70
Biochimica et BioldlysicaAria, ! ! 18( 1991) 70-76 t~ 1991El.~vierSciencePublishelsB.V. All fightsre~rved 0[67-4838/91/$03.50
BBAPRO34052
Substrate specificity of a-chymotrypsin-catalyzed esterification in organic media P e r e C l a p ' s a n d Patrick A d l e r c r e u t z Department of Biotechnol~'..~.'. Cheraical Cen',er. Unirersi~"of Luna~ Lund (Sweden)
(Received 18June i~91)
Keywords: Structureactivityrelationship: a-Chymotwpsin:Subslratespecificily 11 amino acid derivatives were tested as a.chymotrypsin substrates in the esterification reaction with methanol in organic media. The reactions were carried out in water-saturated ethyl acetate and in acetonitrile containing 4% water, a-Chymolrypsin adsorbed on Celite was us-.d as a catalyst. From initial reaction rate measurements, the Michaelis-Menten parameters Vma~ and K~t were determined. All the amino acid derivatives tested were esterified, and the highest values of kc.t/K M were obtained with the N-acylated aromatic amino acids. Correlations between Michaelis-Menten parameters and physical properties of the substrates such as molar refractivity (MR) and log P were deduced. The results show that the specificity of the a-chymotrypsin towards the side chain of the amino acids in organic media is the same as that in aqueous media. However, the specificity towards the N-protecting grovp is opposite to that in water, so the reaction medium affects the interaction of this part of the molecule with the enzyme to a large extent.
Introduction The use of enzymes in organic media has attracted much interest in recent years [1,2]. Some advantages of organic media compared to aqueous solutions are that hydrophobic substrates which are poorly soluble in water can be dissolved and that undesirable hydrolytic side reactions can be suppressed in condensation reactions. In peptide synthesis moderate concentrations of water miscible organic solvents have been used for many years; their main effect is that the acidity of the a-carboxyl group decreases, thereby shifting the equilibrium towards peptide synthesis [3,4]. In recent years organic media with only minute quantities of water have been used and in these reactions the low water activity in the system favours the condensation reaction [5,6,7,8]. Normally enzymes in organic media catalyse the same type of reactions as in aqueous media. However, it has been reported that the substrate specificity of
enzymes in organic media can be reversed compared to the situation in water [9]. The substrate specificity of a-chymotrypsin has been thoroughly studied in aqueous media [10,11,12] and to some extent in organic media [9]. In this study the substrate specificity of a-chymotrypsin in organic media was investigated. The esterification of N-protected amino acids with methanol was used as a model reaction; this reaction obeyed Michaelis-Menten kinetics. The constants Vm~x and K,~ were deduced and K I was determined in cases when substrate inhibition occurred. One aim of the present study was to investigate the specificity of a-chymotrypsin for different amino acids in organic media and another to evaluate different protecting groups for enzymatic peptide synthesis in such media. The results were compared with literature data for reactions in aqueous media in order to distinguish between the intrinsic specificiq, of the enzyme and the effects of the reaction medium. Materials and Methods
Abbreviations:Boc, tert-butylo~carboa¥1; Fmoc,fluorcnylmethyloxycarbonyl: MR, molar refractivity:QSAR,quantitative structure activi~ relationships: 7,, benzylo~carbonyl. Correspondence: P. Adlercreulz, Department of Biotcchnology, Chemical Center. P.O. Box 124 S-221 Of)Lund. Sweden.
Chemicals
a-Chymotrypsin (EC 3A.21.1) from bovine pancreas, with a specific activity of 51 benzoyl-tyrosine ethyl ester units/rag of solid was obtained from Sigma (St Louis, MO, U.S,A.) and was used without further purifica-
tion. CeliteTM (30-80 mesh) was from BDH (Poole, U.K.). N-a-acetyI-L-amino acids, methyl esters and free acids were obtained from Bachem (Feinchemikalien AG, Switzerland) and Sigma (St Louis, MO, U.S.A.). Acetonitrile (HPLC grade) was obtained from Fisons (Loughborough, U,IC). All other chemicals used were of analytical grade.
Preparation of the immobilized enz3,me The enzyme was immobilized by being dried onto the support, a-Chymotrypsin (15 mg solid/ml) was dissolved in 50 mM Tris-HC! buffer (pH 7.8). 1 ml of enzyme solution was mixed with 1 g of support material. After mixing, the preparation was dried under vacuum overnight. The resulting preparation had a protein content of 1.5% (w/w) and its specific hydrolytic activity was 65% of that of free ehymotrypsin [13]. HPLC analysis The amount of N-a-acetyi-L-am,.'no acid methyl esters produced in the enzymatic reactions was measured by HPLC. The columns used were always of C18 type, ODS-2, 10 tt (250 X 4 ram) (Tracer analiticaTM) and Merck Hibar TM LichrosorbTM RP-18, 5 # (125 X4 ram), alone or connected in series to increase the separation. The mobile phase contained different proportions of water and acetonitrile and in all cases 0.05% (v/v) of trifluoroacetic acid was used; the flow rate was 1.0 m]/min. Table 1 summarises the conditions used for all the separations. The amount of product formed was calculated from the peak area using an external standard method. The N-a-protected amino acids and their methyl esters were used as external standards. Kinetic measurements
TABLE I Conditionsfor HPLCanalysis Substrate Column Eluent ACN/H20/ TFA * Ac-Lgu ODS-2 20/80/0.05 Ac-Met ODS-2 +Hibar 17.5/82.5/0.05 Ac-Val OD5-2 15/85/0.05 Ac-Tyr t3DS-2 17/83/0.05 Ac-Trp ODS-2 30170/0.05 Ac.Asp ODS-2 + I-libar 5/95/0,95 At-ASh ODS-2 3/97/0,05 Ac-Phe ODS-2 30/70/0.05 Z-Phe ODS-2 55/45/0.05 Boc-Phe ODS-2 55/45/0.05 Fmoe-Phe ODS-2 70/30/0.05
Detection Retention time ester {rain) 215 12.I4
(am)
215 215 270 278
10,04 8,20 6.07 6.53
215 215 254 ~4 254 265
6.57 4.15 6.18 6,94 692 6.29
* ACN= acetonittile;TFA = trifiuoroaceticacid.
Esterification reactions were carried out in 10 ml stoppered flasks placed in a reciprocal shaker at 25 ° C. For each N-t~-acetyl amino acid and the other derivatives, 9-12 different concentrations were used in a range between 0.2 mM to 30 mM. Stock substrate solutions were made shortly before use by dissolving weighed quantities of the amino acid derivatives in a measured volume of solvent. The solvents used in this study were acetonitrile containing 4% (v/v) of buffer (50 mM Tris-HCl, pH 7.8) and water-saturated ethyl acetate. To 2 ml of a solution containing a controlled amount of substrate, 50-150 mg of immobilized preparation were added. The reaction was started by adding 41 t~! of methanol (giving a concentration of 0.5 M). The rates of enzymatic esterifications were followed by HPLC. Samples (50 p,I) were taken at different time intervals depending on the amino acid. After solvent evaporation, the samples were dissolved in the HPLC eluent and analysed under the conditions described above.
