Contributions to the S'-subsite specificity of papain
Matthias Schuster a, Volker Kasche b and Hans-Dieter Jakubke ~'
a Department of Biochemistry, Biosciences Dirision, Leipzig Unirersity. leipzig (Germany~ and h Bioteclmoh~g,y Department, Technical UnirersiO" Hamburg-l larburg, Hamburg (Germany)
(Received 14 October ltJgl ) (Revised manuscript received 27 December ItJgl )
The product ratio was analyzed for the papain-catalyzed acyl transfer from the specific acyl donor MaI-Phc-Ala-OEtCI to various nuclcophilic amino components, ranging from amino acid amides to tripeptide amides. The data obtained arc discussed in tcrm.~ of binding specificity. From the structure-activity relationships for the S'~-P~ interaction it follows that only three methyl(cne) groups can be accommodated in the S', subsite. Hydrophilic side chains arc bound boner to S'~ than indicated by their hydrophobicities. Negatively charged amino components are inefficient deacylating agents. However, there was no evidence for electrostatic contributions to the nucleophilc binding. Amino components with bulky hydrophobic amino acid residues in the P_~ and in the P.~ position, respectively, are preferentially bound to MaI-Phe-Ala-papain. The results of this study can bc applied to the planning of papain-catalyzed peptide .synthesis reactions.
I n t r o d u c t i o n
The cysteine proteinase papain (EC 3.4.22.3.), con- tained in the latex of Carica papaya, is one of the most thoroughly investigated proteolytic enzymes. The pa- pain-catalyzed hydrolysis of peptidc and ester sub- strates follows the three-step mechanism represented in Scheme I.
From the specificity constants k 2 / K s for the hydro- lysis of 40 diastereomeric alanine peptides Schechter and Berger [1] concluded that the extended active site of papain covers seven distinct binding subsites each accommodating one amino acid residue of a peptide substrate. The subsites St-S 4 interact with four residues N-terminal of the scissile bond (P~-P4), the subsites S~-S~ with three residues C-terminal of it (P(-P;). A preferential binding of Phe ili Pz was reported by the same authors [2]. This preference and the S2-P 2 hydro- gen bond are supposed to determine the specificity of the members of the papain superfamily [3]. Recently,
Correspondence: H.-D. Jakubke, Department of Biocheh+.istry, Bio- sciences Division, University of Leipzig, Talstrasse 33, D-O-7010 Leipzig, Germany,
incremental specificity energies of papain-substrate in- teractions have been reported [4]. Scvcral interactions have been shown to display a strong interdependence.
QSAR studies of hydrolysis experiments [5,6] as well as investigations of papain-catalyzed acyl transfer reac- tions [7-10] were performed to characterize the S'- specificity of papain. Based on the X-ray structure of benzyloxycarbonyl-u-phenylalanyl-t . -alanylmethylcne- papain [11] a hydrophobic region including the side chains of Ala-136, Trp-177 and the ~-carbon of His-159 was suggested [5] to bind the leaving group portion of ester substrates. This region might be identical with the S'j subsite [10] as indicated by the high specificity of papain for hydrophobic residues in P~ [8,10,12-14].
An advantageous approach for studying the S'~ sub- site specificity of serine proteinases is based on the aminolysis of acyl enzymes by nucleophilic amino com- ponents as demonst ra ted for a-chymotrypsin [18], trypsin [19], clastasc [20,21], V8 proteinase [22] and an G l u / A s p - C specific proteinase from Actmomyces sp.
K.~ k. k~ E + S z - '= ' ° ES---I=--~EA + ' E + P 2
1 ,
Pt
Scheme I. Papain-catalyzed hydrolysis of i~ptide and ester sub- strales (E = enzyme: S = acyl donor: ES = Michaelis complex: EA --
Scheme II. Papain-catalyzed acyl transfer reaction (N = nucleophile: EAN = acyl-enzyme-nucleophile complex: P.~ = acyl transfer product).
