Stabilizing effect of penicillin G sulfoxide, a competitive inhibitor of penicillin G acylase: Its practical applications Gregorio Alvaro, Roberto Fernandez-Lafuente, Rosa M. Blanco and Jos~ M. Guisdn I n s t i t u t o de Catdlisis, C . S . I . C . S e r r a n o , Madrid, S p a i n

We have f o u n d that penicillin G sulfoxide (pen G SO) behaves as a general stabilizing agent o f two bacterial penicillin G a~3'lases (PGAs) (from E. coli and from K. citrophila), and this role is related to a strong inhibitor)' effect on the enzymes. The stabilizing effect has been observed during two different inactivation processes: (i) thermal inactivation o f soluble enzymes at alkaline pH, and (ii) inactivation o f immobilized enzymes as a consequence o f covalent muhiinteraction with highly activated agarose aldehyde gels. At the same time, pen G SO behaves as a strong competitive inhibitor o f these two enzymes. The inhibition constant is more than lO-fold lower than the one corresponding to another smaller competitive inhibitor, phenylacetic acid (PAA), the structure o f which is exactly the acyl donor moiety corresponding to pen G SO. In turn, PAA hardly exerts any stabilizing effect on PGAs. The stabilizing effect o f pen G SO allowed the preparation o f derivatives o f these PGAs preserving full catalytic activity in spite o f being 1,400- and 650-fold more stable than the corresponding soluble or one-point attached immobilized enzymes.

Keywords:PenicillinG acylases, competitive inhibitorsof; stabilizingeffect of competitive inhibitors; immobilizationstabilization of penicillin G acylases; enzyme-support multipoint covalent attachment Introduction The stabilizing effect that some substrates and inhibitors may exert on the three-dimensional structures of enzymes is well known.t'2 This protective use of substrates or inhibitors becomes very important in enzyme engineering, because now, to prepare industrial enzyme derivatives, it is necessary to handle enzymes (purification, immobilization, etc.) and to modify them in order to improve their properties (e.g. stabilization through multipoint attachment to the support, chemical modifications, etc.) without distorting their three-dimensional structures. So, the use of inhibitors with stabilizing properties on the active conformation of the e n z y m e is a considerable help in this field of biotechnology. We have developed a rational strategy for immobilization-stabilization of enzymes by multipoint covalent

Address reprint requests to Dr. Guisfinat the Instituto de Catgtlisis. C.S.1.C., Serrano 119, 28006 Madrid, Spain Received 1 November 1989; revised 23 April 1990 210

Enzyme Microb. Technol., 1991, vol. 13, March

attachment to activated preexisting supports. We have proposed the attachment of enzymes, through their amine groups, to monolayers of identical aldehyde groups moderately secluded from support surfaces, e.g. glyoxyl-agarose gels, as a very useful immobilization-stabilization system. 3 Using this system, we have been able to prepare very active and very stable derivatives of a number of enzymes: trypsin, 4 chymotrypsin, 5 and lipase. 6 H o w e v e r , during the experimental procedure for the preparation o f these types o f e n z y m e derivatives, there are two key events that appear to be very dangerous for the preservation of the active e n z y m e conformation: (I) For most of the enzymes tested, this type of immobilization and further e n z y m e - s u p p o r t multiinteraction occurs only at quite alkaline pHs, e.g. pH 10.0, and at this pH some enzymes are quite unstable. (2) To improve e n z y m e stability, we have to promote very intense e n z y m e - s u p p o r t multiinteraction processes. These processes may generate important losses in catalytic activity because of the possibility of the e n z y m e ' s suffering strong or moderate conformational changes. (c> 1991

