Hysteresis and Positive Cooperativity of Iceberg Lettuce Polyphenol Oxidase

Biochemical and Biophysical Research Communications 289, 769 –775 (2001) doi:10.1006/bbrc.2001.6014, available online at http://www.idealibrary.com on

Hysteresis and Positive Cooperativity of Iceberg Lettuce Polyphenol Oxidase Soledad Chazarra, Francisco Garcı´a-Carmona, and Juana Cabanes 1 Departamento de Bioquı´mica y Biologı´a Molecular A, Universidad de Murcia, 30071 Murcia, Spain

Received October 24, 2001

A kinetic study of the diphenolase activity of latent polyphenol oxidase (PPO), purified from Iceberg lettuce (Lactuca sativa L), revealed a sigmoid relationship between the reaction rate and the substrate concentration with a high Hill coefficient (n H ⴝ 3.8). This positive cooperativity had not been previously described for any PPO. Furthermore, the enzyme showed a lag phase in the expression of this activity, suggesting a hysteretic nature of the enzyme. The kinetic behavior, the latency and the lag phase varied at different steps of the purification process. PPO showed hyperbolic or cooperative kinetics depending on the pH assay and the sodium dodecyl sulfate (SDS) concentration. Substrate-induced slow conformational change of the oligomeric enzyme is suggested. The conformational change would be toward a more active enzyme form with higher affinity for the substrate and favoured by acid pH and SDS. © 2001 Elsevier Science Key Words: polyphenol oxidase; kinetics; positive cooperativity; lettuce; latency; activation.

Polyphenol oxidase (PPO) or tyrosinase (EC 1.14.18.1), is a copper-enzyme widely distributed on the phylogenetic scale that catalyses the o-hydroxylation of monophenols (monophenolase activity) and the oxidation of the o-diphenols to o-quinones (diphenolase activity) using molecular oxygen. The o-quinones resulting from the oxidative activity of polyphenol oxidase undergo a complex series of nonenzymatic chemical changes which ultimately yield melanins. The enzyme is responsible for skin, eye, inner ear, and hair melanization (1, 2). In plant, the function of PPO is not yet Abbreviations used: K H, Hill constant; n H, Hill coefficient; PPO, polyphenol oxidase; 4tBC, 4-tert-butylcatechol; ␶, lag period; SDS, sodium dodecyl sulfate. 1 To whom correspondence should be addressed at Departamento de Bioquı´mica y Biologı´a Molecular A, Edificio de Veterinaria, Unidad docente de Biologı´a, Universidad de Murcia, Campus de Espinardo 30071 Murcia, Spain. Fax: 968-364147. E-mail: jcabanes@ um.es.

understood, but there are strong evidences for a defensive role of PPO against pathogens, herbivores, and wounding (3). In fact PPO activity increases in response to biotic and abiotic injury (4, 5). To fully understand the mechanism which triggers the in vivo activation of PPO upon injury, it is necessary to know which agents regulate the activity of this enzyme, and how they work. Diphenolase activity of PPO has been widely studied (6 – 8) and its kinetic has been largely interpreted (9 – 11). One intriguing characteristic of the enzyme, is its ability to exist in a latent state. PPO has been activated by a variety of treatments including acid and base shock (12) and detergents (13–15). The activation process by SDS detergent, was found that implicated a reorganization of protein tertiary structure (14, 16 – 18). Concerning to what might be the biologically relevant counterpart of the detergents, it has suggested that lipids might fulfill this role (19). The enzyme activation process by pH was accompanied by changes in the electrophoretic mobility of the enzyme (20), as well as changes in the Stokes’ radius of the protein (21), which suggests the involvement of conformational changes in the enzyme during activation. Some authors have described that the degree of latency is maintained throughout the purification procedure (22), whereas in other cases it has been found that the enzyme is spontaneously activated as the purification increases (23). However, in this study we have observed, for the first time, that enzyme latency increased as the purity increased, and that changes in the kinetic behavior occurred. Thus, in this paper we have studied the variations in the kinetic behavior of lettuce PPO at different degrees of purity. For the first time, we report positive cooperativity in the expression of its diphenolase activity, which is associated with the appearance of a lag phase, depends on the pH and the SDS detergent concentration. Several explanations for sigmoid kinetics in oligomeric enzymes are based on the idea that the subunits interact with one another. Taking into account the

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tetrameric form of iceberg lettuce PPO (24), we interpret its kinetic behavior in terms of the slow conformational change of an oligomeric enzyme in the Monod et al. model (25) according to the mechanism reported by Kurganov et al. (26). MATERIALS AND METHODS Materials Fresh iceberg lettuce (Lactuca sativa L) was purchased from a local market in Murcia (Spain). 4-tert-butylcatechol (4tBC) was supplied by Fluka (Madrid, Spain). SDS and chlorogenic acid were obtained from Sigma Chemical Co. (Madrid, Spain). Other chemical reagents were of analytical grade.