[23]. It can be used for the characterization of the nucleophile specificity of papain, too, since papain- catalyzed acyl transfer reactions are represented by the same minimal reaction mechanism (Scheme It).
in this study we have analyzed papain-catalyzed acyl transfer reactions to 29 nucleophilic amino compounds ranging from amino acid amides to tripeptide amides. The results have been used to analyze the structure-ac- tivity relationships for the deacylation of MaI-Phe- Ala-papain by amino acid amides and for S~ (i = 1-3) subsite mapping.
Materials and Methods
Chemicals Papain (prepared following the method of Kimmel
and Smith [15])was purchased from Boehringer (Man- nheim, Germany) and used without further purifica- tion. Acyl donor esters were synthesized using chemi- cal standard procedures and showed one peak in HPLC and TLC. Mal-Phe-Ala-OMe (Mp: 155-150°C; [a]~: 65.7 ° (c = 1, methanol); Calcd.: C, 58.61; H, 5.79; N, 8.04; Found: C, 58.33; H, 5.87; N, 7.84). MaI-Phe-Ala- OEtCl (Mp: 98-99°C; [a]~: -45.7 (c = 5, methanol); Calcd.: C, 54.49; H, 5.33; N, 7.06; Found: C, 53.86; H, 5.38; N, 6.91). Nucleophilic amino compounds were either from Bachem (Germany), from Reanal (Hunga- ry) or from our collection.
Hydrolysis experiments The papain-catalyzed hydrolysis of Mal-Phe-Ala-
OMe and MaI-Phe-Ala-OEtCl was performed at 25°C and pH 9.0 in 1 ml 0.2 M KCI (containing 5 mM dithioerythritol). Enzyme concentrations varied be- tween 60-100 nM. Substrate concentrations ranged from 1/5 K M to 5 K M. Papain was preincubated in the presence of 5 mM dithioerythritol for 5 min before adding it to the reaction mixture. The hydrolysis was followed by automatic titration ( ' ITr l , Radiometer, Denmark). Papain stock solutions were prepared daily in degassed water. The normalities of the stock solu- tions were obtained according to the method of Kitsch and Igelstr~m [16]. Kinetic parameters were calculated from the initial velocities and represent the mean of at least two determinations.
Acyi transfer experiments A~..d transfer experiments were performed at 25°(2
in 0.2 M KC! (containing 5 mM dithioerythritol) at pH 9 using a TTT1 pH-stat in 0.5 ml vols. Papain was activated as described above. Stock solutions of the acyl donor esters in DMSO and of the nucleophiles in water were used. The final substrate concentrations were 0.2 mM in partitioning experiments and 2 mM in the progress experiments. The DMSO content of the reaction mixture was 1% or lower. Nucleophile con- centrations were varied between 0.1-200 raM. The reactions were started by addition of 3-5/z i of papain after equilibration of the reaction mixture. After 2-5 rain reaction time 100 #l of the reaction mixture were analyzed by HPLC. Only initial product ratios were used in the calculations.
HPLC analysis HPLC was performed using a LKB gradient system
(Sweden) equipped with a SIC (Japan) chromatocorder and a Pep-S column (LKB Pharmacia Biotechnology, Sweden). Absorbance was monitored at 255 nm. The ratios of the absorption coefficients of the hydrolysis and aminolysis products were obtained as described earlier [17]. Isocratic elution was performed at 25°C using mixtures of 100 mM phosphate buffer (pH 6.5, containing 10 mM tetrabutylammonium hydrogensul- fate) and methanol.
Results and Discussion
Acyl donor substrates In contrast to scrine proteinases papain exhibits an
enhanced catalytic activity towards amide substrates [24]. Therefore, it is necessary to perform acyl transfer reactions using acyl donor esters with highly specific leaving groups in order to suppress secondary reactions of the peptide products. The kinetic parameters of the hydrolysis of Mal-Phe-Ala-OMe and Mal-Phe-Ala- OEtCI are listed in Table I. Due to its higher speci- ficity constant Mal-Phe-Ala-OEtCI was used as acyl donor in our experiments. It guarantees an efficient occupation of the S~ and S 2 subsites. Secondary con- versions of the peptide product were found to be neglectable as long as at least 30% of the initial con- centration of Mal-Phe-Ala-OEtC! is present in the reaction mixture.
