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Modulation of the function of an enzyme immobilized in a conductive polymer by electrochemical changing of the substrate concentration
293
J. Electroad Chem., 347 (1993) 293-301 Elsevier Sequoia S.A., Lausanne
JEC 02474
Modulation of the function of an enzyme immobilized in a conductive polymer by electrochemical changing of the substrate concentration Tetsuya Haruyama, Hiroaki Shinohara, Yoshihito Ikariyama and Masuo Aizawa
l
Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 227 (Japan) (Received 17 July 1992; in revised form 14 September 1992)
A modulation system analogous to in vivo systems controlled by substrate and product inhibition was demonstrated by immobilizing an enzyme catalyst in a conductive polymer film. A phosphate-requiring enzyme, pyruvate oxidase, was selected to provide the system. Reversible electrochemical oxidation and reduction of the polypyrrole accompanying doping and undoping were performed to induce a change in the phosphate concentration in the polymer membrane. This electrochemical changing of the substrate concentration was exploited as a means of modulating the enzyme in the polypyrrole matrix. Local enrichment of phosphate was favourable for the catalytic function of the enzyme immobilized in the conductive polymer. With this approach the apparent enzyme activity was successfully enhanced by lo%-56% even though the anion concentration in the bulk solution was very low.
INTRODUCTION
A variety of r-conjugated organic polymers can be electroconductive in the doped state because of their high bipolaron content, which, being an index of electroconductivity, is dependent on the amount of doped anion [l]. Typical conductive polymers are those obtained from pyrrole [2], acetylene [3], thiophene [4] and various benzene derivatives [5,6]. Polypyrrole film is easily prepared on an electrode surface by electro-oxidative polymerization in both aqueous and nonaqueous media. The electropolymerized polypyrrole is oxidized or neutralized depending on the applied potential. The oxidation and neutralization processes
l
To whom correspondence
0022-0728/93/$06.00
should be addressed.
0 1993 - Elsevier Sequoia S.A. All rights reserved
294
involve anion doping or undoping at the polymer membrane. We have reported that these processes are accompanied by changes in the anion and counterion concentrations in the vicinity of the conductive polymer [7,8]. From another point of view the electrochemical behaviour can be employed for effective anion trapping and release. It seems probable that anionic substances can be enriched in a localized area of the r-conjugated polymer matrix and consumed by an enzyme immobilized in the polymer. Therefore a novel method of modulating the anion concentration within and in the surroundings of the polymer membrane may be developed. Changing the substrate concentration is one of the fundamental methods of modulating enzyme-catalysed reactions. Needless to say, modulation caused by a variety of metabolites occurs most frequently in the complicated pathways of cell metabolism. We present here an analogous approach to the catalytic control of enzymes by exploiting the electrochemical change of anion concentration provided by the doping and undoping of a r-conjugated polymer. We wish to make full use of the electrochemical properties of polypyrrole by localizing an enzyme in the polypyrrole matrix. A Henri-Michaelis-Menten-type enzyme shows a linear relationship when the substrate concentration is much lower than the K, value. This relationship can be derived from the Henri-Michaelis-Menten equation. In this paper pyruvate oxidase (EC 1.2.3.3) catalysing the following reaction-pyruvate + Pi + 0, + acetyl phosphate + CO, + H,O,, -is taken as a typical enzyme, since the enzyme requires inorganic phosphate Pi as one of the substrates. We will show the capability of polypyrrole as an anion localizer through the electrochemical modulation of the catalytic function of pyruvate oxidase. EXPERIMENTAL
Materials Pyruvate oxidase (pyruvate: oxygen oxidoreductase, phosphorylating; EC 1.2.3.3., 2.9 u (units) mg- ’ solid) from the Pediococcus species was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The pyrrole used was a product of Tokyo Kasei Co. (Tokyo, Japan) and was purified by filtration through an aluminium oxide 90 (Merck Chemical Co.) column and distilled before use. Other chemicals were guaranteed reagent grade. Apparatus Polypyrrole film was prepared over the surface of a disk-type glassy carbon electrode (diameter 3 mm). Electra-oxidative polymerization was carried out using an HA-301 potentiostat and an HF-201 coulomb meter from Hokuto Denko Co. (Tokyo, Japan). Differential pulse voltammograms and cyclic voltammograms were obtained by a P-1100 polarographic analyser from Yanako Co. (Kyoto, Japan). The enzyme activity was measured using a UV-visible spectrophotometer from the Japan Spectroscopic Co. (Tokyo, Japan). Electrochemical operations were carried
295