Calculations Initial reaction rates were deduced by linear regression for each concentration of substrate. MiehaelisMenten parameters, Vm~x and K M were deduced by fitting the experimental data directly to the MichaelisMenten equation using a computer program (Graphpad version 1.0) based on a non-linear regression method [14]. In case of substrate inhibition the following equation was used [15]:
Vm~x[S] [S] 2 KM + is] ÷ fc---~This equation has earlier been applied to chymotr~sin-catalyzed esterification in organic media [16]. The inhibition constant K t was determined using the plots of 1/L' VS. 1/S and I / v vs. S. All the results presented in this work were calculated on the basis of the amount of a-chymotrypsin in the imlnobi|ized preparation; no correction was made for the possible presence of inactive enzyme.
Results and Discussion Eleven amino acid derivatives were tested as achymotrypsin substrates in acetonitrile or ethyl acetate with controlled amounts of water. These solvents were chosen because earlier studies have shown that they are good solvents for enzymatic reactions [6]. Furthermore, since enzymes behave differently in water misci-
ble and water immiscible solvents, one solvent of each group was chosen. Since the main aim of this study was to investigate the interactions between the amino acid substrates and the enzyme, a saturating concentration of the other substrate, methanol, should be used. Preliminary kinetic measurements showed that the Kst values for methanol in the reactions s,.udied were around 200 mM. A methanol concentration of 1 or 2 M would then be suitable to saturate the enzyme. However, in the type of experimental system used, one should also consider other possible effects of methanol. Methanol can be assumed t.o accumulate in the mieroenvironment of the enzyme (together with water) and interact with the enzyme in a nonspecific way due to its properties as a solvent. High amounts of solvents in the microenvironment of the enr/me can cause decreased enzymatic activity. Decreasing enzyme activities was indeed observed already at methanol concentrations above 500 mM and therefore a methanol concentration of 500 rnM was used in the rest of the experiments. The reaction rates were determined during the initial part of the reaction. With some substrates, low reaction rates were obtained and consequently long reaction times were needed. However, constant reaction rates were observed, also in the slowest reactions; no indications of enzyme inactivation were observed. This is in agreement with earlier results, which showed that chymotr~sin operating under similar conditions
was stable for several days [5]. The reaction rates were proportional to the amount of enzyme preparation employed in the range used (50-150 mg immobilized preparation). S u b s t r a t e speci/icity
In the ester±float±on of N-acetyl amino acids in organic media .'.he highest values of k , ~ , / K M were obtained with the aromatic amino acid derivatives (Table 11). This is not surprising since a-chymotrypsin in aqueous media cata!yses the hydrolysis of peptide bonds with aromatic or bulky aliphatic amino acid residues as the earbox'yl coml~aent. All the amino acid derivatives tested were ester±fled at varying reaction rates. This shows that a-chymotrypsin can be used for synthetic reactions involving several different amino acids. The Vm~ values for the different substrates differed widely while the K M values varied much less; most K M values were around 1 mM (Table 1I). The rates of the reactions in organic media depend on several factors, includie, g the solvent and the water content. In water miscible solvents such as acetonitrile, the rate normally increases with increasing water content. The water content used in this study (4%) is comparatively low because this gives the possibility of reaching high yields in reactions where hydrolysis is a competing reaction [6]. When water immiscible solvents are used the reaction rate normally increases with increasing water content until the medium is
TABLE It Kinetics of the ester±fleet±on of N-prolected amino acids with methanol tO.5 ItD in ,rater-saturated ethyl acetate and aeetonitrile with 4% ~vater catalyzed by a-ch~'mot~'psin deposited on celite
Substrate Ac-l..eu Ac-Leu At-Met Ac-Met Ac-Val Ac-Val Ac-Asp At-Asp Ac-Asn Ae-Asn Ac-Trp, Ac-Trp Ac-Tyr Acffyr Ac-Phe Ac-iPhe Z-Phe Z-P,~e Boc-Phe Boe-Phe Fmoc-Phe Fmo'~-Phe
Solveat Acetonitrile Ethyl acetate Acetonitrile Ethyl acetate Acetonitrile Ethyl acetate Acetonitrile Ethyl acetate Acetonitrile Ethylacetate Acetonitrile Ethyl acetate Acetonitrile Ethylacetate Acetonitrile Ethyl acetate Acelonilrile Ethylacetate Aeetonitrile Ethyl acetate Acetonitrile Ethyl acetate
Vm~~
K sI
K,
k ,~, ~Ks!
(retool h - t mmolenzyme- t )
(mM)
(mM)
(s - 1 M" I)
1.60 _+0.07 2.56 +_0.07 5.6 _+0.4 4.9 _+0.2 0.064 _+0.007 0.0178-+0.0007 0.45 _+0.02 0.17 _+0.(Vl 1.86 __+0.11 2.7 +_0. I 48.3 +0.9 106 _+2 01 _+3 258 _+1 17.4 _+0.7 57 ±1 0.80 +0.02 3.3 .4,0.2 0.312 +_0.1J04 0.43 _+0.02 0.254 ±0.904 0.31 _+0.01
i.5 4-0.3 1.1 +-0.2 i.2 ±0.1 1.0 :~0.1 17.3 ±0.5 0.95 _+0.02 0.%±0.03 0.52_+0.03 7.5 ± 1.4 8.9 _ 1.3 1.1 +0.1 0.9 ±0.1 1.8 .4,0.3 0.8 ±0.1 1.6 +_0.3 1.2 _+0.2 1.6 _+0.3 5.3 _+0.7 1.9 _4-0.1 0.6 +0.2 1.2 4-_0.1 2.0 _+0.2
28 _+I 4.0+_0.4 28.6+ 0.2 30 +-2 17 ± ! -
0.31 0.69 1.3 1.4 0.0010 0.0052 0.139 0.089 0.07 0.08 12 33 14 90 3.1 13 0.14 0.18 0.044 0.2 0.058 0.044
saturated with water; at higher water contents the reaction rate is often fairly constant. In the present investigation the substrates were dissolved in watersaturated ethyl acetate; results obtained with buffersaturated (50 mM Tris-HCI, pH 7.8) ethyl acetate were not significantly different. For instance, using the esterification of Ac-Phe as a model reaction the value of k~,,,/K M for the reaction in buffer saturated ethyl acetate was 6% lower than that obtained with water saturated solvent, but this difference was smaller than the experimental errors. The reaction rates observed in water-saturated ethyl acetate and in acetoaitrile containing 4% water were in the same range. Some substrates, for example Ac-Tyr, Ac.Trp and Ac-Phe, were more rapidly converted ip ethyl acetate while others reacted more rapidly in aeetonitrile (Table i1). With Ac-Val and Ac-Asp substrate inhibition was observed in both solvents and with At-Met in aeetonitrile only. The reason for substrate inhibition under these conditions is not knmvn. The substrate inhibition by Ac-Asp may be due to nonproductive binding between the enzyme and the earboxyl group in the side chain. It is possible that protection of thi~ carbo~l group would give a better substrate, as was the case when aspartic acid amide was used as the nucleophile in a a-chymotrypsin-catalysed reaction in organic media [71. in order to determine the influence of the N-protecting group, three groups in addition to acetyl were tested in the esterification of phenylalanine: Z (benzyloxycarbonyl), Boc (tert-butyloxyearbonyl) and Fmoc (fluorenylmethyloxycarbonyl). These protecting groups are the most widely used in peptide synthesis. Much higher rates were observed with acetyl as protecting group than with the others (Table I!). Unfortunately, acetyl is not a very s-fitable protecting group in peptide synthesis since it is not easily removed, it would be of interest to find a protecting group that, like aeetyl, gives high reaction rates in organic media and which is also practical concerning protection and deprotection reactions. All components of the reaction mixture can influence the rate of the enzyme catalyzed reaction. Since the aim was to study the interactions between the enzyme and its substrales, the support should be chosen so that it does not interfere with these interactions. It is known that some supports can influence the partitioning of substrates and thereby greatly influence the reaction rate [17]. in preliminary experiments Celite was used as support. It is known that only minimal amounts of water are adsorbed on Celite [13] and measurements showed no detectable adsorption of the substrates used in this study. To further investigate the possible interference of (?elite, experiments were carried out with enzyme powder, without support, as catalyst. Because of problems concerning aggregation
of the enzyme particles and attachm,nt of thosz to the reactor walls, the accuracy in these experiments was somewhat less than in those using Celite as enzyme support. The k,~, values were lower than those observed with Celite, probably because some enzyme molecules inside the aggregates formed were inactive. However, there was no significant difference in the K M values obtained with an.d without Celite, which indicates that Celite did not interfere with enzyme-substrate interactions. In a second set of experiments, the adsorption of substrates on Celite was measured under the conditions prevailing in the kinetic measurements. The analysis of the substrates before Celite addition and of substrates and products during the reaction revealed that the sum of ~he concentrations of substrates and products (containing the amino acid) was constant, indicating that no adsorption occurred. In the rest of the experiments, ~-chymotrypsin on Celite was used as catalyst.