T A B L E 1
Kinetic constants for the papain-catalyzed Iwdrolysis of Mal-Phe-Ala- OMe and MaI-Phe-Aia-OEtCI (25°C; pH 9.0; 0.2 M KCl).
Substrate k cat KM k~.-,t/KM (s- I ) (mM) ( M - t s - I)
Deacylation kinetics The ratio of the initial rates of hydrolysis d[PE]/dt
and aminolysis d[P3l/dt of the acyl donor depends on the nucleophile concentration as given by Eqn. 1
d[P2]/dt p
d[P3l /d t [N] ( I )
were p is the partition constant [25] characterizing the efficiency of the nucleophile under investigation in the deacylation of the acyl enzyme. Eqn. 2 gives the depen- dence of p on [N] according to Scheme II:
ks p = ~-~INI+ 4Ks (2)
k 3 is constant for a given acyl enzyme. Therefore, the term KNka/k4 reflects the energy of activation of the reaction of the free acyl enzyme with the nucleophile. As follows from thermodynamic considerations [26] and has been demonstrated in a-chymotrypsin- catalyzed reactions [27], the S' specificity of a pro- teinase is expressed in the deacylation step of acyl transfer reactions. The second-order rate constants k4/KN measured for a series of nucleophiles in aeyl transfer reactions using the same aeyl donor are pro- portional to the specificity constants k2/Ks for the acylation of the enzyme by the corresponding peptide products provided that the overall equilibrium con- stants for the hydrolysis of these peptide products are identical. Since the equilibrium constants of peptide hydrolyses differ only slightly, KNk3/k4 can serve as a measure of the nucleophile specificity of a serine or cysteine proteinase.
ks /k 4 in Eqn. 2 gives the ratio of the rates of hydrolysis and aminolysis of the acyl-enzyme-nucleo- phile complex.
"Recently [17], we have introduced an integrated form of Eqn. 2 given by Eqn. 3,
[P2] ks ka " + - - ' K N ln{[N]0/([Nl0- [Pd)}/ iP~] (3)
[P3] k4 k4 " "
which is valid as long as conversions of the product P3 can be neglected. According to Eqn. 3 a plot of the product ratio [Pz]/[l"3] vs. In{ [Nl . / ( [N]0 - [P3])I/[P31 yields a straight line with the slope KNk3/k4 and an intercept on the y-axis at ks /k 4.
Fig. 1 shows the plot according to Eqn. 3 for the deacylation of Mal-Phe-Ala-papain by different nucleophilic amino components. Obviously, the ks /k 4 values are too small to be measured exactly. Since this applies to the other nucleophiles under investigation, too, hereafter only Ksk3/k 4 will be discussed.
25r
T T 20 / .f :.:
l sL[ ! .~:" .... :~
a_ : ÷ i " t.._a - / ~
a. °~ 10 / t...a ~~]
/
3o- 75 . . . . . 3 0 4 0 s o . . . . 6 0
In [N] 0- [P3 ]
Fig. 1. Plot of the product ratio IP2I/IP.d versus ln{[N]0/([N] o- [P3])}/[P3] for the deacylation of MaI-Phe-Ala-papain by H-Leu-NH 2 ( • ~ • ), H-VaI-NH 2 ( v v ), H-AIa-NH,_ (o o)
and H-Nva-NH 2 (D ~ []).
Discussing the data it must be considered that KNk3/k 4 involves a contribution of both binding and the reactivity of the EAN complex.