out with a three-electrode system consisting of a glassy carbon disk (diameter 3 mm) as the working electrode, a platinum plate (10 x 10 mm*) as the counterelectrode and an Ag/AgCl/KCl(sat.) reference electrode. Electrochemical synthesis of enqme-entrapped polypyrrole membrane electrode The reaction mixture for pyrrole polymerization contained 0.1 M pyrrole and 0.3 M sodium ethanesulphonate as the supporting electrolyte in deoxygenated distilled water. The film was grown by electro-oxidation of pyrrole at a constant potential of 0.75 V(Ag/AgCl). The film thickness was controlled by the charge passed for electropolymerization [l]. Here a charge density 1 C/cm-* was passed for the film development. The entrapment of the enzyme in the polypyrrole film over the electrode was performed in a manner similar to the above-described method. The electropolymerization was carried out in the presence of 10 U per 0.5 ml pyruvate oxidase, 1 mg per 0.5 ml nicotinamide adenine dinucleotide (NAD) and 1.5 mg per 0.5 ml thiaminediphosphate (TPP). Determination of catalytic function of pyruvate oxidase with and without potential application The enzyme reaction was initiated by immersing the enzyme electrode in a magnetically stirred solution containing a substrate mixture. After incubation for a given time interval at 30°C the enzyme electrode was removed from the reaction mixture to terminate the enzyme reaction. Each reaction mixture, buffered at pH 7.5, contained phosphate of different concentration. In the case of the activity determination of the potential-applied enzyme electrode a conventional threeelectrode system was employed. An Ag/AgCl reference electrode was connected to the reaction chamber through a salt bridge. One end of the salt bridge was sharpened to make the leakage of the other anion (Cl-1 from the salt bridge as negligible as possible. The applied potential was stepped from -350 mV (Ag/AgCl) to each desired potential. The enzyme activity was determined by measuring the acetylphosphate production [9]. The measurement was based on the method of McComb, although it was modified partially. RESULTS
AND DISCUSSION
Characterization of the enzyme-entrapped electrode The enzyme activity was first determined at zero applied potential. Under this experimental condition the reaction mixture initially contained about 1.1 mM oxygen and 100 mM pyruvate. Since the phosphate concentration was much lower than those of pyruvate and dissolved oxygen, the phosphate concentration hardly seemed to be a rate-determining factor. A linear relationship in the double-reciprocal plot was obtained for both free and immobilized pyruvate oxidase. The
TABLE 1 Enzyme parameters for free and immobilized pyruvate oxidase
V,,= /PM min-’ Lpi /mM
Immobilized pyruvate oxidase
Free pyruvate oxidase
54.5 9.8
63.4 5.6
The reaction mixture contained 100 mM pyruvate. Each reaction mixture was buffered by phosphate at pH 7.5. The enzyme reaction was initiated by the addition of enzyme or by immersing the enzyme electrode in the reaction mixture. After incubation at 30°C the enzyme reaction was terminated by the addition of acid or by removing the electrode.
K, and V,, values are given in Table I. The entrapped enzyme also exhibited a typical Michaelis-Menten-type behaviour. The apparent K, value of the polypyrrole-entrapped enzyme was slightly larger than the K, value of free pyruvate oxidase and the entrapped enzyme retained reasonable activity. The apparent difference in K, seems to have been caused by the considerable decrease in diffusion of substrates within the solid phase matrix. Belanger et al. have reported the detrimental effects of hydrogen peroxide generated by glucose oxidase on the conductivity of polypyrrole caused by the strong oxidizing agent with ring opening and loss of conjugation [lo]. However, the detrimental effect seems to be negligible in our system where the enzyme activity of pyruvate oxidase (2.9 u/mg-‘1 is much smaller than that of glucose oxidase (100 u/mg-‘). In addition, the reaction of pyruvate oxidase was performed in a substrate- (phosphate-) limited condition, i.e. the enzyme underwent acetylphosphate production at low phosphate concentration far from the optimum condition. Relation of applied potential to enzyme activity in poiypyrrole matrix The enzyme requires pyruvate and oxygen as its substrates, besides inorganic phosphate. In each enzyme reaction a substrate mixture containing an excess amount of pyruvate (100 mM) was exploited. Molecular oxygen was continuously supplied from the air, since the reaction chamber was open to atmospheric oxygen. On the other hand, the phosphate concentration was less than 100 PM, which is far less than the K, value. Hence the pyruvate and oxygen concentrations were not rate-determining factors, whereas the concentration in the vicinity of the conductive polymer on potential application seemed to be enriched as described in our previous papers [7,8]. From the cyclic voltammogram of enzyme-incorporated polypyrrole (data not shown) we concluded that phosphate was repeatedly doped and undoped depending on the applied potential. Therefore a decrease and an increase in phosphate anion concentration were caused in the polypyrrole matrix by the doping and undoping processes respectively. The enzyme-entrapped polypyrrole may work as a localizer of the anion when the polymer is repeatedly doped and undoped. Tanguy et al. 1111described the details of the anion-doping