Butterworth-Heinemann

Stabilizing effects of enzyme inhibitors: G. Alvaro et al. Indeed, we have shown this use of competitive inhibitors by testing the stabilizing effect of benzamidine in the preparation of highly stabilized trypsin(amine)agarose (aldehyde) derivatives. 7 Further, we have now applied our immobilizationstabilization strategy to the preparation of penicillin G acylase (PGA) derivatives. In this case, the two points remarked on above were extraordinary relevant, and hence it was particularly important to find stabilizing agents for these enzymes. We have tested two possible agents: phenylacetic acid (PAA), a product of penicillin G hydrolysis by this enzyme, which has been reported as a competitive inhibitor; 8 and penicillin G sulfoxide (pen G SO), a close analogue of the substrate penicillin G (pen G). Pen G SO is a derivative of pen G, which is obtained by oxidation of the antibiotic with peracetic acid. 9 Plaskie et al. ~° have reported that pen G SO is not hydrolysed by PGA from E. coli, in spite of its very close structural similarity with pen G, the best substrate for this enzyme. For this reason, we have supposed that this compound should also be an interesting competitive inhibitor of these enzymes, with good properties as a stabilizing agent. In this paper we report a brief study of inhibition of two different PGAs (from E. coli and from K. citrophila) by PAA and by pen G SO. Also, we have tested the possible stabilizing effect of these inhibitors on the active conformations of these two PGAs. We have studied two different inactivation processes: (i) irreversible thermal inactivation of soluble enzymes at alkaline pH, and (ii) multiinteraction of immobilized PGAs with highly activated agarose-aldehyde gels. Finally, we have compared the activity/stability of derivatives of these two PGAs prepared in the presence or in the absence of pen G SO.

per minute in identical experimental conditions, but at 37°C. The ratio between catalytic activities at 37°C and 25°C was essentially the same for the two enzymes used: approximately 1.7. The presence of 9 mM pen G SO in some suspensions of immobilized enzyme or solutions of soluble enzyme did not affect the observed reaction rates, because only very small aliquots of these solutions/suspensions were added to the assay mixture (usually 50 ~1). Therefore, the [substrate]/[inhibitor] ratio in the assay mixture was very high (approximately 300), and under these conditions the inhibitory effect of pen G SO was practically negligible (see Results).

Kinetic studies For determination of the kinetic constant of the two soluble enzymes, we have used an assay very similar to the standard one described in the previous paragraph. Now substrate concentrations ranged between 3 /~M and 3 mg. In this range, the kinetics of pen G hydrolysis fit to a Michaelis-Menten equation. ~1 Inhibition by PAA or by pen G SO was studied by performing three sets of experiments with different inhibitor concentrations and varying substrate concentrations in the range indicated above.

Irreversible thermal inactivations Soluble enzymes at alkaline pH. We have incubated samples of soluble PGAs, 8 U ml-1 of whole solution, in 50 mM bicarbonate buffer pH 10.0 at 20°C. At different times, aliquots were withdrawn and assayed by following the standard procedure described above. For each enzyme tested (the one from E. coli and the one from K. citrophila), different incubations were performed in the presence of: (a) 9 mM pen G SO, (b) 200 mM PAA, (c) no inhibitor.

Materials and m e t h o d s

Materials Semipurificd extracts of PGA from E. coli were generously donated by Boehringer Mannheim GmbH (Penzberg, FRG). Extracts of the enzyme from Kluyvera citrophila and the substratc analogue pen G SO were a gift from Antibioticos S.A. (Le6n, Spain). Sepharose CL 6B was purchased flem Pharmacia Fine Chemicals (Uppsala, Sweden), and all other reagents and substrates were from Sigma Chem. Co. (St. Louis, MO, USA).