Quantitatit:e structure actit:ity relationships The interaction between an enzyme and its substrates is a complex phenomenon and in order to make meaningful predictions of kinetic parameters in the enzymatic reactions many different factors must normally be taken into consideration. However, it is sometimes possible to construct fairly simple models describing enzyme-substrate interactions, and this has been done for chymotrypsin. For a series of close~,y related amino acid esters the value of the specificity factor kc.~t/K M for the hydrolysis in aqueous media correlated well with the hydrophobicity of the amino acid side chain [18]. However, in a larger investigation by Grieco, Hansch and co-workers [19,20] quantitative structure activity relationships (QSAR) of ehymotrypsin were studied, and it was shown that the molar refractivity described the vaAations in keat/K M better than the hydrophobicity. The molar refractivity is a measure of the 'bulk' of the substituent and i~ also lelated to the polarizability [21]. The values of log P (P is the partition constant in the octanol-water twophase system and is often used as a measure of the hydrophobicity) [22,23] and the molar refractivity (MR) for the different amino acid side chains and N-protecting groups are given in Table i1I. Grieco eta!. [20] included data for different amino acid side chains, N-protecting groups and ester groups. An equation for the dependence of kcat/K M on the molar refractivity of the different parts of the substrate molecule as well as other parameters was developed. Other equationz correlating 1/K M with MR and other parameters were also developed but the fit was not quite as good as for k cat/ K r~. In the present investigation eight different amino acids were used. The values of Iog(kc~t/K M) were plotted against the molar refractivity and log P for the
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Log P Fig. 1. The values of log(kc~t/K~t) for the ester[float[on of N-acet~'lamino acids v,ith methanol (0.5 M) ~. the molar refractivity,~a)or log P (b) of the amino acid side chain. The substrates were dissolved in water-~lur'zted ethyl acetate and the reactions were catalysed by a-chymotrypsin deposited on Celite. The molar refractMties and log P values for the dill•toni amino acid side chains are listed in Table I!1. The unit for k~dt/Kxl values iss -z M -t,
side chains. Fig. 1 shows the results obtained in ethyl acetate; similar results were obtained in acetonitrile. The correlation between the molar refractivity and log(k,~t/K M) was ~ m e w h a t better than that between log P and log(k,.JKM), although the amount of data is too limited to draw any conclusions with certainty. As ~entioned above the K M values varied comparatively little; the variation in kcat/K M was mainly due to the differences in ken t values. The simple l,~near correlation between log(keat/K M) and molar refractivity or log P must, of course, be corrected when the potential substrates contain structural elements that are too large or do not fit in the active site of the enzyme. Valine derivatives, for example, are poor substrates for a-chymotrypsin because of the isoprolryl group directly bound to the a-carbon atom. When Grieco et al. [20] developed the formula for the correlation of kcat/K M with molar refractivity and other parameters a special correction factor was introduced for substrates having an isopropyl group on ",he a-carbon atom. Corrections for large groups were done by introducing a negative term in (MR): for the
side chain [20]. Because of the large deviation valine was not included in Fig. I. In order to distinguish between effects of the direct enzyme-substrate interaction and the effects of the reaction medium, the lo~kcat/KM) values for the esterification reactions were plotted against literature values of log(k,~ffK M) for the reversed reactions (ester hydrolysis) in aqueous media (Fig. 2). It is dearly seen that for these reactions the specificity of a-chyraotrypsin in organic media is closely related to its specificity in aqueous media. The present results agree with those reported for the ester[ileal[on of N-acetylamino acids in ethanol catalyzed by subtilisin Cadsberg [24]. Subtilisin is known to have a preference for aromatic amino acids in aqueous media and the same specificity was observed in ethanol. The derivatives of histidine and arginine were not ester[fled [24]. Some researchers have reported pronounced differences in the substrate specificity of proteinases in aqueous and organic media, respectively [9,25]. In the
TABLE Iii
Molar refractitities [211 and log P [231 vahtesfor different amino acid side chains and N-protectbzggroups used in this ~tud)' Group
Molar refi activity
Log P
CH2CH(CH 3)2 (CH.,):SCH 3 CH 2C~,Hs CHrCoH4OH CH 2COOH CH zCONH2 CH2-indoyl
19.60 22.77 30.01 31.80 1!.88 14.41 42.30
2.169 1252 2.416 2.073 - 0.424 - 1.44 2.'/8
CH(CH3)z
14.98
COCH~ COOCH_,C~H~ COOCICH 3)3
!1.18 36.40 ~.82 62.60
1.64 - 1~00
COOCH,-fluorenyl
1.12 0.96 2.32
"~
0
rd)
.2 -3-
~,
-4¢ .
.
.
.
.
L o g k = t / K . in water
Fig. 2. Co,lrelation of the substrat¢ specificityof a-chymotrypsin in organic and aqueous media. The values of tog(kc.~t/KM) for the eslerification of N-acetyl amino acids with methanol vs. Iog(k~,/K M) for the reversed reaction, hydrolysisof N-acetylamino acid methyl esters(data from Griceo et al. [20}).The organic reaction media were water-saturated ethyl acetate ( i ) a n d acelonitrile with 4% water (o); the enz$-meused in organic media was deposited on Celite. The unit for kca~/K~.t values is s- t M- '.