process in a chemically synthesized polypyrrole. There are two trapping states, i.e. “deep trapping” and “shallow trapping”. A shallowly trapped ion is defined as an anion trapped (doped) weakly by the polypyrrole chains. When the potential is increased, these weakly bound anions undergo deep trapping. It is likely that the deeply trapped ions may be released in the polymer, and some portion of them become weakly fixed, depending on the applied potential. These weakly trapped anions may be utilized as a substrate for the enzyme. Effect of potential application on catalytic activity of pyruvate oxidase The enzymatic activity of pyruvate oxidase was assessed by the rate of acetylphosphate production. An increase in catalytic activity was observed on application of a potential higher than the rest potential as shown in Fig. 1. In addition to the enzyme, this polymer electrode contained flavin adenine dinucleotide (FAD) and TPP, and the flavin coenzyme is directly reduced at the electrode when the electrode potential is lower than the redox potential of FAD. In the figure no significant change in catalytic activity was observed at potentials from -0.3 to -0.1 V. The potential of -0.3 V coincided roughly with the redox potential of FAD. Hence it is concluded that the change in catalytic activity was not caused by the formation of the reduced form of FAD. In previous work [12,13] the reversible electrochemical oxidation and reduction of prosthetic groups of some enzymes, such as pyrrolo-quinoline quinone (PQQ) and FAD, were shown to be essential for the electrochemical control of PQQ enzymes and FAD enzymes using a conductive polypyrrole-film-coated electrode. However, the present approach to catalytic activity modulation is clearly different from the acceleration of
&plied potential / mVvs. AgoAgcl Fig. 1. Potential dependence of activity of pyruvate oxidase. Potential stepping was performed from -350 mV (&/AgcI). The enzyme reaction was studied in the same way as described in the footnote to Table 1.
298
the electron transfer processes of these enzymes. In this modulation of the catalytic activity of pyruvate oxidase the enzymatic activity increased in the potential range above - 100 mV (Ag/AgCl), where polypyrrole was oxidized and phosphate was doped into the polypyrrole. From another point of view the change in the oxidation state of polypyrrole is estimated to have been compensated by the doping of the anion. These results suggest that the increase in enzymatic activity is presumably due to the change in phosphate concentration caused by electrochemical doping and undoping. A significant increase in enzymatic activity was observed in the doped state, which may indicate a localized concentration of phosphate. Pyruvate oxidase activity vs. phosphate concentration relationship Since phosphate is an essential substrate of pyruvate oxidase, the enzymatic activity increased significantly depending on the phosphate concentration. At high phosphate concentration an enhancement in catalytic activity on application of a potential was scarcely observed (Fig. 2(A)), because the enzyme was surrounded by an excess amount of phosphate. However, in the case of lower phosphate concentration the application of a potential elicited a lo%-56% enhancement in the rate of acetylphosphate production as shown in Fig. 2(B). A linear relation between the rate of acetylphosphate production and the phosphate concentration was obtained at phosphate concentrations lower than 100 PM (0.01 K,) for both the potentialapplied enzyme-immobilized polypyrrole electrode and the potential-free electrode, although the rate for the former system is greater than for the latter.
Phosphate
concentration
/ nY
Fig. 2. Dependence of production rate of acetylphosphate on phosphate concentration. Each reaction mixture contained 100 mM pyruvate. A potential of 100 mV was applied to the enzyme-immobilized electrode (0 -0) and the production rate was compared with that observed without potential application (0 -----~I. The relation at low phosphate concentration is shown in (B). Conditions were those given in the Experimental Section and Table 1.
299
However, at phosphate concentrations higher than 10 K, the acetylphosphate production rate was almost the same for the two systems (Fig. 2). The marked increase in the catalytic activity of the enzyme of lower phosphate concentration (less than 100 PM) seemed to have been caused by the localized increase in phosphate anion concentration in the vicinity of the enzyme immobilized in the conductive polymer. The enzymatic activity is generally proportional to the concentration of substrate in the case of a Michaelis-Menten-type enzyme as long as the substrate concentration is less than about 0.1 K,. However, at sufficiently high substrate concentration compared with K, the enzyme activity is independent of the substrate concentration. It is in the low substrate concentration region that the catalytic ratio of the enzyme is modulated by potential application. The results described here suggest that the polypyrrole membrane can be exploited as an electrochemical localizer of anions and utilized as a material for modulating the enzyme function. Effect of competing electrolytes on the modulation of catalytic activity
We have studied the effect of coexisting anions on the enzymatic activity, since small molecular anions can be dopants of polypyrrole. Neither chloride nor p-toluenesulphonate shows any inhibitory effect on free pyruvate oxidase; therefore we have taken these two species as competing anions. Figure 3 shows the effect of competing anions on the enzymatic activity of pyruvate oxidase immobi-
2 Concentration
6 of ccmpeting
10 anion
/ PM
Fig. 3. Competing effect of coexisting anions on acetylphosphate production. A potential of 100 mV (Ag/AgCl) was applied to the enzyme-immobilized electrode. p-Toluenesulphonate (01 or chloride (A 1 competed with phosphate. The enzyme reaction and potential application were performed as described in the text.
