Pronase-catalysed hydrolysis of amino acid amides

Pronase-catalysed hydrolysis of amino acid amides I.A. Yamskov, T.V. Tichonova and V.A. Davankov A.N. Nesmeyanov Institute o f Organo-Element Compounds, USSR Academy o f Sciences, 117813 Moscow, USSR (Received 23 April 1985; revised 19 September 1985) Pronase from S t r e p t o m y c e s griseus exhibits a high activity and enantioselectivity in the hydrolysis of

three amino acid amides: Ieucine amide, phenylalanine amide and tryptophan amide. The influence of pH, temperature and metal ions on pronase (EC 3.4.24.4) amidase activity and enantioselectivity has been studied. Keywords: Pronase; hydrolysis; amino acid amides

Introduction Pronase (EC 3.4.24.4) is known to show high activity and enantioselectivity in hydrolysis of esters of such amino acids as leucine, tryptophan and phenylalanine, t': This enzyme preparation can therefore be used for hydrolysing racemic amino esters to arrive immediately at optically active amino acids. The main active component in the above process of hydrolysis of amino esters appears to be leucine aminopeptidase. It can be assumed that employing amino amides which are more specific substrates for leucine aminopeptidase can raise the rate and enantioselectivity of the hydrolysis and increase the optical purity of the amino acid produced. Amino esters would limit the range of conditions for such a resolution process to neutral pH values and low temperatures (because of high rates of spontaneous hydrolysis of the esters even in weakly alkaline media) whereas amino amides prove stable in alkaline solutions. Therefore, in the present paper, hydrolysis of amino amides by pronase has been examined in more detail.

A ctivity The activity of pronase, A, (in /.trnol min -~ g - ' ) was estimated as initial rate of substrate hydrolysis which was followed polarimetrically in a 10 cm cell at a wavelength of 436 nm and temperature of 25°C and calculated according to the following formula: /x~ [S]o 1 At Cto--O~t [E]o

A

where: Aa//Xt is the rate of change in the optical activity of the solution at the initial stage of reaction (where the dependence of the optical rotation c¢ on time t is linear); [S]o is the initial substrate concentration; [E]o is the initial enzyme concentration (all the experiments were carried out in the range of linear dependence of the hydrolysis rate on [E]o); ao is the optical rotation of the initial amino amide solution; at is the optical rotation of the amino acid produced at 100% substrate conversion. The contribution of the enzyme to the optical rotation of the reaction mixture is not large: it does not exceed 1-2% in the hydrolysis of L-substrates and 2 0 - 4 0 % in that of the D-substrates where the experiments had to be carried out at a high enzyme concentration.

Materials and methods In the present work pronase was purchased from Merck (art 7433, activity 70000 PUKg - t ) and used without any additional purification. The stock enzyme solutions of concentrations 1 g 1-1 and 20 g 1-2 were prepared in water and used the same day.

Substrates and o t h e r reagents Acetates of L- and D-tryptophan and L- and Dqeucine amides as well as L- and D-phenylalanine amides were synthesized as described) The stock substrate solutions were prepared in a 0.1 M diethanolamine-HC1 buffer and in a 0.1 M Tfis-HC1 buffer. Salts such as CaCI2.6H20 and COC12.6H20 analytical grade were used. 0141 --0229/86/040241 --04 $03 .O0 © 1986 Butterworth & Co. (Publishers) Ltd

Results Preliminary examination of the pronase-catalysed reaction has shown that in the case of L-phenylalanine amide and L-leucine amide at pH 8.0 and 10.0, the hydrolysis rate does not depend on the substrate concentration in the range examined, 14-50 mM (no check of this kind has been made for tryptophan amide). Therefore, all the subsequent experiments were carried out at a substrate concentration of 20raM. No data were available for hydrolysis of D-isomers. The pH dependence of the hydrolysis rates of the three above amino amides reveal two maxima (Figures 1 - 3 ) , both located in the alkaline pH region. The first was at pH 8.0-8.1 for the three amides and the second one was at

Enzyme Microb. Technol. 1986, vol. 8, April

241

Papers pH 9.5 for L-leucine0amide, at pH 9.2-11.0 for L-phenylalanine amide and at pH 9.5 10.5 for u-tryptophan amide. In the case of L-phenylalanine amide and tryptophan amide, the hydrolysis rates at both maxima are almost the same, whereas in the case of L-leucine amide the enzyme activity at pH 9.5 is substantially lower than that at pH 8.0. The hydrolysis rates of #-form amino amides reveal a more dramatic fall at the alkaline pH compared to those for corresponding L-substrates. In view of this fact the enantioselectivity of the process characterized by the ratio AL/A D with passage through a mininmm (at pH 8.1-8.8 for