7~
hydrolysis of N-acetylamino acid esters catalyzed by a-chymotrypsin or subtilisin in water the phenylalanine derivative was a much petter substrate (higher k , t / K M) than those of histidine and serine. However, when the same substrates were used in a transesterification reaction with n-propanol in octane the phenylalanine derivative was the worst substrate [9]. Similar observations have been made using polyethylene glycol modified a-chymotwpsin and subtilisin [25]. In the hydrolysis of N-benzoylamino acid esters in water the iysine derivative was a poor substrate or was not converted at all. On the other hand, in a peptide synthesis reaction in benzene it was a better substrate than the tyrosin derivative which was a very good substrate in water [25]. In the present investigation the derivatives of aspartic acid and asparagine were fairly poor substrates in organic media and the corresponding esters can therefore be expected to be poor substrates in water (no literature data were found). it can be concluded that in the esterifieation of N-acetyl-L-amino acids in organic media the substrate specificity of a-chymotrypsin and subfilisin concerning the amino acid side chain is similar to that for ester hydrolysis reactions in water. However, when esters of N-protected amino acid esters are used for transesterification or peptide synthesis reactions in organic mt:dia, the substrate specificity is reversed compared to when the same substrates are hydrolyzed in water. One reason for this discrepancy might be that the transesterification and peptide synthesis reactions described were carded out in solvents (benzene and octane) with considerably higher hydrophobicity than those used in the esterification reactions (ethanol, acetoaitrile and ethyl acetate). However, in order to fully understand these observations, more research is needed concerning the influence of the reaction medium on the rates of the separate steps in the reactions. Four different N-protecting groups were used for phenylalanine. Literature values for the corresponding
TABLE IV
Vahtes of the specificity constant log ~k,~t / ~,',,~)far the hydrolysis of different N-protected amino acid metl,yl estrrs hi am~eoltsmedia (data from Grie,'o et at. [20]) Protecting group
log( k ca~ / K,,.t)
Acetyl Benzoyl
Val
Phe
Tyr
0.13 I. 15
4.62 5.t)5
5.56 6.70
hydrolysis reactions in aqueous media were not found for all protecting groups. However, kinetic constants for some pairs of acetyl- and benzoyl-derivatives have been published (Table IV). In all these examples the exchange of the acetyl group to the benzoyl group, which corresponds to an increase in both log P and molar refractivity, resulted in an increase in keat/K m (Table 4). Furthermore, in the formula of Grieco etal. [20] the coefficient for the molar refractivity of the protecting group is positive, which means that k~t/K M increases with increasing molar refractivity. In organic media the opposite trend was observed. The value of k¢at/K M decreased with increasing log P or molar refractivity of the protecting group. Fig. 3 shows a plot of the data obtained in ethyl acetate. The results can be ir.terpreted l:y considering the interaction between the enzyme and the N-protecting group of the substrate as being fairly nonspecific. In water large, hydrophobic protecting groups are favorable and a large proportion of the interaction is probably hydrophobic in nature. However, in organic media hydrophobic interactions are less important and more hydrophilie protecting groups like acetyl are more favorable, Accordingly, as far as the protecting group is concerned the effect of the reaction medium is quite strong and a reversal of substrate specificity was observed when changing from aqueous to organic medium.
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Fig. 3. The values of Iog(k=~/K~I) for the esterification of N-protected phenyldanine with methanol (0.5 M) ~s. the molar refractivity (a) or log P (b) of the protecting group. The substrates were disso|ved in water saturated ettsyl acetate and the reactions were catalysed by ~-chymotrypsin deposited on (:'elite. The molar refractivities and log P values for the different protecting groups are li.~ted in Table !1I. The unit for kc~t/K~ values iss -~ M- t.
76 in conclusion, the substrate specificity of a-chymotrypsin in the esterification of N-protected amino acids with methanol in organic media is goverened by the intrinsic specificity o f the enzyme as far as the amino acid side chain is concerned and to a large extent by the reaction medium as far as the protecting group is concerned. Acknowledgements This project was supported by the Biotechnology Research Foundation (SBF) and the National Swedish Board for Technical Development (STUF). P. C l a p , s wishes to express his gratitude to Consejo Superior de lnvestigaciones Cientificas (C.S.I.C,) (Spain) for its financial support. The authors wish to thank Professor 13o Mattiasson for valuable discussions and Scott Bloomer for linguistic advice. References i Laurie, C., Tramper, J. and Lilly. M.D. (eds.) (1987) Biocalalysis in Organic Media. Elsevier. Amsterdam. 2 Zaks, A. and Russell, AJ. (1988) J. BiolechnoL8, Z';9-270. 3 Homandberg, G.A, Matlis, J.A. and Laskowski, M., Jr. (1978) Biochemistry 17. 5220-5227. 4 Jakubke. H-D.. Kuhl. P. and K6nneeke, A. (1985)Angew. Chem. Int. Ed. Engl. 24, 85-93. 5 Reslow, M., Adlerereutz. P. and Mattiasson, B. (1988) Eut. J. Biocnem. 177. 313-318. 6 Clap,s, P.. Adlercreutz, P. and Mattiasson, B. (1990) J. Biotechnol. 15, 323-338.
7 Claims, P_ Adlercreutz, P. and Maniasson, B. (1990) Biot¢chnol. AppL Biochem, i2. 376-386. 8 Kise, H., Fujimoto, K. and Nodtomi, H. (1988) J. BiozechnoL8. 279-290. 9 Zaks, A. and Klibanov, A. (1986) J. Am. Chem. So(:. 108, 27672768. 10 Wailc, H,R. and Niemann, C. (1%2) Biochcmislry l, 250-253. 11 Baumann. W.K.. Bizzozero. S.A., Dutler, H, (1973) Eur. J. Biochcm. 39, 381-391. 12 Morgenstem, L, Recanatini, M., Klein, T.E., Steinmelz, W., Yang, C.-Z., Langridge, R, and Hansch, C. (1987) J. Biol. Cbhem. 26~ 10767-10772. 13 Reslow, M.. Adlercreutz, P. and Mattiasson, B. (1988) Eur. J. Biochem. 172. 573-578. 14 Sagnela, G.A. (1985) Trends Biochem. Sci, 10. 100-103. 15 Bailey, J.E. and Ollls D.F. (1986) BiochemicalEngineering Fundamentals. McGraw-Hill Inc.. Singapore. 1,50zamiri, M, Adlercrautz, P. and Maniasson. B. (1991) Biotechnol. Appl. Biochem. 13, 54-64. 17 Omala, T., lida, T., Tanaka. A. and Fukui, S. (1979) Eur. J. Appl. Microbiol. Biolechnol. 8, 143-155. 18 Dorovska, V.N, VarfoIomeyev,S.D., Kazanskaya. N.F., Klyo~ov, A.A. and Martinek, K. (1972) FEBS Len, 23, 122-124. 19 Hansch, C. (1977) J. Me,d. Chem. 20, 1420-1435. 20 Grieco, C., Hansch, C., Silipo. C., Srnilh, R.N., Vittoria, A. and Yamada, K. (t979) Arch. Biochem. Biophys. 194, 542-551. 21 tlansch, C, Leo, A., Unger, S.H., Klm, K.H., Nikaitani, D. and Lien. EJ. (1973)£ Med. Chem. 16, 1207-1216. 22 Laane, C, Boeren, S. and Vos, IC (1985) Trends Bioteehnol. 3, 251-~2. 23 Rekker, R.F. (1977l The hyd~ophobicfragmental constant. Elsevier, Amsterdam. 24 K!se, H. (I990) Bioorg. Chem. 18. 107-115. G. ertner, H. and Puigserver. A. (1989) Eur. J. Biochem. 181, 207-213.