lized in the conductive polymer, where the coexisting anion was chloride or p-toluenesulphonate. Less bulky anions are more easily doped into polypyrrole than are bulkier ones [14]; therefore the competing effect of chloride should be larger than that of p-toluenesulphonate when these anions compete with phosphate in the enzyme reaction. In other words, chloride is more easily doped into the polypyrrole matrix than is phosphate, while p-toluenesulphonate is less easily doped into the matrix than is phosphate. The results show that the acetylphosphate production decreased more markedly when chloride competed with phosphate than when p-toluenesulphonate did. We have ascertained that p-toluenesulphonate was less easily doped than phosphate anion because of the bulkiness of the dopant anion. These results strongly suggest that the increase in catalytic activity is mainly caused by the increase in concentration of the dopant ion (phosphate). Recently, a variety of approaches for controlling enzyme activity have been presented, some of which are based on controlling the electron transfer between enzymes and solid state materials as described previously [7,12,13,15]. In addition, we have adopted a novel strategy for electronic communication between NAD enzymes and electrode materials by introducing a molecular interface where enzyme, coenzyme and electron mediator are incorporated in a conductive polymer to facilitate electron transfer from enzyme to electrode or vice versa [16,17]. However, the method described here is an analogous approach to in uiuo regulation of enzyme activity, since the enzyme activity is generally controlled by the increase (or decrease) in substrate or product in cellular metabolism. With polypyrrole as a material of phosphate enrichment in a localized area, the polypyrrole electrode can be successfully utilized as a regulator of biomolecules whose functions are dependent on the phosphate concentration. The present approach can be extended to other enzymes which require small anions such as carbonate and sulphate. The modulation system may be applied to the regulation of a bioreactor with a multienzyme system, since the controlling principle is analogous to in uivo regulation. The localized enrichment of phosphate anion in polypyrrole may be applied to the stimulation of the PHO gene of some micro-organisms, since the gene is switched on at the strict threshold phosphate concentration of about 700 PM [18]. The PHO gene expression is responsive to environmental phosphate. Although the enzymatic activity is only slightly increased by the electrochemical enrichment of phosphate, it may be sufficient to stimulate the PHO gene when a PHO-gene-carrying micro-organism is incubated at a phosphate concentration slightly lower than the threshold value. With this in mind we are currently investigating the possibility of stimulating the PHO gene of Succharomyces cereoisiae. REFERENCES 1 A.F. Diaz, J.I. Castillo, J.A. Logan and L.W. Yaung, J. Electroanal. Chem., 129 (1981) 115. 2 K. Kanazawa, A.F. Diaz, R.H. Geiss, W.D. Gill, J.F. Kwak, J.A. Logan, J.F. Robott and G.B. Street, J. Chem. Sot., Chem. Commun., (1974) 854.