Enzymatic assays Soluble and immobilized enzyme were assayed with pen G as substrate, titrating the liberated PAA with 0.01 M NaOH, dissolved in 0.1 M NaC1, by using a pHstat (model TTT80 Radiometer, Denmark). Standard experiments were carried out at pH 8.0 at 25°C, using 50 ml of 3 mM pen G, dissolved in 0.1 M NaC1, as the assay mixture. Between 0.1 and 0.4 units (U) of enzyme were used in each experiment. A unit is defined as the amount of enzyme that hydrolyses 1 /zmol of pen G

Immobilized derivatives at pH 8.0 and very high temperature. We have incubated samples of immobilized PGA-agarose derivatives at pH 8.0 and high temperature. Derivatives of the enzyme from E. coli were incubated at 58°C and the ones of the enzyme from Kluyvera at 62°C. At different times, aliquots were withdrawn and assayed at pH 8.0 and 25°C according to the standard procedure.

Preparation o f PGA (amine)-agarose (aldehyde) derivatives Activation of agarose gels. GlyoxyI-Sepharose CL 6B gels (Agarose-O-CHz-CHO) were prepared by etherification of agarose gels with glycidol (2,3-epoxypropanol) and further oxidation of the resulting glyceryl-agarose (Agarose-O-CHz-CHOH-CH2OH) with periodate. A detailed description of this activation method has been given in a previous paper) Preparation of penicillin G acylase-agarose derivatives. It has been also performed essentially as previously described. 12 Enzyme Microb. Technol., 1991, vol. 13, March

211

Papers Reaction m&ture (immobilization suspension): 100 U of PGA were added to 100 ml of 50 mM bicarbonate buffer pH 10.0 (in some cases containing a high concentration of inhibitors of the enzyme: see Results) containing 10 ml of very highly activated glyoxyl-Sepharose CL 6B (a gel containing 70 p~Eq of aldehyde group per milliliter, which corresponds to a surface density of 17 aldehyde residues per 1000 A 2 of gel surface3). At the same time, a blank was prepared by adding 10 U of acylase to 10 ml of the same buffer but containing 1 ml of inactivated gel (very highly activated glyoxyl agarose previously reduced with sodium borohydride to eliminate the reactive aldehyde groups). Then the immobilization suspension and blank were very gently stirred inside a high-low temperature incubator at 20°C (except where otherwise indicated). At different times, aliquots of supernatant and whole suspension of immobilization suspension or blank were withdrawn and assayed by following the standard assay described above. Endpoint of the enzyme-support muhiinteraction process: After 3 h of contact time between the enzyme, already immobilized, and the activated support, derivatives were reduced with borohydride in the same "optimal conditions" previously found for trypsin-agarose derivatives. ~3 In this way the Schifrs bases formed between the enzyme and the support were reduced into very stable secondary amine bonds, and the remaining aldehyde groups in the gel were converted into inert hydroxyl ones.

Results

Inhibition o f penicillin G acylases We have studied the inhibitory effect of PAA and pen G SO on pen G hydrolysis by these two PGAs. Our results of PAA inhibition of the enzyme from E. coli, obtained with semipurified extracts, perfectly agree with the ones previously reported in literature, obtained with pure enzyme: 8 inhibition is competitive and the values for KM, pen G, and K], PAA, are very similar to the ones previously reported. We have found that PAA is also a competitive inhibitor of the enzyme from Kluyvera, and KM as well as K l for this enzyme fall in the same range as those obtained for the enzyme from

E. coli (Table 1). In addition, we have also found pen G SO behaves as a competitive inhibitor of the two enzymes. Again,

Table 1

Kinetic constants of the t w o PGAs

Microbial source K M, pen G Ki, PAA K I, pen G SO

E. coil

K. citrophila

20 a 200 ~ 12

11 150 8

a Data taken f r o m ref. 8 and reproduced in o u r l a b o r a t o r y with s e m i p u r i f i e d extracts. Experimental procedure f o r obtaining these constants is described in Materials and methods. Constants are given in p~M

212

E n z y m e M i c r o b . T e c h n o l . , 1991, v o l . 13, M a r c h

100 $ >.-'75 i.> "~ 50

~ZS

o

2

t,

6

8

I

I

10

TIME, hours

Figure 1 Time courses of irreversible inactivation of soluble PGAs at pH 10.0 and 20°C. Circles: enzyme from E. coil. Squares: enzyme from K. citrophila. Open symbols: enzyme incubated in the presence of 9/~M of pen G SO. Half-filled symbols: enzyme incubated in the presence of 200 mM PAA

the values of K 1corresponding to the two enzymes are very similar, and they are quite lower than KM for pen G but are of the same magnitude (Table 1). The inhibitory effect of pen G SO is greater than that of PAA: K] values are more than one order of magnitude lower for pen G SO than for PAA (approx. 20-fold).