Biochimica et BioldlysicaAria, ! ! 18( 1991) 70-76 t~ 1991El.~vierSciencePublishelsB.V. All fightsre~rved 0[67-4838/91/$03.50
BBAPRO34052
Substrate specificity of a-chymotrypsin-catalyzed esterification in organic media P e r e C l a p ' s a n d Patrick A d l e r c r e u t z Department of Biotechnol~'..~.'. Cheraical Cen',er. Unirersi~"of Luna~ Lund (Sweden)
(Received 18June i~91)
Keywords: Structureactivityrelationship: a-Chymotwpsin:Subslratespecificily 11 amino acid derivatives were tested as a.chymotrypsin substrates in the esterification reaction with methanol in organic media. The reactions were carried out in water-saturated ethyl acetate and in acetonitrile containing 4% water, a-Chymolrypsin adsorbed on Celite was us-.d as a catalyst. From initial reaction rate measurements, the Michaelis-Menten parameters Vma~ and K~t were determined. All the amino acid derivatives tested were esterified, and the highest values of kc.t/K M were obtained with the N-acylated aromatic amino acids. Correlations between Michaelis-Menten parameters and physical properties of the substrates such as molar refractivity (MR) and log P were deduced. The results show that the specificity of the a-chymotrypsin towards the side chain of the amino acids in organic media is the same as that in aqueous media. However, the specificity towards the N-protecting grovp is opposite to that in water, so the reaction medium affects the interaction of this part of the molecule with the enzyme to a large extent.
Introduction The use of enzymes in organic media has attracted much interest in recent years [1,2]. Some advantages of organic media compared to aqueous solutions are that hydrophobic substrates which are poorly soluble in water can be dissolved and that undesirable hydrolytic side reactions can be suppressed in condensation reactions. In peptide synthesis moderate concentrations of water miscible organic solvents have been used for many years; their main effect is that the acidity of the a-carboxyl group decreases, thereby shifting the equilibrium towards peptide synthesis [3,4]. In recent years organic media with only minute quantities of water have been used and in these reactions the low water activity in the system favours the condensation reaction [5,6,7,8]. Normally enzymes in organic media catalyse the same type of reactions as in aqueous media. However, it has been reported that the substrate specificity of
enzymes in organic media can be reversed compared to the situation in water [9]. The substrate specificity of a-chymotrypsin has been thoroughly studied in aqueous media [10,11,12] and to some extent in organic media [9]. In this study the substrate specificity of a-chymotrypsin in organic media was investigated. The esterification of N-protected amino acids with methanol was used as a model reaction; this reaction obeyed Michaelis-Menten kinetics. The constants Vm~x and K,~ were deduced and K I was determined in cases when substrate inhibition occurred. One aim of the present study was to investigate the specificity of a-chymotrypsin for different amino acids in organic media and another to evaluate different protecting groups for enzymatic peptide synthesis in such media. The results were compared with literature data for reactions in aqueous media in order to distinguish between the intrinsic specificiq, of the enzyme and the effects of the reaction medium. Materials and Methods
Abbreviations:Boc, tert-butylo~carboa¥1; Fmoc,fluorcnylmethyloxycarbonyl: MR, molar refractivity:QSAR,quantitative structure activi~ relationships: 7,, benzylo~carbonyl. Correspondence: P. Adlercreulz, Department of Biotcchnology, Chemical Center. P.O. Box 124 S-221 Of)Lund. Sweden.
Chemicals
a-Chymotrypsin (EC 3A.21.1) from bovine pancreas, with a specific activity of 51 benzoyl-tyrosine ethyl ester units/rag of solid was obtained from Sigma (St Louis, MO, U.S,A.) and was used without further purifica-
tion. CeliteTM (30-80 mesh) was from BDH (Poole, U.K.). N-a-acetyI-L-amino acids, methyl esters and free acids were obtained from Bachem (Feinchemikalien AG, Switzerland) and Sigma (St Louis, MO, U.S.A.). Acetonitrile (HPLC grade) was obtained from Fisons (Loughborough, U,IC). All other chemicals used were of analytical grade.
Preparation of the immobilized enz3,me The enzyme was immobilized by being dried onto the support, a-Chymotrypsin (15 mg solid/ml) was dissolved in 50 mM Tris-HC! buffer (pH 7.8). 1 ml of enzyme solution was mixed with 1 g of support material. After mixing, the preparation was dried under vacuum overnight. The resulting preparation had a protein content of 1.5% (w/w) and its specific hydrolytic activity was 65% of that of free ehymotrypsin [13]. HPLC analysis The amount of N-a-acetyi-L-am,.'no acid methyl esters produced in the enzymatic reactions was measured by HPLC. The columns used were always of C18 type, ODS-2, 10 tt (250 X 4 ram) (Tracer analiticaTM) and Merck Hibar TM LichrosorbTM RP-18, 5 # (125 X4 ram), alone or connected in series to increase the separation. The mobile phase contained different proportions of water and acetonitrile and in all cases 0.05% (v/v) of trifluoroacetic acid was used; the flow rate was 1.0 m]/min. Table 1 summarises the conditions used for all the separations. The amount of product formed was calculated from the peak area using an external standard method. The N-a-protected amino acids and their methyl esters were used as external standards. Kinetic measurements
TABLE I Conditionsfor HPLCanalysis Substrate Column Eluent ACN/H20/ TFA * Ac-Lgu ODS-2 20/80/0.05 Ac-Met ODS-2 +Hibar 17.5/82.5/0.05 Ac-Val OD5-2 15/85/0.05 Ac-Tyr t3DS-2 17/83/0.05 Ac-Trp ODS-2 30170/0.05 Ac.Asp ODS-2 + I-libar 5/95/0,95 At-ASh ODS-2 3/97/0,05 Ac-Phe ODS-2 30/70/0.05 Z-Phe ODS-2 55/45/0.05 Boc-Phe ODS-2 55/45/0.05 Fmoe-Phe ODS-2 70/30/0.05
Detection Retention time ester {rain) 215 12.I4
(am)
215 215 270 278
10,04 8,20 6.07 6.53
215 215 254 ~4 254 265
6.57 4.15 6.18 6,94 692 6.29
* ACN= acetonittile;TFA = trifiuoroaceticacid.
Esterification reactions were carried out in 10 ml stoppered flasks placed in a reciprocal shaker at 25 ° C. For each N-t~-acetyl amino acid and the other derivatives, 9-12 different concentrations were used in a range between 0.2 mM to 30 mM. Stock substrate solutions were made shortly before use by dissolving weighed quantities of the amino acid derivatives in a measured volume of solvent. The solvents used in this study were acetonitrile containing 4% (v/v) of buffer (50 mM Tris-HCl, pH 7.8) and water-saturated ethyl acetate. To 2 ml of a solution containing a controlled amount of substrate, 50-150 mg of immobilized preparation were added. The reaction was started by adding 41 t~! of methanol (giving a concentration of 0.5 M). The rates of enzymatic esterifications were followed by HPLC. Samples (50 p,I) were taken at different time intervals depending on the amino acid. After solvent evaporation, the samples were dissolved in the HPLC eluent and analysed under the conditions described above.
