undergoes a subsequent rate-limiting change to an inactive conformation.
suggesting solvent-induced conformational effects have been made, although for much higher solvent concentrations. For example, several alcohols have been found to induce a conformational change in pepsinogen rendering it inactivatableby acid (Neumann and Shinitzky, 1971), and the increase in the rate of deacylation of ecyl-chymotrypsins in the presence of isopropyl alcohol and dioxane has been attributed to such an effect (Faller and Sturtevant, 1966). Materials and Methods Materials. Cbz-glycine pnitrophenyl 1 ester was obtained from Cyclo (lot N 1629) and recrystallized several timesfrom ethyl acetate-hexane, mp 126.5-127.5 ’. Cbz-L-lysine pnitrophenyl ester, also from Cyclo (lot N 1645), was recrystallized from acetonitrile, mp 152-154’. Cbz-glycine, Cyclo (lot N 1781A), mp 118-120°, and N-benzoyl-barginhe ethyl ester, Mann (lot N 4143), mp 131-132O, were used without further purification. Chromatoquality 2-butanol (MCB), Spectroquality 2propanol (MCB), reagent grade 1-butanol (MCB), 2-pentanol (Baker), 2-butylamine (Aldrich), and 2-butanone (Aldrich) were distilled prior to use (the central 6Ox of the distillate being used). Acetonitrile was Spectroquality (MCB) and used without further purification. Doubly distilled water was used and all buffer materials and EDTA (Baker) were reagent grade, 2-Butyl and 2-propyl Cbz-glycinate were synthesized by acid-catalyzed reactions according to the method of Greenstein and Winitz (19611). The 2-propyl ester formed white crystals, mp U).5-53.O0, and showed the expected nuclear magnetic resonance (nmr) and mass spectra. The 2-butyl ester was obtained as a pale yellow oil which was homogeneous on paper chromatography (in ethyl acetate-hexane 36:M; and absolute methanol). Nmr and mass spectral evidence were consistent with the desired structure. I Abbreviations used are: Cbz, carbobenzoxy; Cbz-GlyONph, Cbzglycine p-nitrophenyl ester: Cbz-LysONph, Cbx-L-lyrine pnitmpbcnyl ester; Cbz-GlyOBu and Cbz-OlyOPr, 2-butyl and 2.propyl ester of Cbz-Gly, respectively; Cbz-Oly, Cbz-glycine; BzAr#OEt, N-benzoyl+ arginine ethyl enter; Nbt. S,~’-dithiobis(2-nitrobentoicacid).
COWIOkYATXO#AL C H A Y O I I I N ? A ? A I N
1.80 X 1.00 X 8.70 >f 4.42 X 3.13 x 3.39 x 1.62 X
10-* 5.83 X IO-, 10-1 1-43 X 1 0 - 7 le8 3.62 X 10-7 lo-' 1.87 X 16-7 5.83 x 10-8 IO-' lo-' 4.73 x Io-' 10-4 5.83 X 10-8
5.1 X 2.3 X 2.4 X 1.0 x 7.2 x 9.4 x 4.0 X
10-1 10-1 10-1 10-1 10-1
28.2 22.5
21.7 22.6 23.0 27.7 25.0
Io-'
Ib'
Av 24.3 mum 1: Tjpical lirst-ordcr plots for the %butanol-induced inactivation of papain. %Butnnolconcentrations: (0)1.62 X M; (0)4.42 X 10-8 M; (A) 1.0 X IO-* M. pH 5.2, p = 0.05 M, 25 0.1°,2.5x lW4mEDTA.