Stabilizing effect o f the inhibitors against enzyme denaturation in alkaline media In Figure 1 we observe that in the cases of both the enzyme from E. coli and that from K. citrophila, the time courses of inactivation at pH 10.0 and 20°C are exactly the same in the presence or in the absence of 200 mM of PAA. On the contrary, we observe a dramatic stabilizing effect promoted by the presence of pen G SO in the incubation of the enzyme under alkaline media. For example, the enzyme from E. coli remains almost fully active after 8 h incubation in the presence of pen G SO, but it preserves less than 10% of residual activity when incubated in the same conditions but in the presence of PAA or in the absence of inhibitors. The concentrations of inhibitors used in these tests on their stabilizing effect were three orders of magnitude higher than the values of K~ found at pH 8.0 and 25°C, that is, 200 mM PAA and 9 mM pen G SO. Since the values of the kinetics constants, K[ and KM, at pH 10.0 are of the same order of magnitude (results not shown), we can assume that at these concentrations the inhibitors are adsorbed in every instant on the active centers of more than 99% of PGA molecules during the incubation at pH 10.0. In fact, PGA is completely inhibited at pH 10.0 when using high substrate concentrations (approx. 10 KM) in the presence of these concentrations of inhibitors.

Presence of penicillin G sulfoxide during the immobilized enzyme-activated support multiinteraction process In Figure 2, we observe that pen G SO also exerts a dramatic stabilizing effect on these two PGAs during their processes of multiinteraction with the highly acti-

Stabilizing effects o f e n z y m e i n h i b i t o r s : G. A l v a r o e t al.

vated glyoxyl-agarose gels. Derivatives prepared in the presence of the inhibitor remained fully active during 3 h of immobilized enzyme-activated support multiinteraction. Immobilization is complete after the first 10 min (the activity of supernatant of immobilization suspension becomes zero and those of whole suspension and of the supernatant of the blank remained at 100% level). Further immobilized enzyme-activated support multiinteraction seems to take place, because stabilization of these derivatives increases dramatically when they are reduced with borohydride after different incubation times (from 20 minutes to 3 h), as we have previously reported.12 On the contrary, derivatives prepared in the absence of pen G SO lose a great percentage of their catalytic activity. This loss of activity is greater for the enzyme from E. coli and it is also greater at 20°C than at 15°C (Figure 2). Immobilization was complete after 10 min, and derivatives reduced at that moment resulted in reasonable stability under these experimental conditions (see broken lines in Figure 2). Hence, it seems very clear that the loss of catalytic activity observed for unreduced immobilized enzyme-activated support conjugates (continuous lines) after the first 10 min is a direct consequence of covalent multiinteraction of the