Calculations Initial reaction rates were deduced by linear regression for each concentration of substrate. MiehaelisMenten parameters, Vm~x and K M were deduced by fitting the experimental data directly to the MichaelisMenten equation using a computer program (Graphpad version 1.0) based on a non-linear regression method [14]. In case of substrate inhibition the following equation was used [15]:
Vm~x[S] [S] 2 KM + is] ÷ fc---~This equation has earlier been applied to chymotr~sin-catalyzed esterification in organic media [16]. The inhibition constant K t was determined using the plots of 1/L' VS. 1/S and I / v vs. S. All the results presented in this work were calculated on the basis of the amount of a-chymotrypsin in the imlnobi|ized preparation; no correction was made for the possible presence of inactive enzyme.
Results and Discussion Eleven amino acid derivatives were tested as achymotrypsin substrates in acetonitrile or ethyl acetate with controlled amounts of water. These solvents were chosen because earlier studies have shown that they are good solvents for enzymatic reactions [6]. Furthermore, since enzymes behave differently in water misci-
ble and water immiscible solvents, one solvent of each group was chosen. Since the main aim of this study was to investigate the interactions between the amino acid substrates and the enzyme, a saturating concentration of the other substrate, methanol, should be used. Preliminary kinetic measurements showed that the Kst values for methanol in the reactions s,.udied were around 200 mM. A methanol concentration of 1 or 2 M would then be suitable to saturate the enzyme. However, in the type of experimental system used, one should also consider other possible effects of methanol. Methanol can be assumed t.o accumulate in the mieroenvironment of the enzyme (together with water) and interact with the enzyme in a nonspecific way due to its properties as a solvent. High amounts of solvents in the microenvironment of the enr/me can cause decreased enzymatic activity. Decreasing enzyme activities was indeed observed already at methanol concentrations above 500 mM and therefore a methanol concentration of 500 rnM was used in the rest of the experiments. The reaction rates were determined during the initial part of the reaction. With some substrates, low reaction rates were obtained and consequently long reaction times were needed. However, constant reaction rates were observed, also in the slowest reactions; no indications of enzyme inactivation were observed. This is in agreement with earlier results, which showed that chymotr~sin operating under similar conditions
was stable for several days [5]. The reaction rates were proportional to the amount of enzyme preparation employed in the range used (50-150 mg immobilized preparation). S u b s t r a t e speci/icity
In the ester±float±on of N-acetyl amino acids in organic media .'.he highest values of k , ~ , / K M were obtained with the aromatic amino acid derivatives (Table 11). This is not surprising since a-chymotrypsin in aqueous media cata!yses the hydrolysis of peptide bonds with aromatic or bulky aliphatic amino acid residues as the earbox'yl coml~aent. All the amino acid derivatives tested were ester±fled at varying reaction rates. This shows that a-chymotrypsin can be used for synthetic reactions involving several different amino acids. The Vm~ values for the different substrates differed widely while the K M values varied much less; most K M values were around 1 mM (Table 1I). The rates of the reactions in organic media depend on several factors, includie, g the solvent and the water content. In water miscible solvents such as acetonitrile, the rate normally increases with increasing water content. The water content used in this study (4%) is comparatively low because this gives the possibility of reaching high yields in reactions where hydrolysis is a competing reaction [6]. When water immiscible solvents are used the reaction rate normally increases with increasing water content until the medium is
TABLE It Kinetics of the ester±fleet±on of N-prolected amino acids with methanol tO.5 ItD in ,rater-saturated ethyl acetate and aeetonitrile with 4% ~vater catalyzed by a-ch~'mot~'psin deposited on celite
Substrate Ac-l..eu Ac-Leu At-Met Ac-Met Ac-Val Ac-Val Ac-Asp At-Asp Ac-Asn Ae-Asn Ac-Trp, Ac-Trp Ac-Tyr Acffyr Ac-Phe Ac-iPhe Z-Phe Z-P,~e Boc-Phe Boe-Phe Fmoc-Phe Fmo'~-Phe
Solveat Acetonitrile Ethyl acetate Acetonitrile Ethyl acetate Acetonitrile Ethyl acetate Acetonitrile Ethyl acetate Acetonitrile Ethylacetate Acetonitrile Ethyl acetate Acetonitrile Ethylacetate Acetonitrile Ethyl acetate Acelonilrile Ethylacetate Aeetonitrile Ethyl acetate Acetonitrile Ethyl acetate
Vm~~
K sI
K,
k ,~, ~Ks!
(retool h - t mmolenzyme- t )
(mM)
(mM)
(s - 1 M" I)
1.60 _+0.07 2.56 +_0.07 5.6 _+0.4 4.9 _+0.2 0.064 _+0.007 0.0178-+0.0007 0.45 _+0.02 0.17 _+0.(Vl 1.86 __+0.11 2.7 +_0. I 48.3 +0.9 106 _+2 01 _+3 258 _+1 17.4 _+0.7 57 ±1 0.80 +0.02 3.3 .4,0.2 0.312 +_0.1J04 0.43 _+0.02 0.254 ±0.904 0.31 _+0.01
i.5 4-0.3 1.1 +-0.2 i.2 ±0.1 1.0 :~0.1 17.3 ±0.5 0.95 _+0.02 0.%±0.03 0.52_+0.03 7.5 ± 1.4 8.9 _ 1.3 1.1 +0.1 0.9 ±0.1 1.8 .4,0.3 0.8 ±0.1 1.6 +_0.3 1.2 _+0.2 1.6 _+0.3 5.3 _+0.7 1.9 _4-0.1 0.6 +0.2 1.2 4-_0.1 2.0 _+0.2
28 _+I 4.0+_0.4 28.6+ 0.2 30 +-2 17 ± ! -
0.31 0.69 1.3 1.4 0.0010 0.0052 0.139 0.089 0.07 0.08 12 33 14 90 3.1 13 0.14 0.18 0.044 0.2 0.058 0.044
saturated with water; at higher water contents the reaction rate is often fairly constant. In the present investigation the substrates were dissolved in watersaturated ethyl acetate; results obtained with buffersaturated (50 mM Tris-HCI, pH 7.8) ethyl acetate were not significantly different. For instance, using the esterification of Ac-Phe as a model reaction the value of k~,,,/K M for the reaction in buffer saturated ethyl acetate was 6% lower than that obtained with water saturated solvent, but this difference was smaller than the experimental errors. The reaction rates observed in water-saturated ethyl acetate and in acetoaitrile containing 4% water were in the same range. Some substrates, for example Ac-Tyr, Ac.Trp and Ac-Phe, were more rapidly converted ip ethyl acetate while others reacted more rapidly in aeetonitrile (Table i1). With Ac-Val and Ac-Asp substrate inhibition was observed in both solvents and with At-Met in aeetonitrile only. The reason for substrate inhibition under these conditions is not knmvn. The substrate inhibition by Ac-Asp may be due to nonproductive binding between the enzyme and the earboxyl group in the side chain. It is possible that protection of thi~ carbo~l group would give a better substrate, as was the case when aspartic acid amide was used as the nucleophile in a a-chymotrypsin-catalysed reaction in organic media [71. in order to determine the influence of the N-protecting group, three groups in addition to acetyl were tested in the esterification of phenylalanine: Z (benzyloxycarbonyl), Boc (tert-butyloxyearbonyl) and Fmoc (fluorenylmethyloxycarbonyl). These protecting groups are the most widely used in peptide synthesis. Much higher rates were observed with acetyl as protecting group than with the others (Table I!). Unfortunately, acetyl is not a very s-fitable protecting group in peptide synthesis since it is not easily removed, it would be of interest to find a protecting group that, like aeetyl, gives high reaction rates in organic media and which is also practical concerning protection and deprotection reactions. All components of the reaction mixture can influence the rate of the enzyme catalyzed reaction. Since the aim was to study the interactions between the enzyme and its substrales, the support should be chosen so that it does not interfere with these interactions. It is known that some supports can influence the partitioning of substrates and thereby greatly influence the reaction rate [17]. in preliminary experiments Celite was used as support. It is known that only minimal amounts of water are adsorbed on Celite [13] and measurements showed no detectable adsorption of the substrates used in this study. To further investigate the possible interference of (?elite, experiments were carried out with enzyme powder, without support, as catalyst. Because of problems concerning aggregation
of the enzyme particles and attachm,nt of thosz to the reactor walls, the accuracy in these experiments was somewhat less than in those using Celite as enzyme support. The k,~, values were lower than those observed with Celite, probably because some enzyme molecules inside the aggregates formed were inactive. However, there was no significant difference in the K M values obtained with an.d without Celite, which indicates that Celite did not interfere with enzyme-substrate interactions. In a second set of experiments, the adsorption of substrates on Celite was measured under the conditions prevailing in the kinetic measurements. The analysis of the substrates before Celite addition and of substrates and products during the reaction revealed that the sum of ~he concentrations of substrates and products (containing the amino acid) was constant, indicating that no adsorption occurred. In the rest of the experiments, ~-chymotrypsin on Celite was used as catalyst.