*
Papain, as a crystalline suspension, was obtained from Worthington, activated with mercaptoethanoland purified by aftinity chromatography (Blumberg et ut., 1970). The' active enzyme was stored at 4O. Enzymatic activity was determined by a kinetic assay using BzArgOEt (Bender et at., 1%; Mole and Horton, 1973). Kinetic Meawements. Measurements of the rate of release of pnitrophenol were made with either a Cary 14 or Beckman Kintrac spectrophotometerat 347.6 nm. The hydrolysis of the 2-butyl and 2-propyl esters was determined using a pH. Stat (Radiometer). All kinetic measurements were m d e at 25.0 0.lo, pH 5.2, p = 0.05 M, and [EDTA] = 2.5 X IO-' M. Stock solutions of substrate were prepared in acetonitrile such that the final acetonitrile concentrationwas 3.2'7,. The effect of pH and temperature on the rate of inacthation was measured as follows. The enzyme was incubated in buffer (citrate, acetate, phosphate, or borate) of appropriate pH and temperature, containing 2-butanol, 2.5 X IO-' M EDTA, 1 X lo-' M cysteine, and p = 0.05 M. Aliquots were removed at appropriate time intervals and immediately assayed with Cbz-LysONph under the standard assay conditions (pH 5.2, 2.5 X 10-4 M EDTA, 25.0 O.loj. Controls were run to determine the rate of inactivation in the absence of 2-butanol. Typical enzyme concentrationswere 1.5 X IO-' M. Effect of Substrate on Inactioation. In these experiments 2-butanol was added during the papain-catalyzed hydrolysis of Cbz-LysONph under zero-order conditions. The rate of inactivation was obtained by comparison of the curve resulting from addition of 2-butanol to the identical reaction with the omission of 2-butanol. The rate of inactivation was also determined under identical conditions but with the substrate omitted. Typical concentrationsused were Eo = 3.6 X IO-' M, 2-butanol = 8.7 x 1 0 - ' ~ Cbz-LysONph , = 3.2 X lo-' M . Spectruf Efmts. The uv absorption difference spectrum was obtained using the method of Herskovits (1967). Fluorescent measurements were made on an Hitachi-Perkin-Elmer Model MPF-2A fluorescence spectrophotometer with excitation at 290 nm. CD measurements were made on a Jasco Model J20 spectropolarimeter.
*
*
Results Initial Obserwtions of the Efect of 2-Butanol on the Rate of Papain-Catalyzed Hydrolyses. When the papain-catalyzed hydrolysis of Cbz-glycine pnitrophenyl ester was studied in
"k0bsd is the observed Rtst-orda rate constant fiH the inactivation reaction, CoXTected for non-2-butanol loss of activity occurring under the same conditions. pH 5.2, p 0.05~,25.0*0.1~,2.5XW'MEDTA.
-
the presence of 1 X IO-* M 2-butano1, the reaction was found to be inhibited in a rather peculiar manner. The reaction began at a reduced rate and within a few seconds the rate of formation of pnitrophenol decreased very rapidly and the reaction appeared to stop. Very similar results were obtained using Cbz-L-lysine pnitrophenyl ester as substrate, and with 2-butanol over a range of concentrations from 1 x 10-1 to 5 x 1w'M. In order to determine whether the 2-butyl ester of Cbzglycine was being formed by transesterification and acting as a competitive inhibitor we examined the effect of Cbz-GlyOBu and Cbz-GlyOPr on papain-catalyzed hydrolyses. C ~ P GlyOBu was found to have an inhibitory effect on the papaincatalyzed hydrolysis of Cbz-LysONph; however, the effect was much less than that observed with the correspondingconentration of 2-butanol. and the kinetics were inconsistent with competitive and noncompetitive inhibition. By studying the effect of papain on Cbz-GlyOBu using the pH-Stat it was observed that Cbz-GlyOBu was a substrate far papain. The reaction did not follow Michaelis-Menten kinetics, presumably because the 2-butanol liberated by the reaction