100 I :

_-

--

"2- El

so

100

~ so

®

0

I

1

I

2 TIHE, hours

I

3

0

I

1

I

2

1

3

TIHE, hours

2 Time courses of immobilization-multiinteraction of PGAs on agarose-aldehyde gels. Residual activity is the activity of whole immobilization suspension as compared (in percentage) with the activity of soluble enzyme offered to the support. General conditions for preparation of derivatives are given in Materials and methods. (A) PGA from Kluyvera citrophila, temperature of reaction 20°C; (B) derivative prepared in presence of pen G SO; (E3)derivative prepared without inhibitor; (O) irreversible inactivation, at pH 10 and 20°C, of a derivative prepared without inhibitor but reduced with borohydride after only 10 min of immobilization. (B) PGA from E. coil: temperature of immobilization was 20°C (triangles) or 15°C (diamond). Closed symbols: derivatives prepared in the presence of inhibitor. Open symbols: derivatives prepared without inhibitor. Circles: irreversible inactivation (at pH 10 and 20°C) of a derivative prepared at 20°C without inhibitor but reduced with borohydride after only 10 min of immobilization. Letters associated with four curves (A, B, C, and D) indicate the time courses of preparation of the derivatives whose final properties (activity and stabilization) are presented in Table 2 Figure

Table 2 Activity/stabilization of PGA-agarose derivatives prepared in the presence or absence of pen G SO Microbial source

E. coil [pen G SO] 9 /~M --

K. citrophila Activity/stability

100/1400 (A) 70/1800 (C)

100/650 (B) 60/800 (D)

Activity is expressed as % corresponding to soluble enzyme that has been immobilized, and it was determined according to the standard assay described in Materials and methods. Stabilization is defined as the ratio between tl/2 corresponding to derivative and the one corresponding to one-point attached enzyme (which was also identical to that of soluble enzyme). These ha If-life times were obtained in experiments of irreversible thermal inactivation at pH 8.0 performed as described in Materials and methods. Concentration of inhibitor is that used in the immobilization suspension, that is, the one present during the process of multiinteraction of the enzyme with the support. Contact time immobilized enzyme-activated support was 3 h in all cases. Temperature was 20°C except for the derivative from E. coliwithout in hibitor, which was 15°C. Time courses of preparation of these derivatives are represented in Figure2: letters between parentheses in thetable correspond with those in the figure

enzyme and the support, which is not possible in the case of reduced derivatives. Inactivation observed in the first 10 rain should also be mainly caused by the same process, because inactivation of soluble enzyme or of immobilized intermediates existing during these first 10 min is almost negligible in such a short period of time, as observed for soluble enzyme in Figure 1. Anyway, since the loss of catalytic activity observed during the first 10 min is small, as compared with the overall inactivations, we can assume that the overall inactivations observed in Figure 2 in the absence of pen G SO are mainly due to covalent multiinteraction of immobilized enzyme and activated support. In Table 2 we report activity/stability data for PGA-agarose derivatives prepared with or without the presence of pen G SO. Derivatives prepared in the absence of inhibitor result in less active but more stable derivatives than those prepared in the presence of pen G SO. Borohydride reduction of the derivatives and further washings of the derivatives do not affect their activities, because these are exactly the same as those measured in the immobilization suspension (Figure 2). Discussion

Phenyl acetic acid, which is a small molecule constituted only by the acyl donor moiety of pen G SO (and also of the substrate pen G) is a weak competitive inhibitor of the enzymes. This weak inhibitor hardly exerts any stabilizing effect on the enzyme structure for the two inactivation processes studied in this paper. On the contrary, pen G SO, which also contains a nucleophile moiety very similar to that of the substrate pen G, behaves as a quite strong competitive inhibitor of the two PGAs we have tested (K~ values are lower E n z y m e M i c r o b . T e c h n o l . , 1991, v o l . 13, M a r c h