Fig. 2 shows the Bronsted plot for the deacylation of Mal-Phe-Ala-papain by a number of L-amino acid amides. Apparently, there is no clear trend (r = -0 .22) for a relationship between free activation energy and pKg. This finding corresponds to results of Brubacher and Bender [8] who .~nvestigated nucleophiles with p K~ values ranging from 5.3 to 10.56 in the deacylation of trans-cinnamoyl-papain. Since the pK a values in Fig. 2 range only front ', .3-8.4 considerable differences in the reactivity of the acyl-enz~e-nucleophile complexes are not to be expected. Furthermore, it must be consid- ered, that the pK~ values reflect not only the nucle- ophilicity, but also solvent influences on the protona- tion equilibria. Otherwise, the decrease of p K a in the sequence H-Giy-NH 2 > H-AIa-NH z > H-Nva-NH 2 > H-NIe-NH z, i.e. as a consequence of the introduction
% N 0q
2t
T
0 4 ~ Nle I
0.0 .'- Met• Trip mArg
-0 ,4 - Phe
- 0 8 His
-1.2 ~- Thr
-16 i
-2.0 i
7.3 7 6
Nva Leu •
lie
Val
Abu
Ala
Glu • l
I
7 9 8 2 8 5
PKa
Fig. 2. Brenstedt plot for the deacylation of MaI-Phc-Ala-papain by various nucleophilic amino acid amides (pK=, at 25°C, 0.2 M NaCI),
210
of electron donating, but hydrophobic substituents, cannot be explained.
Consequently, we will discuss the deaeylation data obtained in terms of binding.
Acyl transfer to amino acid amides In Table I! the KNk3/k4 for the deacylation of
Mal-Phe-Ala-papain by various amino acid amides are compiled. The data cover four orders of magnitude indicating a marked specificity of the S'j binding sub- site. The distorted binding of D-amino acid amides to acyl papains characterises the steric requirements of this subsite [8]. The positively charged H-Arg-NH2 is bound approx. 50-times better than the negatively charged H-Glu-NH,. Generally, a prefered binding of hydrophobic residues is evident. A comparison with the data for the deacylation of trans-cinnamoyl-papain (also shown in Table II) indicates that the hydrolysis/ aminolysis ratio depends on the acyl donor too.
Fig. 3 shows a plot of -lg(KNk3/k 4) against the relative hydrophobicity ~- (derived from the hydropho- bic fragmental constants [28]) of the side chains of various amino acid amides.
There is a linear dependence of the logarithm on the hydrophobicity of unbranched hydrophobic side chains up to H-Nva-NHz(filled squares; including H- GIy-NH 2 lacking a side chain). The additional methy- lene group in Nie-NH 2 does not increase the binding
TABLE !I
K s.k,~ / k ~ of the papain-catalyzed acyl transfer to rarious nucleophilic amino acid amides.
" 25°C; pH 9.0; 0.2 M KCI. t, Calculated using data from Ref. q. ¢ pH 9.09; 250C. 'J pH 9.2; 25*C.
\
"6 T
0 8 ' "
04 -
0 0 -
- 0 4
- 0 8
- 1 2
- 1 6 -
- 2 0
. . . . . . . . . . . . . . . . . . ;
Nova Leu ~1~ i
i Met lie Tr
_- ph; ! His / Val
Tar=' / ~ t a "
Gly 1 / /
- o i 00 b 4 b : 6 i 2 .... 16 2 0 2 4
1"[
Fig. 3. Plot of - I g ( K s k ~ / k 4 ) for the deac3"lation of MaI-Phe-Ala- papain vs. hydrophobicity 7r of the side chains of the deacylating amino acid amides (,', = uncharged hydrophilic side chain; • = unbranched hydrophobic side chain or no side chain; ra = branched hydrophobic side chain; [] = main chain of a branched hydrophobic
side chain).
energy indicating that only three of the methyl(ene) groups are bound in S' !. Considering their hydro- phobicities nucleophiles with branched side chains (un- filled squares) show poor binding. If it is assumed that the contact of the S'm subsite with the side chain of the nucleophile involves only the main chain of the latter with maximally three methylene groups, then for each of H-Leu-NHz, H-Ile-NH2, H-Nle.NH z and H-Val- NH 2 one methyl group does not contribute to the nucleophile binding. Consequently, the hydrophobicity of the appropriate side chains to be considered for the interaction of the nucleophile with the acyl enzyme may be reduced as denoted by the arrows in Fig. 3. The rigidity of their side chains prevents a treatment of H-Trp-NH z and H-Phe-NH 2 using the same approach.