301 3 C.K. Chang, M.A. Druy, SC. Gau, A.J. Heeger, A.G.L. MacDiarmid, Y.W. Park and H. Shiraka, Solid State Ionics, 37 (1990) 149. 4 T. Yamamoto, K. Sanechika and A. Yamamoto, J. Polym. Sci., Polym. Lett. Edn., 18 (1980) 9. 5 L.W. Shacklette, R.R. Cance, D.M. Ivory, G.G. Miller and R.H. Baughman, Synth. Met., 1 (1979) 307. 6 G.E. Wnek, J.C.W. Chien, F.E. Karasa and C.P. Lillya, J. Polym. Sci., Polym. Lett. Edn., 20 (1979) 1441. 7 H. Shinohara, G.F. Khan, Y. Ikariyama and M. Aizawa, J. Electroanal. Chem., 304 (1991175. 8 S. Yabuki, H. Shinohara and M. Aizawa, J. Chem. Sot., Chem. Commun., (1989) 945. 9 E.A. McComb, Anal. Chem., 29 (1957) 819. 10 D. Belanger, J. Nadreau and G. Fortier, J. Electroanal. Chem., 274 (19891 143. 11 J. Tanguy, N. Menmilliod and M. Hoclet, Electrochem. Sci. Technol., 134 (4)(1987) 795. 12 H. Shinohara, T. Chiba and M. Aizawa, Sensors and Actuators, 13 (1988) 79. 13 S. Yabuki, H. Shinohara, Y. Ikariyama and M. Aizawa, J. Electroanal. Chem., 277 (1990) 179. 14 H. Shinohara, M. Aizawa and H. Shirakawa, J. Chem. Sot., Chem. Commun., (1986) 87. 15 G.F. Khan, E. Kobatake, H. Shinohara, Y. Ikariyama and M. Aizawa, Anal. Chem., 64 (1992) 1254. 16 T. Ishizuka, E. Kobatake, Y. Ikariyama and M. Aizawa, Tech. Dig. 10th Sensor Symp. Institute of Electrical Engineers of Japan, Tokyo, 1991, p. 73. 17 Y. Ikariyama, T. Ishizuka, H. Shinohara and M. Aizawa, Denkti Kagaku, 58 (12) (1990) 1097. 18 K.A. Bostian, J.M. Lemire and H.O. Halvorson, Mol. Ceil. Biol., 3 (19831 839.
J. Electroad Chem., 347 (1993) 293-301 Elsevier Sequoia S.A., Lausanne
JEC 02474
Modulation of the function of an enzyme immobilized in a conductive polymer by electrochemical changing of the substrate concentration Tetsuya Haruyama, Hiroaki Shinohara, Yoshihito Ikariyama and Masuo Aizawa
l
Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 227 (Japan) (Received 17 July 1992; in revised form 14 September 1992)
A modulation system analogous to in vivo systems controlled by substrate and product inhibition was demonstrated by immobilizing an enzyme catalyst in a conductive polymer film. A phosphate-requiring enzyme, pyruvate oxidase, was selected to provide the system. Reversible electrochemical oxidation and reduction of the polypyrrole accompanying doping and undoping were performed to induce a change in the phosphate concentration in the polymer membrane. This electrochemical changing of the substrate concentration was exploited as a means of modulating the enzyme in the polypyrrole matrix. Local enrichment of phosphate was favourable for the catalytic function of the enzyme immobilized in the conductive polymer. With this approach the apparent enzyme activity was successfully enhanced by lo%-56% even though the anion concentration in the bulk solution was very low.
INTRODUCTION
A variety of r-conjugated organic polymers can be electroconductive in the doped state because of their high bipolaron content, which, being an index of electroconductivity, is dependent on the amount of doped anion [l]. Typical conductive polymers are those obtained from pyrrole [2], acetylene [3], thiophene [4] and various benzene derivatives [5,6]. Polypyrrole film is easily prepared on an electrode surface by electro-oxidative polymerization in both aqueous and nonaqueous media. The electropolymerized polypyrrole is oxidized or neutralized depending on the applied potential. The oxidation and neutralization processes
l
To whom correspondence
0022-0728/93/$06.00
should be addressed.
0 1993 - Elsevier Sequoia S.A. All rights reserved
294
involve anion doping or undoping at the polymer membrane. We have reported that these processes are accompanied by changes in the anion and counterion concentrations in the vicinity of the conductive polymer [7,8]. From another point of view the electrochemical behaviour can be employed for effective anion trapping and release. It seems probable that anionic substances can be enriched in a localized area of the r-conjugated polymer matrix and consumed by an enzyme immobilized in the polymer. Therefore a novel method of modulating the anion concentration within and in the surroundings of the polymer membrane may be developed. Changing the substrate concentration is one of the fundamental methods of modulating enzyme-catalysed reactions. Needless to say, modulation caused by a variety of metabolites occurs most frequently in the complicated pathways of cell metabolism. We present here an analogous approach to the catalytic control of enzymes by exploiting the electrochemical change of anion concentration provided by the doping and undoping of a r-conjugated polymer. We wish to make full use of the electrochemical properties of polypyrrole by localizing an enzyme in the polypyrrole matrix. A Henri-Michaelis-Menten-type enzyme shows a linear relationship when the substrate concentration is much lower than the K, value. This relationship can be derived from the Henri-Michaelis-Menten equation. In this paper pyruvate oxidase (EC 1.2.3.3) catalysing the following reaction-pyruvate + Pi + 0, + acetyl phosphate + CO, + H,O,, -is taken as a typical enzyme, since the enzyme requires inorganic phosphate Pi as one of the substrates. We will show the capability of polypyrrole as an anion localizer through the electrochemical modulation of the catalytic function of pyruvate oxidase. EXPERIMENTAL