213

Papers than K M for pen G). This strong competitive inhibitor also acts as a very interesting stabilizing agent of this enzyme against two very different inactivation processes: irreversible thermal inactivation at alkaline pH and inactivation promoted by multipoint attachment on aldehyde-agarose gels. A very clear correlation between inhibitory and stabilizing effects may be established and pen G SO may be considered as a general stabilizing agent of PGAs. We can assume the following mechanism for this general stabilization promoted by substrates and strong competitive inhibitors: The presence of these substances strongly adsorbed on the active center of the enzyme protects its structure from inactivation, because now the conformational changes induced by an inactivating agent will also be associated with a very important loss of energy of adsorption for the inhibitornative enzyme complex. So, the stabilizing effect is much greater when inhibition is stronger and the inhibitor interacts with a larger area of the active center of the enzyme (in this case, subsites responsible for acyl donor and nucleophile recognition). As an example of the important practical applications of the use of general stabilizing agents, we like to remark that, in the present case, pen G SO has become very useful for preparing very active and very stable derivatives of PGAs. Derivatives prepared in the presence of this inhibitor preserved full activity (100% of catalytic activity corresponding to soluble enzyme that has been immobilized), in spite of being 1,400 (PGA from E. coli) and 650 (PGA from K. citrophila) times more stable than the corresponding soluble or onepoint immobilized enzymes. Indeed, derivatives prepared in the absence of inhibitor resulted in less active but more stabilized enzymes. Similar results were previously obtained with trypsin and its competitive inhibitor benzamidine. 7 According to the assumptions made in the previous paragraph, the presence of the inhibitor prevents the enzyme from additional attachments to the support, which are associated with important conformational changes in enzyme structure, and so multipoint attachment in the presence of inhibitor results less intense but less distorting. Because of the number of possible industrial applications of PGAs (hydrolysis of pen G, synthesis of semi-

214

Enzyme Microb. Technol., 1991, vol. 13, March

synthetic antibiotics, resolution of racemic mixtures, protection/deprotection of amino acids and sugars, etc. 14), we expect that new modifications of these enzymes will be tried in the future. The use of pen G SO as a general protecting agent for this enzyme constitutes an important breakthrough for such attempted manipulations.

Acknowledgements We thank Boehringer Mannheim GmbH (Penzberg, FRG) for its generous gift of PGA from E. coli and Antibioticos S.A. (Le6n, Spain) for its generous gifts of PGA from Kluyvera citrophila and pen G SO. We are also very grateful to Dr. J. L. Garcia (C.S.I.C. Spain) and Prof. Alan Russel (University of Pittsburgh, PA) for very useful discussions. This work was supported by the Spanish CICYT (project No. BIO88-27601) and the Commission of the European Communities [Biotechnology Action Programme, contract No. 395.E (JR)].

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Lumry, R. in The Enzymes (Boyer, P. D., Lardy, H. and Myrbfick, K., eds.) 2nd ed. (1959) Vol. 1, pp. 157-231; Academic Press, New York (1977) pp. 303-320 Grisolia, S. Physiol. Rev. 1964, 44, 657-712. Guisfin, J. M. Enzyme Microb. Technol. 1988, 10, 375-382 Blanco, R. M., Calvete, J. J. and Guisfin, J. M. Enzyme Microb. Technol. 1989, 11, 353-359 Guisfin, J. M., Ceinos, M. C. and Blanco, R. M. Spanish Patent No. 8902176 (1989) Otero, C., Ballesteros, A. and Guisfin, J. M. Appl. Biochem. Biotechnol. 1988, 19, 163-176 Blanco, R. M. and Guisgm, J. M. Enzyme Microb. Technol. 1988, 10, 227-232 Kutzbach, C. and Rauenbusch, E. Hoppe-Seylers Z. Physiol. Chem. 1974, 354, 45-53 Essery, J. M., Dabado, K., Gottsein, W. J., Hallstrand, A. and Cheney, L. C. J. Org. Chem. 1965, 30, 4388-4389 Plaskie, A., Roets, E. and Vanderhaeghe, H. J. Antibiot. 1978, 31, 783-788 Alvaro, G. Ph.D. Thesis, Universidad Autdnoma de Madrid, 1988 Alvaro, G., Fernandez-Lafuente, R. and Guisfin, J. M. Appl. Biochem Biotechnol. (in press) Blanco, R. M. and Guisfin, J. M. Enzyme Microb. Technol. 1989, 11,360-366 Waldmann, H. Liebigs Ann. Chem. 1988, 12, 1175-1180