The straight line in Fig. 3 corresponds to the rela- tionship between -Ig(KNk3/k4) and the side chain hydrophobicity of nine unbranched and branched hy- drophobic amino acid amides (filled and half-filled squares) based on the binding model discussed above. The slope is 1.41 and the correlation 0.97, respectively. A slope of 1.27 was reported for the dependence of lg(1./K m) for the hydrolysis of phenyl esters of N-ben- 7oy!g!>'cine on the hydrophobicity of the meta-sub- stitucnts of the ester moiety [6]. The similarity of both coefficients leads to the assumption that the same binding subsite has been investigated. Our binding model is supported by the fact that the hydrophobic pocket formed by Trp-177, Ala-136 and the y-carbon of His-159 which is assumed to accomodate the meta- substituent of phenyl esters is only of moderate size as follows from model inspection [6].
Berti et al. [4] considered the formation of an ad- sorptive complex between an acyl papain and H-GIy- NH 2 unlikely because of the absence of a side chain.
TABLE I!1
K s k.~ / k j of the papain-catalyzed acyl transfer from Mal-Phe-Ala- OEICI to raritms alanine peptides (25°(?; pH 9.0; 0.2 M KCl).
This view contradicts the linearity of the hydrophobic- ity plot in Fig. 3. The figure indicates that H-Gly-NH 2 is bound in the same mode as more hydrophobic nude- ophiles.
The possibility of an involvement of Gin-19 in the S~ subsite resulting in hydrogen-bonding capabilities has been discussed by Alecio et al. [10], but could not be proved experimentally. The reactivity of H-Thr-NH 2 and H-His-NH 2 (triangles) cannot be explained by the overall hydrophobicity of their side chains. Additional polar interactions could contribute to the binding en- ergy of these nudeophiles. However, the results must be treated with caution, since it is uncertain which parts of the side chains interact with the acyl enzyme. The data cannot serve as strong evidence for the oe- curence of hydrogen bonds.
Acyl transfer to alanme peptides The KNk~/k 4 for the deacylation of Mal-Phe-Ala-
papain by a series of alanine peptides are compiled in Table III. An elongation of the peptide chain improves the binding, as described before by Bender et al. [9]. H-AIa-AIa-NH2 is 40-times more reactive than the negatively charged H-AIa-AIa-OH. This observation corresponds to the low deacylation efficiency of H-GIu- NH2.
"In order to screen the possibility of ionic interac- tions between acyl enzyme and nucleophile the deacy- lation of Mal-Phe-Ala-papain by H-AIa-AIa-NH 2 and H-AIa-Aia-OH was investigated at varying concentra- tions of KCI. The result is given by Fig. 4. The linearity of both plots can be attributed to salting out of the nucleophiles according to Eqn. 4 [29]:
lg L = Ig fo + xlsaltl (4)
where f " and fs are the activity coefficients c~f the nucleophile in the absence and in the presence of salt, respectively, and X is a coefficient describing the salt- ing out effect. No deviations from this relationship can be recognized at low salt concentrations. Therefore, a contribution of an electrostatic repulsion to the binding of H-AIa-AIa-OH can be excluded. This finding corre- sponds to the absence of negative charges in the sup-
211
1 8 -
1 4 "
1 0 :
061 i
0 . 2 !