Quantitatit:e structure actit:ity relationships The interaction between an enzyme and its substrates is a complex phenomenon and in order to make meaningful predictions of kinetic parameters in the enzymatic reactions many different factors must normally be taken into consideration. However, it is sometimes possible to construct fairly simple models describing enzyme-substrate interactions, and this has been done for chymotrypsin. For a series of close~,y related amino acid esters the value of the specificity factor kc.~t/K M for the hydrolysis in aqueous media correlated well with the hydrophobicity of the amino acid side chain [18]. However, in a larger investigation by Grieco, Hansch and co-workers [19,20] quantitative structure activity relationships (QSAR) of ehymotrypsin were studied, and it was shown that the molar refractivity described the vaAations in keat/K M better than the hydrophobicity. The molar refractivity is a measure of the 'bulk' of the substituent and i~ also lelated to the polarizability [21]. The values of log P (P is the partition constant in the octanol-water twophase system and is often used as a measure of the hydrophobicity) [22,23] and the molar refractivity (MR) for the different amino acid side chains and N-protecting groups are given in Table i1I. Grieco eta!. [20] included data for different amino acid side chains, N-protecting groups and ester groups. An equation for the dependence of kcat/K M on the molar refractivity of the different parts of the substrate molecule as well as other parameters was developed. Other equationz correlating 1/K M with MR and other parameters were also developed but the fit was not quite as good as for k cat/ K r~. In the present investigation eight different amino acids were used. The values of Iog(kc~t/K M) were plotted against the molar refractivity and log P for the
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Log P Fig. 1. The values of log(kc~t/K~t) for the ester[float[on of N-acet~'lamino acids v,ith methanol (0.5 M) ~. the molar refractivity,~a)or log P (b) of the amino acid side chain. The substrates were dissolved in water-~lur'zted ethyl acetate and the reactions were catalysed by a-chymotrypsin deposited on Celite. The molar refractMties and log P values for the dill•toni amino acid side chains are listed in Table I!1. The unit for k~dt/Kxl values iss -z M -t,
side chains. Fig. 1 shows the results obtained in ethyl acetate; similar results were obtained in acetonitrile. The correlation between the molar refractivity and log(k,~t/K M) was ~ m e w h a t better than that between log P and log(k,.JKM), although the amount of data is too limited to draw any conclusions with certainty. As ~entioned above the K M values varied comparatively little; the variation in kcat/K M was mainly due to the differences in ken t values. The simple l,~near correlation between log(keat/K M) and molar refractivity or log P must, of course, be corrected when the potential substrates contain structural elements that are too large or do not fit in the active site of the enzyme. Valine derivatives, for example, are poor substrates for a-chymotrypsin because of the isoprolryl group directly bound to the a-carbon atom. When Grieco et al. [20] developed the formula for the correlation of kcat/K M with molar refractivity and other parameters a special correction factor was introduced for substrates having an isopropyl group on ",he a-carbon atom. Corrections for large groups were done by introducing a negative term in (MR): for the
side chain [20]. Because of the large deviation valine was not included in Fig. I. In order to distinguish between effects of the direct enzyme-substrate interaction and the effects of the reaction medium, the lo~kcat/KM) values for the esterification reactions were plotted against literature values of log(k,~ffK M) for the reversed reactions (ester hydrolysis) in aqueous media (Fig. 2). It is dearly seen that for these reactions the specificity of a-chyraotrypsin in organic media is closely related to its specificity in aqueous media. The present results agree with those reported for the ester[ileal[on of N-acetylamino acids in ethanol catalyzed by subtilisin Cadsberg [24]. Subtilisin is known to have a preference for aromatic amino acids in aqueous media and the same specificity was observed in ethanol. The derivatives of histidine and arginine were not ester[fled [24]. Some researchers have reported pronounced differences in the substrate specificity of proteinases in aqueous and organic media, respectively [9,25]. In the
TABLE Iii
Molar refractitities [211 and log P [231 vahtesfor different amino acid side chains and N-protectbzggroups used in this ~tud)' Group
Molar refi activity
Log P
CH2CH(CH 3)2 (CH.,):SCH 3 CH 2C~,Hs CHrCoH4OH CH 2COOH CH zCONH2 CH2-indoyl
19.60 22.77 30.01 31.80 1!.88 14.41 42.30
2.169 1252 2.416 2.073 - 0.424 - 1.44 2.'/8
CH(CH3)z
14.98
COCH~ COOCH_,C~H~ COOCICH 3)3
!1.18 36.40 ~.82 62.60
1.64 - 1~00
COOCH,-fluorenyl
1.12 0.96 2.32
"~
0
rd)
.2 -3-
~,
-4¢ .
.
.
.
.
L o g k = t / K . in water
Fig. 2. Co,lrelation of the substrat¢ specificityof a-chymotrypsin in organic and aqueous media. The values of tog(kc.~t/KM) for the eslerification of N-acetyl amino acids with methanol vs. Iog(k~,/K M) for the reversed reaction, hydrolysisof N-acetylamino acid methyl esters(data from Griceo et al. [20}).The organic reaction media were water-saturated ethyl acetate ( i ) a n d acelonitrile with 4% water (o); the enz$-meused in organic media was deposited on Celite. The unit for kca~/K~.t values is s- t M- '.