was exerting its inhibitory effect. The limited solubility of Cbz-GlyOPr precluded precise determination of KI.Using Cbz-GlyONph as substrate, Ki was estimated to be approximately 1 X 10-: M ([Cbz-GlyOPrl = 1.0 X 1 0 - 4 M). CbzGlyOPr was hydrolyzed slowly by papain. Time-Dependence of 2-Butanol-Induced Inactivation. The effect of various concentrationsof 2-butanol at zero.time on the papain-catalyzed hydrolysis of CbtLysONph was investigated. At concentrations below 1 X lo-* M there was no detectable inhibitory erect at r 0. At higher concentrations such an effect was noted, e.g., at 1.0 M 2-butanol inactivation was complete in less than 1 sec. The effect of incubating papain with 2-butanol for various time intervals prior to adding substrate was studied. The data, Figure 1, show that the inactivation of papain is time dependent and that the rate of inactivation increases with increasing concentration of 2-butanol. Examination of the rates of inactivation of papain showed them to be first order in enzyme concentration for at least 90'7,of the reaction (Figure 1). The observed tirst-order rate constants for inactivation as a function of 2-butanol concentration (corrected for m - 2 butanol inactivation) are shown in Table 1. 5
B I O C H E M I S T R Y , VOL. 13, NO. 6, 1 9 7 4
1191
-om -0.006
230
260
270
290
X (nm)
Uv absorption difference spectrum of Zbutlnd-inactivated papain. %Butanol = 1.25 x 10-1 M, papain = 8.33 X IO-' M,PH5.2, 25.0°.
PIQURE 4:
+
- ~ " x lo3
~ i o ~ l2:l eArrhenius plot for the 2-butanol-induced inactivation of papain. Conditions: 1.5 x 10-2 M 2-butanoh 5.0 x IO-' M papain, 2.5 X IO-~MEDTA, 1 X IWMcySkine.
At concentrations of 2-butanol higher than 5 X lo-* M (and enzyme concentrations 4 x 10-0 M) the rate of inactivation was too rapid to determine by the procedure used. At much higher enzyme and 2-butanol concentrations the inactivation was slower although still first order for 90% of the reaction. For 4.3 X 10-1 M 2-butanol and 7.5 X 1W6M papain kobad = 0.28 min-1. From Table I one would calculate a value.of 10.2 min-1. At 1 x 1 0 - 6 M 2-butanol there was no detectable 2-butanol-induced inactivation over a 100-min
period. Irreversibility of the Inactimtion. In order to determine whether the inactivation was reversible, the effect of diluting the 2-butanol concentration to a negligible value and of removing the 2-butanol from the enzyme by gel filtration was examined. In a typical experiment 2-butanol (6.4 X lo-* M) was incubated with papain (1.5 X 10-6 M) at 25.0'. pH 5.2, until inactivation was complete. The solution was then diluted to a 2-butanol concentration of 2.1 X 1 0 - 6 M and papain = 4.9 X 10-0 M. The solution was then assayed at intervals over a 60-min period, but no activity was detected. Since control experiments indicated that the final concentration of 2-butano1 did not cause detectable inactivation over the time period involved, and a control experiment omitting the 2-butanol showed retention of >90% enzymatic activity, the results indicate the inactivation is irreversible. This result was corroborated by the gel filtration experimnts in which Sephadex G-25 was used to separate the inativated enzyme from 2-butanol. The eluted enzyme fraction showed no activity over a 40-min period, whereas activity was present in the control experiment in which the 2-butanol was omitted. However, after a 20-hr period at 0' the 2-butanol-treated enzyme fraction showed the return of 15% of the activity of the control at that time.
I
6
I
I
I
7
8
9
PH
The pH dependence of 2-butanol-induced inactivation of papain. The points are experimental. the curve is calculated for p& = 4.0. 2-Butanol = 1.5 X lo-* M, papain = 3.0 X M, EDTA = 2.5 X 10-4 M, 25.0 .f 0.1 FfOURE 3:
'.