Materials Pyruvate oxidase (pyruvate: oxygen oxidoreductase, phosphorylating; EC 1.2.3.3., 2.9 u (units) mg- ’ solid) from the Pediococcus species was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The pyrrole used was a product of Tokyo Kasei Co. (Tokyo, Japan) and was purified by filtration through an aluminium oxide 90 (Merck Chemical Co.) column and distilled before use. Other chemicals were guaranteed reagent grade. Apparatus Polypyrrole film was prepared over the surface of a disk-type glassy carbon electrode (diameter 3 mm). Electra-oxidative polymerization was carried out using an HA-301 potentiostat and an HF-201 coulomb meter from Hokuto Denko Co. (Tokyo, Japan). Differential pulse voltammograms and cyclic voltammograms were obtained by a P-1100 polarographic analyser from Yanako Co. (Kyoto, Japan). The enzyme activity was measured using a UV-visible spectrophotometer from the Japan Spectroscopic Co. (Tokyo, Japan). Electrochemical operations were carried
295
out with a three-electrode system consisting of a glassy carbon disk (diameter 3 mm) as the working electrode, a platinum plate (10 x 10 mm*) as the counterelectrode and an Ag/AgCl/KCl(sat.) reference electrode. Electrochemical synthesis of enqme-entrapped polypyrrole membrane electrode The reaction mixture for pyrrole polymerization contained 0.1 M pyrrole and 0.3 M sodium ethanesulphonate as the supporting electrolyte in deoxygenated distilled water. The film was grown by electro-oxidation of pyrrole at a constant potential of 0.75 V(Ag/AgCl). The film thickness was controlled by the charge passed for electropolymerization [l]. Here a charge density 1 C/cm-* was passed for the film development. The entrapment of the enzyme in the polypyrrole film over the electrode was performed in a manner similar to the above-described method. The electropolymerization was carried out in the presence of 10 U per 0.5 ml pyruvate oxidase, 1 mg per 0.5 ml nicotinamide adenine dinucleotide (NAD) and 1.5 mg per 0.5 ml thiaminediphosphate (TPP). Determination of catalytic function of pyruvate oxidase with and without potential application The enzyme reaction was initiated by immersing the enzyme electrode in a magnetically stirred solution containing a substrate mixture. After incubation for a given time interval at 30°C the enzyme electrode was removed from the reaction mixture to terminate the enzyme reaction. Each reaction mixture, buffered at pH 7.5, contained phosphate of different concentration. In the case of the activity determination of the potential-applied enzyme electrode a conventional threeelectrode system was employed. An Ag/AgCl reference electrode was connected to the reaction chamber through a salt bridge. One end of the salt bridge was sharpened to make the leakage of the other anion (Cl-1 from the salt bridge as negligible as possible. The applied potential was stepped from -350 mV (Ag/AgCl) to each desired potential. The enzyme activity was determined by measuring the acetylphosphate production [9]. The measurement was based on the method of McComb, although it was modified partially. RESULTS
AND DISCUSSION
Characterization of the enzyme-entrapped electrode The enzyme activity was first determined at zero applied potential. Under this experimental condition the reaction mixture initially contained about 1.1 mM oxygen and 100 mM pyruvate. Since the phosphate concentration was much lower than those of pyruvate and dissolved oxygen, the phosphate concentration hardly seemed to be a rate-determining factor. A linear relationship in the double-reciprocal plot was obtained for both free and immobilized pyruvate oxidase. The
TABLE 1 Enzyme parameters for free and immobilized pyruvate oxidase
V,,= /PM min-’ Lpi /mM
Immobilized pyruvate oxidase
Free pyruvate oxidase
54.5 9.8
63.4 5.6
The reaction mixture contained 100 mM pyruvate. Each reaction mixture was buffered by phosphate at pH 7.5. The enzyme reaction was initiated by the addition of enzyme or by immersing the enzyme electrode in the reaction mixture. After incubation at 30°C the enzyme reaction was terminated by the addition of acid or by removing the electrode.