- 0 2
L2 ........ .....
i I I
I O0 0 5 10 15 2.0 2 5 3 0
[KCt] (M) Fig. 4. Dependence of Ig(K N k .~/k 4) for the deacylation of MaI-Phe- Ala-papain by H-Ala-Ala-OH ([]) and H-Ala-AIa-NH z ( • ) on the
concenlrafion of KCI.
posed nucleophile binding area according to the X-ray structure. An explanation for the low binding tendency of H-AIa-AIa-OH compared to H-Aia-AIa-NH, could be either the lack of the C-terminal amide function which can serve as donor for hydrogen bonds or a different binding mode within the EAN complex. A different binding mode could be also the reason for the inefficient binding of H-Glu-NH 2 in comparison to H-Arg-NH 2.
Acyi transfer to di- and tripeptides The Knk3 /k 4 for the deacylation of Mal-Phe-Ala-
papain by various di- and tripeptide amides are listed in Table IV. With the exception of H-Ala-AIa-NH 2, which is the most efficient deacylating agent within the series of dipeptide amides (H-AIa-X-NHz), the data indicate a preference of the acyl enzyme for hydropho- bic bulky amino acid residues in P2 and in P~.
However, one should be cautious to transfer these conclusions to the binding specificity of the free en- zyme. The recently described strong interdependencies between the binding subsites of papain (induced fit) [4] have not been considered within the present study.
TABLE IV K,~,k 3 / k ~ of the papain-catalyzed ao'! transfer from Mal-Phe-AIa- OEtC! to a series of di- and tripeptides (25°C: pH 9. 0: 0. 2 M KCI).
Our data allow a refined characterization of the nucleophile specificity of papain: - The S' L subsite can accommodate maximally three methyl(ene) groups of hydrophobic side chains. - In the case of branched hydrophobic side chains only the main chain interacts with the S'~ subsite. - The hydrophobicity of the S] subsite is very similar to that of the binding region accomodating the meta -
substituent of phenyl esters of hippuric acid. Thus, this region might be identical with the S~ subsite. - Hydrophilic uncharged side chains exhibit a stronger binding to S' I than would be expected from their hydrophobicity. Thus, the occurrence of polar S'I-P [ interactions cannot be excluded. - The deacylation of MaI-Phe-Ala-papain by H-AIa- AIa-OH in the presence of varying concentrations of KCI gave no evidence for an electrostatic repulsion between acyl enzyme and negatively charged nucle- ophiles. - From our data a preferential binding of nucleophiles with bulky hydrophobic amino acid residues in P~ and in P~ may be concluded. However, the validity of this conclusion for other substrate systems has to be proved.
Our studies show that acyl transfer data can be applied succesfully to the analysis of structure-activity relationships.
A c k n o w l e d g e m e n t s
We wish to thank Dr. Hans-J6rg Hofmann for help- ful advice concerning special problems of structure-ac- tivity relationships and Dr. Peter Hailing for reading and correcting the manuscript.
R e f e r e n c e s
1 Schechler, I. and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-167.
2 Schechter, I. and Berger, A. (1968) Biochem. Biophys. Rcs. Commun. 32, 888-902.
3 Asb6th, B., Majer. Z. and Polg~r, L. (1988) FEBS Lett. 233, 339-341,
4 Berti, P.J.. Faerman, C.H. and Storer. A.C. (1991) Biochemistry 30, 1394-1402.
5 Smith, R.N., Hansch. C., Kim, K.H,, Omiya, B., Fukumura, G,, Selassie, C.D., jow, P.Y.C., Bianey, J.M. and Langridge, R. (1982) Arch. Biochem. Biophys. 215, 319-328.
6 Compadre, C.M., Hansch, C., Klein, T,E. and Langridge, R. (1990) Biochim. Biophys. Acta 1038, 1578-163.
7 Mycek, M.J. and Fruton, J.S. (1957) J. Biol. Chem. 226, 165-171. 8 Brubacher, LJ. and Bender, M.L. (1966) J. Am. Chem. Soc. 88,