7~
hydrolysis of N-acetylamino acid esters catalyzed by a-chymotrypsin or subtilisin in water the phenylalanine derivative was a much petter substrate (higher k , t / K M) than those of histidine and serine. However, when the same substrates were used in a transesterification reaction with n-propanol in octane the phenylalanine derivative was the worst substrate [9]. Similar observations have been made using polyethylene glycol modified a-chymotwpsin and subtilisin [25]. In the hydrolysis of N-benzoylamino acid esters in water the iysine derivative was a poor substrate or was not converted at all. On the other hand, in a peptide synthesis reaction in benzene it was a better substrate than the tyrosin derivative which was a very good substrate in water [25]. In the present investigation the derivatives of aspartic acid and asparagine were fairly poor substrates in organic media and the corresponding esters can therefore be expected to be poor substrates in water (no literature data were found). it can be concluded that in the esterifieation of N-acetyl-L-amino acids in organic media the substrate specificity of a-chymotrypsin and subfilisin concerning the amino acid side chain is similar to that for ester hydrolysis reactions in water. However, when esters of N-protected amino acid esters are used for transesterification or peptide synthesis reactions in organic mt:dia, the substrate specificity is reversed compared to when the same substrates are hydrolyzed in water. One reason for this discrepancy might be that the transesterification and peptide synthesis reactions described were carded out in solvents (benzene and octane) with considerably higher hydrophobicity than those used in the esterification reactions (ethanol, acetoaitrile and ethyl acetate). However, in order to fully understand these observations, more research is needed concerning the influence of the reaction medium on the rates of the separate steps in the reactions. Four different N-protecting groups were used for phenylalanine. Literature values for the corresponding
TABLE IV
Vahtes of the specificity constant log ~k,~t / ~,',,~)far the hydrolysis of different N-protected amino acid metl,yl estrrs hi am~eoltsmedia (data from Grie,'o et at. [20]) Protecting group
log( k ca~ / K,,.t)
Acetyl Benzoyl
Val
Phe
Tyr
0.13 I. 15
4.62 5.t)5
5.56 6.70
hydrolysis reactions in aqueous media were not found for all protecting groups. However, kinetic constants for some pairs of acetyl- and benzoyl-derivatives have been published (Table IV). In all these examples the exchange of the acetyl group to the benzoyl group, which corresponds to an increase in both log P and molar refractivity, resulted in an increase in keat/K m (Table 4). Furthermore, in the formula of Grieco etal. [20] the coefficient for the molar refractivity of the protecting group is positive, which means that k~t/K M increases with increasing molar refractivity. In organic media the opposite trend was observed. The value of k¢at/K M decreased with increasing log P or molar refractivity of the protecting group. Fig. 3 shows a plot of the data obtained in ethyl acetate. The results can be ir.terpreted l:y considering the interaction between the enzyme and the N-protecting group of the substrate as being fairly nonspecific. In water large, hydrophobic protecting groups are favorable and a large proportion of the interaction is probably hydrophobic in nature. However, in organic media hydrophobic interactions are less important and more hydrophilie protecting groups like acetyl are more favorable, Accordingly, as far as the protecting group is concerned the effect of the reaction medium is quite strong and a reversal of substrate specificity was observed when changing from aqueous to organic medium.
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Fig. 3. The values of Iog(k=~/K~I) for the esterification of N-protected phenyldanine with methanol (0.5 M) ~s. the molar refractivity (a) or log P (b) of the protecting group. The substrates were disso|ved in water saturated ettsyl acetate and the reactions were catalysed by ~-chymotrypsin deposited on (:'elite. The molar refractivities and log P values for the different protecting groups are li.~ted in Table !1I. The unit for kc~t/K~ values iss -~ M- t.
76 in conclusion, the substrate specificity of a-chymotrypsin in the esterification of N-protected amino acids with methanol in organic media is goverened by the intrinsic specificity o f the enzyme as far as the amino acid side chain is concerned and to a large extent by the reaction medium as far as the protecting group is concerned. Acknowledgements This project was supported by the Biotechnology Research Foundation (SBF) and the National Swedish Board for Technical Development (STUF). P. C l a p , s wishes to express his gratitude to Consejo Superior de lnvestigaciones Cientificas (C.S.I.C,) (Spain) for its financial support. The authors wish to thank Professor 13o Mattiasson for valuable discussions and Scott Bloomer for linguistic advice. References i Laurie, C., Tramper, J. and Lilly. M.D. (eds.) (1987) Biocalalysis in Organic Media. Elsevier. Amsterdam. 2 Zaks, A. and Russell, AJ. (1988) J. BiolechnoL8, Z';9-270. 3 Homandberg, G.A, Matlis, J.A. and Laskowski, M., Jr. (1978) Biochemistry 17. 5220-5227. 4 Jakubke. H-D.. Kuhl. P. and K6nneeke, A. (1985)Angew. Chem. Int. Ed. Engl. 24, 85-93. 5 Reslow, M., Adlerereutz. P. and Mattiasson, B. (1988) Eut. J. Biocnem. 177. 313-318. 6 Clap,s, P.. Adlercreutz, P. and Mattiasson, B. (1990) J. Biotechnol. 15, 323-338.
7 Claims, P_ Adlercreutz, P. and Maniasson, B. (1990) Biot¢chnol. AppL Biochem, i2. 376-386. 8 Kise, H., Fujimoto, K. and Nodtomi, H. (1988) J. BiozechnoL8. 279-290. 9 Zaks, A. and Klibanov, A. (1986) J. Am. Chem. So(:. 108, 27672768. 10 Wailc, H,R. and Niemann, C. (1%2) Biochcmislry l, 250-253. 11 Baumann. W.K.. Bizzozero. S.A., Dutler, H, (1973) Eur. J. Biochcm. 39, 381-391. 12 Morgenstem, L, Recanatini, M., Klein, T.E., Steinmelz, W., Yang, C.-Z., Langridge, R, and Hansch, C. (1987) J. Biol. Cbhem. 26~ 10767-10772. 13 Reslow, M.. Adlercreutz, P. and Mattiasson, B. (1988) Eur. J. Biochem. 172. 573-578. 14 Sagnela, G.A. (1985) Trends Biochem. Sci, 10. 100-103. 15 Bailey, J.E. and Ollls D.F. (1986) BiochemicalEngineering Fundamentals. McGraw-Hill Inc.. Singapore. 1,50zamiri, M, Adlercrautz, P. and Maniasson. B. (1991) Biotechnol. Appl. Biochem. 13, 54-64. 17 Omala, T., lida, T., Tanaka. A. and Fukui, S. (1979) Eur. J. Appl. Microbiol. Biolechnol. 8, 143-155. 18 Dorovska, V.N, VarfoIomeyev,S.D., Kazanskaya. N.F., Klyo~ov, A.A. and Martinek, K. (1972) FEBS Len, 23, 122-124. 19 Hansch, C. (1977) J. Me,d. Chem. 20, 1420-1435. 20 Grieco, C., Hansch, C., Silipo. C., Srnilh, R.N., Vittoria, A. and Yamada, K. (t979) Arch. Biochem. Biophys. 194, 542-551. 21 tlansch, C, Leo, A., Unger, S.H., Klm, K.H., Nikaitani, D. and Lien. EJ. (1973)£ Med. Chem. 16, 1207-1216. 22 Laane, C, Boeren, S. and Vos, IC (1985) Trends Bioteehnol. 3, 251-~2. 23 Rekker, R.F. (1977l The hyd~ophobicfragmental constant. Elsevier, Amsterdam. 24 K!se, H. (I990) Bioorg. Chem. 18. 107-115. G. ertner, H. and Puigserver. A. (1989) Eur. J. Biochem. 181, 207-213.