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phenylalanine amide, at pH 8.4 9.6 for leucine amide and at pH 8.6 for tryptophan amide) increases towards the higher pH range (Figures ] 3). It should be noted that pronase displays a considerably higher enantioselectivity in the hydrolysis of leucine amide compared to tryptophan amide; phenylalanine amide in this respect occupies an intermediate position (Table I). It has been shown 1'2 that the presence of Ca2+ and Co 2+ ions can enhance the activity and enantioselectivity of pronase in the hydrolysis of amino esters. Therefore, the influence of Ca> and Co 2+ ions on the pronase-catalysed hydrolysis of amide substrates was also examined. Solutions of Ca> or Co> salts were added to the stock solution of pronase (1 g 1-') and the activity of the mixture was examined after several periods of incubation. As seen from Figure 4, the rate of hydrolysis of Ldeucine amide at pH 8.0 by a pronase solution which was made 10 mm in Ca 2+ ions increased during the first 15-20 min to a twofold value and then decreased again to about 70-80% of the pronase activity observed in the absence of Ca2+ ions. This level of activity was maintained for at least six additional hours. A similar phenomenon was also observed on adding 5 mM Co 2+ ions but the rate of L-leucine amide hydrolysis only reached a maximum of 150-160%. Quite different behaviour was observed when the activity of Ca2.- and Co2*-modified pronase solutions was estimated on the rate of hydrolysis of L-tryptophan amJde (at pH 8.0). On adding Co g÷ ions, the enzymatic activity with

Figure 1 pH dependence of enantioselectivity and activity of pronase in the hydrolysis of L- and D-phenylalanine amides: o, L-phenylalanine amide; • D-phenylalanine amide; ~, A L/AD

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Table 1

Figure 3 pH dependence of enantioselectivity and activity of pronase in the hydrolysis of L- and D-tryptophan amides: ~,, Ltryptophan ami de; e, D-tryptophan a mide; ~, A L/A D

Activity and enantioselectivity of pronase-catalysed hydrolysis of amino acid amides and esters Methyl ester

Amide Amino acid

pH

AL

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A L/A D

pH

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Enzyme Microb. Technol. 1986, vol, 8, April

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Pronase on amino acid amides: I.A. Yamskov eta/.

respect to L-tryptophan amide increased by a factor of 8.6-8.8 in the course of 1-1.5 h (Figure 4) and this high level of activity remained for one or two days (a longer period was not considered). When Ca 2+ ions were introduced into the pronase solution, no changes in the Ltryptophan amidase activity are observed. The study of thermostability of the pronase solutions (concentration 1 g 1-] ) has shown that pronase maintained 100% activity with respect to L-leucine amide up to a

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Figure 4 Pronase activity versus incubation time with Ca d* and Co =* ions. Pronase solution concentration, 1 g 1-1 , Ca2. ion concentration, 1 0 m M ; Co 2+ ion concentration, 5 r a M . Activity was determined from the rate of hydrolysis of (m, +) L-tryptophan amide, [S]o = 2 0 m M pH 8.0 and (o, &) L-leucine amide, [S]o = 20 mM pH 8.0. (m, o) Ca2+-containing pronase solution; (+, A) Co 2+_ containing pronase solution

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temperature as high as 60-65°C provided 10 mM Ca 2+ or 5 mM Co 2+ ions were present in the solution (Figure 5). On the contrary, aqueous pronase solutions maintained an invariable activity only up to a temperature of 50°C; at 60°C the measured activity was found to be less than 80% of the initial value. The above stability tests were made at pH 8.0. For comparison, we checked the activity of Ca2+-containing pronase solution with respect to L-leucine amide at pH 9.7 where it was observed to drop at 65°C to a value of 38% of the initial activity at this pH. The activity of all three pronase solutions with respect to L-tryptophan amide (.pH 8.0) dropped continuously with a rise in temperature (Figure 6), the most dramatic drop being observed in the case of the Co2+-containing pronase solution which maintained only half its initial activity at 60°C. The examination of complete kinetic curves for L- and DL-phenylalanine amides at the pH optimum for enzyme activity with respect to the b-substrate showed that at pH 8.2 the hydrolysis of L-phenylalanine amide proceeded up to a conversion of 89%. If a racemic substrate was used, the presence of D-phenylalanine amide inhibits the initial rate of hydrolysis of L-substrate to a large extent, but does not markedly influence its final conversion. The same picture was also seen at pH 10.0 where the conversion of Lphenylalanine amide was 87%. The comparison of complete kinetic curves for the hydrolysis of L- and DL-leucine amides both at pH 8.0 and pH 10.0 showed that the conversion always approached a level of 100%; even in the case of the racemic substrate, the presence of Dqeucine amide appeared to exert no marked effect either on the conversion of the Losubstrate or on the initial rate of its hydrolysis.