K, and V,, values are given in Table I. The entrapped enzyme also exhibited a typical Michaelis-Menten-type behaviour. The apparent K, value of the polypyrrole-entrapped enzyme was slightly larger than the K, value of free pyruvate oxidase and the entrapped enzyme retained reasonable activity. The apparent difference in K, seems to have been caused by the considerable decrease in diffusion of substrates within the solid phase matrix. Belanger et al. have reported the detrimental effects of hydrogen peroxide generated by glucose oxidase on the conductivity of polypyrrole caused by the strong oxidizing agent with ring opening and loss of conjugation [lo]. However, the detrimental effect seems to be negligible in our system where the enzyme activity of pyruvate oxidase (2.9 u/mg-‘1 is much smaller than that of glucose oxidase (100 u/mg-‘). In addition, the reaction of pyruvate oxidase was performed in a substrate- (phosphate-) limited condition, i.e. the enzyme underwent acetylphosphate production at low phosphate concentration far from the optimum condition. Relation of applied potential to enzyme activity in poiypyrrole matrix The enzyme requires pyruvate and oxygen as its substrates, besides inorganic phosphate. In each enzyme reaction a substrate mixture containing an excess amount of pyruvate (100 mM) was exploited. Molecular oxygen was continuously supplied from the air, since the reaction chamber was open to atmospheric oxygen. On the other hand, the phosphate concentration was less than 100 PM, which is far less than the K, value. Hence the pyruvate and oxygen concentrations were not rate-determining factors, whereas the concentration in the vicinity of the conductive polymer on potential application seemed to be enriched as described in our previous papers [7,8]. From the cyclic voltammogram of enzyme-incorporated polypyrrole (data not shown) we concluded that phosphate was repeatedly doped and undoped depending on the applied potential. Therefore a decrease and an increase in phosphate anion concentration were caused in the polypyrrole matrix by the doping and undoping processes respectively. The enzyme-entrapped polypyrrole may work as a localizer of the anion when the polymer is repeatedly doped and undoped. Tanguy et al. 1111described the details of the anion-doping
process in a chemically synthesized polypyrrole. There are two trapping states, i.e. “deep trapping” and “shallow trapping”. A shallowly trapped ion is defined as an anion trapped (doped) weakly by the polypyrrole chains. When the potential is increased, these weakly bound anions undergo deep trapping. It is likely that the deeply trapped ions may be released in the polymer, and some portion of them become weakly fixed, depending on the applied potential. These weakly trapped anions may be utilized as a substrate for the enzyme. Effect of potential application on catalytic activity of pyruvate oxidase The enzymatic activity of pyruvate oxidase was assessed by the rate of acetylphosphate production. An increase in catalytic activity was observed on application of a potential higher than the rest potential as shown in Fig. 1. In addition to the enzyme, this polymer electrode contained flavin adenine dinucleotide (FAD) and TPP, and the flavin coenzyme is directly reduced at the electrode when the electrode potential is lower than the redox potential of FAD. In the figure no significant change in catalytic activity was observed at potentials from -0.3 to -0.1 V. The potential of -0.3 V coincided roughly with the redox potential of FAD. Hence it is concluded that the change in catalytic activity was not caused by the formation of the reduced form of FAD. In previous work [12,13] the reversible electrochemical oxidation and reduction of prosthetic groups of some enzymes, such as pyrrolo-quinoline quinone (PQQ) and FAD, were shown to be essential for the electrochemical control of PQQ enzymes and FAD enzymes using a conductive polypyrrole-film-coated electrode. However, the present approach to catalytic activity modulation is clearly different from the acceleration of
&plied potential / mVvs. AgoAgcl Fig. 1. Potential dependence of activity of pyruvate oxidase. Potential stepping was performed from -350 mV (&/AgcI). The enzyme reaction was studied in the same way as described in the footnote to Table 1.
298
the electron transfer processes of these enzymes. In this modulation of the catalytic activity of pyruvate oxidase the enzymatic activity increased in the potential range above - 100 mV (Ag/AgCl), where polypyrrole was oxidized and phosphate was doped into the polypyrrole. From another point of view the change in the oxidation state of polypyrrole is estimated to have been compensated by the doping of the anion. These results suggest that the increase in enzymatic activity is presumably due to the change in phosphate concentration caused by electrochemical doping and undoping. A significant increase in enzymatic activity was observed in the doped state, which may indicate a localized concentration of phosphate. Pyruvate oxidase activity vs. phosphate concentration relationship Since phosphate is an essential substrate of pyruvate oxidase, the enzymatic activity increased significantly depending on the phosphate concentration. At high phosphate concentration an enhancement in catalytic activity on application of a potential was scarcely observed (Fig. 2(A)), because the enzyme was surrounded by an excess amount of phosphate. However, in the case of lower phosphate concentration the application of a potential elicited a lo%-56% enhancement in the rate of acetylphosphate production as shown in Fig. 2(B). A linear relation between the rate of acetylphosphate production and the phosphate concentration was obtained at phosphate concentrations lower than 100 PM (0.01 K,) for both the potentialapplied enzyme-immobilized polypyrrole electrode and the potential-free electrode, although the rate for the former system is greater than for the latter.