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B I O C H E M I S T R Y ,VOL. 13, NO. 6, 1 9 7 4
Effeteof Substrate on Inactivation. In order to determine the effect of the prewnce of substrate on the 2-butanolinduced inactivation, the 2-butanol was added during the course of the catalytic readion under substratesaturating conditions (S>> Km,zcfro-order kinetics). The rate of inactivation under these conditions was then compared with that obtained under identical conditions, but without substrate, For Cbz-LysQNph as substrate kobsd for inactivation WRS found to be 3.5 min-1 while under identical conditions in the absence of Cbz-LysONph kobsd was 0.19 min-1. The substrate therefore caused a 20-fold increase in the rate of inactivation. In the presence of the less specific substrate CbzGlyONph a threefold increase in the rate of inactivation was found. Effect of Temperature and pH.Figure 2 shows the effect of temperature on the first-order rate constant for inactivation. The values calculated for AH* and AS were found to be 10.1 1.0 kcal/mol and -29 eu, respectively, at 25'. The effect of pM on the rate of inactivation is shown in Figure 3 where the solid line is drawn for a pK of 4.0.These experiments were carried out in both the presence and absence of cysteine and no differences were observed. Effectof2-Butanol on the Uv,Fluorescence,and C D Spectra ofpapain. The effect of 2-butanol on the uv absorption properties of papain was examined using difference spectra. No significant difference spectrum was observed although a small positive deviation occurred at wavelengths below 250 nm (Figure 4). The addition of 2-butanol (final concentration 5.0 X lo-' M) to papain (2.7 X 10-6 M) caused time-dependent changes in the fluorescence emission spectrum at pH 5.2. The changes paralleled the rate of inactivation and ceased when inactivation was complete. The addition of another aliquot of 2-butanol caused no further change. The spectrum of the fully inactivated enzyme showed a 7.5 increase in intensity and a red shift in the A,, from 336.5 to 337.8 nm compared to the native enzyme (uncorrected values). No significant effects of 2-butanol on the CD spectrum of papain were observed in the 310-250-nm range. At lower wavelengthsa decreasein ellipticity was observed (Figure 5). Efecr of Related Compounds on Papain. Several compounds of related structure to 2-butanol were examined for a similar effect. The results are shown in Table XI and indicate that the inactivating effect is peculiar to four-carbon chain compounds, the nature and position of the substituent having only minor effect. Varying the ionic strength (0.003-0.3 M) and EDTA concentration (0-2.5 x 10-4 M) had no significant effect on the rate of inactivation. No uptake or release of protons was observed when 2-butanol was added to papain at pH 4.5. Various tests were performed in order to make sure that the inactivating effect was due to 2-butanol and not to some
*
*
C O N I O R Y A T I O N A L CHAblOEL) IN P A ? A I X
TABU
n: Rata of Xnrctivatiw of
pounds..
.
"IRE
*tb"d PH 5.2,
5: Circuhr dichroism dit€amcc spectrum for Zbutnndpapain. [E] 1.0 X lo-' M, 2-butand p 1.5 X lo-' M, 25.0°; path length; 1.0 cm for 250-310 nm, 0.1 cm for
215-250nm.
impurity. For example, several experiments were carried out in the presence of cysteine in which the concentration was varied from 0-5 x 10-4 M. The presence of cysteine had a negligible effect on the rate of inactivation. Different lots of 2-butanol were also used and gave identical results. Sdfiydryf Group Titration. The effect of 2-butanol-induced inactivation on the concentration of titratable thiol groups in papain was determined using N b s as the titrant (Ellman, 1959). The control reaction in which 2-butanol was omitted showed 0.9 SH group per mole of active enzyme whereas the 2-butanol-inactivated enzyme showed no titratable thiol. The 2-butanol solutions in the absence of enzyme had no effecton Nbs2.
2-B~m01 l-Butan01 2-Butanone 2-Butylamine 2-Propanol 2-Penmol
3.2 X 3.2 X 5.0 x 3.2x 5.0X 8.3 x
1W' 7.5 X 10-*
1.0
la* 2.6 X 1 0 - 8 18-1 0.43
0.35 0.34
1W*
0.28
10-2
2.1 0
16-8 5.1
0.5
x
10-2
0
x lo-*
7 x IO-*
-
0,025