Discussion The comparative study of the enzymatic hydrolysis of three

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T h e r m o s t a b i l i t y of L - t r y p t o p h a n amidase activity of pronase. Pronase solution in: ©, 5 m M CoCI 2 , I , water, ~, 10 m M CaCI 2 . Thermostabi#ity was checked as described in F/gufe 5. A c t i v i t y of pronase w i t h respect to L - t r y p t o p h a n amide was measured at pH 8.0

E n z y m e M i c r o b . T e c h n o l . 1 9 8 6 , vol. 8, A p r i l

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Papers amino amides has shown L-leucine amide to be the best amide substrate for pronase with respect to the enzyme's activity and enantioselectivity (Table 1). This finding resembles an earlier observation that L-leucine esters are the best substrates of several amino esters examined. It should be noted that whereas the rates of hydrolysis of L-leucine amide and L-leucine methyl ester are practically the same, in the case of L-tryptophan a similar substitution of an ester substrate for the corresponding amide leads to a significant decrease in the rate of enzymatic hydrolysis. Pronase is known to contain at least ten proteolytic components: five serine-type proteases, two Zn2+-endo peptidases, two Zn>-leucine aminopeptidases and Zn >carboxypeptidase (see references in ref. 1). Hydrolysis of the ester and amide substrates may be catalysed by both the serine proteases and the leucine aminopeptidases. From the data obtained so far it is difficult to decide which individual enzyme component of the pronase preparation plays the major role in hydrolysis of one or another substrate. However, the existence of two maxima on the pHdependence curve of pronase activity and the different thermostability of pronase components which hydrolyse substrates at these two pH optima (Figure 5) indicates at least two enzymes participating in the hydrolysis of amides. It is known that the leucine aminopeptidases are the most thermostable components of pronase. 4's At 70°C pronase fully retains its leucine aminopeptidase activity, all other components of pronase lost 90% of their activity at this temperature. In our experiments with the amide substrates at pH 8.0 pronase retained 70% of its activity. It is possible that the main contribution to the pronase activity towards amide substrate at pH 8.0 is due to leucine aminopeptidases. Pronase components hydrolysing amides at the more alkaline pH (9.7) have low thermostability. Thus at this pH amide hydrolysis is probably due to proteases other than leucine aminopeptidases. As mentioned above, the utilization of racemic amino amides for producing optically active amino acids makes it

244

Enzyme Microb. Technol. 1986, vol. 8, April

possible to carry out the process of enzymatic hydrolysis in a rather broad temperature and pH range due to higher stability of amides to spontaneous hydrolysis compared to amino esters. The most important result of the present work is that the enzymatic complex pronase exhibits a high activity and enantioselectivity in the hydrolysis of leucine, phenylalanine and tryptophan amides (Table 1), whereby one should note the rise in enantioselectivity in the pH region higher than 9.5. An additional positive factor is the high conversion of L-substrates (i.e. the absence of inhibition by the products), as well as the absence of any noticeable influence of D-substrates upon the degree of conversion of L-amino amides. As for DL-leucine amide, the D-enantiomer has practically no influence even on the initial rate of hydrolysis. Another important advantage in the use of pronase is the high thermostability of this preparation, increasing even more in the presence of Ca 2+ ions which has practically no effect on the amidase activity of pronase. The utilization of Co2+-containing solutions of pronase which acquire a substantially elevated activity with respect to L-tryptophan an-tide is very promising at temperatures less than 40°C (Figure 6). Thus, the hydrolysis of amino amides in the presence of pronase is characterized by high rate, significant enantioselectivity and high degree of conversion of substrates, which makes this enzyme complex suitable for preparing optically active amino acids. References 1 Yamskov, 1. A., Tikhonova, T. V. and Davankov, V. A. Enzyme Microb. Technol.. 1981, 3,137-140 2 Yamskov,I. A., Tikhonova, T. V. and Davankov, V. A.t:'nzyme Microb. TeehnoL 1981, 3,141-144 3 Smith, E.L. andSlonim, N.B.J. BioLChem. 1948,176,83884l 4 Naxahashi,Y. and Yanagita, M. J. Biochem. (Tokyo) 1967,62, 633-641 5 Vosbeck, K. D., Chow, K.-F. and Awad, W. M, J. Biol. Chem. 1973, 248, 6029--6034