Phosphate
concentration
/ nY
Fig. 2. Dependence of production rate of acetylphosphate on phosphate concentration. Each reaction mixture contained 100 mM pyruvate. A potential of 100 mV was applied to the enzyme-immobilized electrode (0 -0) and the production rate was compared with that observed without potential application (0 -----~I. The relation at low phosphate concentration is shown in (B). Conditions were those given in the Experimental Section and Table 1.
299
However, at phosphate concentrations higher than 10 K, the acetylphosphate production rate was almost the same for the two systems (Fig. 2). The marked increase in the catalytic activity of the enzyme of lower phosphate concentration (less than 100 PM) seemed to have been caused by the localized increase in phosphate anion concentration in the vicinity of the enzyme immobilized in the conductive polymer. The enzymatic activity is generally proportional to the concentration of substrate in the case of a Michaelis-Menten-type enzyme as long as the substrate concentration is less than about 0.1 K,. However, at sufficiently high substrate concentration compared with K, the enzyme activity is independent of the substrate concentration. It is in the low substrate concentration region that the catalytic ratio of the enzyme is modulated by potential application. The results described here suggest that the polypyrrole membrane can be exploited as an electrochemical localizer of anions and utilized as a material for modulating the enzyme function. Effect of competing electrolytes on the modulation of catalytic activity
We have studied the effect of coexisting anions on the enzymatic activity, since small molecular anions can be dopants of polypyrrole. Neither chloride nor p-toluenesulphonate shows any inhibitory effect on free pyruvate oxidase; therefore we have taken these two species as competing anions. Figure 3 shows the effect of competing anions on the enzymatic activity of pyruvate oxidase immobi-
2 Concentration
6 of ccmpeting
10 anion
/ PM
Fig. 3. Competing effect of coexisting anions on acetylphosphate production. A potential of 100 mV (Ag/AgCl) was applied to the enzyme-immobilized electrode. p-Toluenesulphonate (01 or chloride (A 1 competed with phosphate. The enzyme reaction and potential application were performed as described in the text.
lized in the conductive polymer, where the coexisting anion was chloride or p-toluenesulphonate. Less bulky anions are more easily doped into polypyrrole than are bulkier ones [14]; therefore the competing effect of chloride should be larger than that of p-toluenesulphonate when these anions compete with phosphate in the enzyme reaction. In other words, chloride is more easily doped into the polypyrrole matrix than is phosphate, while p-toluenesulphonate is less easily doped into the matrix than is phosphate. The results show that the acetylphosphate production decreased more markedly when chloride competed with phosphate than when p-toluenesulphonate did. We have ascertained that p-toluenesulphonate was less easily doped than phosphate anion because of the bulkiness of the dopant anion. These results strongly suggest that the increase in catalytic activity is mainly caused by the increase in concentration of the dopant ion (phosphate). Recently, a variety of approaches for controlling enzyme activity have been presented, some of which are based on controlling the electron transfer between enzymes and solid state materials as described previously [7,12,13,15]. In addition, we have adopted a novel strategy for electronic communication between NAD enzymes and electrode materials by introducing a molecular interface where enzyme, coenzyme and electron mediator are incorporated in a conductive polymer to facilitate electron transfer from enzyme to electrode or vice versa [16,17]. However, the method described here is an analogous approach to in uiuo regulation of enzyme activity, since the enzyme activity is generally controlled by the increase (or decrease) in substrate or product in cellular metabolism. With polypyrrole as a material of phosphate enrichment in a localized area, the polypyrrole electrode can be successfully utilized as a regulator of biomolecules whose functions are dependent on the phosphate concentration. The present approach can be extended to other enzymes which require small anions such as carbonate and sulphate. The modulation system may be applied to the regulation of a bioreactor with a multienzyme system, since the controlling principle is analogous to in uivo regulation. The localized enrichment of phosphate anion in polypyrrole may be applied to the stimulation of the PHO gene of some micro-organisms, since the gene is switched on at the strict threshold phosphate concentration of about 700 PM [18]. The PHO gene expression is responsive to environmental phosphate. Although the enzymatic activity is only slightly increased by the electrochemical enrichment of phosphate, it may be sufficient to stimulate the PHO gene when a PHO-gene-carrying micro-organism is incubated at a phosphate concentration slightly lower than the threshold value. With this in mind we are currently investigating the possibility of stimulating the PHO gene of Succharomyces cereoisiae. REFERENCES 1 A.F. Diaz, J.I. Castillo, J.A. Logan and L.W. Yaung, J. Electroanal. Chem., 129 (1981) 115. 2 K. Kanazawa, A.F. Diaz, R.H. Geiss, W.D. Gill, J.F. Kwak, J.A. Logan, J.F. Robott and G.B. Street, J. Chem. Sot., Chem. Commun., (1974) 854.
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