Methods Enzyme extraction. The purification of iceberg lettuce PPO from the chloroplast membranes was performed as previously described (24) following these steps: Step 1: Chloroplast sonication. Step 2: Ammonium sulfate fractionation (purification factor 1.2). Step 3: Chromatography on Sephacryl HR S-200 (purification factor 5.4); most of the PPO activity appeared in a high molecular weight fraction, which was used for following purification step. Step 4: Ion exchange chromatography on DEAE (purification factor 44.8); PPO activity appeared in two peaks; the major one eluted near 150 mM KCL was used as highly purified enzyme. Enzyme assay. The diphenolase activity of the enzyme was determined using two different substrates, 4tBC and chlorogenic acid. In both cases activity was determined at 25°C by spectrophotometrically monitoring, at 400 nm, the appearance of the o-benzoquinone product of the reaction (␧ ⫽ 1150 and 1018 M ⫺1cm ⫺1, respectively). Enzyme assays were performed in 50 mM sodium phosphate buffer, pH 6.5, varying the substrate concentration in the reaction medium. The steady-state rate was defined as the slope of the linear zone of the product accumulation curve. The lag period (␶) was estimated as the intercept on the abscissa axis obtained by extrapolation of the linear part of the product accumulation curve (9). pH studies were carried out using 50 mM sodium acetate and sodium phosphate buffers and SDS studies were performed in 50 mM sodium phosphate buffer pH 6.5. Experiments were performed in triplicate. Protein determination. Protein concentration was determined according to the Bradford Bio-Rad protein assay using bovine serum albumin as standard (27).

RESULTS AND DISCUSSION Hysteresis and Cooperativity During the purification process, the diphenolase activity was assayed in each step. The experimental records are shown in Fig. 1. With the exception of a curve obtained by using the initial extract (step 1), it can be observed that the enzyme activity increased with time reaching a steady-state after a discernable lag phase. This lag phase varied depending on the enzyme purity (curves b– d) and the substrate concentration (curves d– e). The existence of a lag phase is a well-known phenomenon in the expression of the monophenolase activity of PPO (9, 28 –30), but in the diphenolase activity of PPO has only been previ-

FIG. 1. Product accumulation curves for diphenolase activity at different purification steps. The reaction medium contained 6 mM 4-tBC in 50 mM sodium phosphate buffer, pH 6.5. (a) Step 1: initial extract [13 ␮g/ml]. (b) Step 2: extract concentrated with ammonium sulfate [11 ␮g/ml]. (c) Step 3: high molecular weigh fraction obtained by chromatography on Sephacryl HR S-200 [2.5 ␮g/ml]. (d) Step 4: fraction eluted at 150 mM ClK by chromatography on DEAE [0.3 ␮g/ml]; (e) same as (d) except that in this case the 4tBC concentration was 4 mM. The broken lines represent the extrapolation of the linear part of the product accumulation curve used for calculating the lag period by the intercept on the abscissa.

ously described for grape (31, 32) and broad bean (33) enzymes. Furthermore, the kinetic plot of v against [S] 0 turned out to be not hyperbolic, showing an intermediate plateau (Fig. 2) when a relatively high range of substrate concentration was used. An explanation for the existence of an intermediate plateau on the v-[S] 0 curve is the superimposition of hyperbolic and sigmoidal curves. These results were obtained at pH 6.5 for the latent enzyme. The enzyme activated by detergent (0.7 mM SDS) at pH 6.5 or by acid pH (pH 4.5) displays the usual hyperbolic dependence of v on [S] 0, as we have described previously (8). The results obtained using chlorogenic acid, the natural substrate of PPO in lettuce (34, 35), were the same as those obtained with 4tBC (results not shown). However, 4tBC was used in all the experiments since the quinone thus obtained is much more stable (36), which enables us to affirm that the results are not an artifact of the enzyme assay. Figure 2 shows the results obtained for the extract concentrated with ammonium sulfate (step 2, Fig. 2A), for the high molecular weight fraction obtained on Sephacryl HR S-200 (step 3, Fig. 2B) and for the fraction eluted at 150 mM KCl on DEAE-Mensep (step 4, Fig. 2C). As the purification increased, the initial hyperbola decreased until it disappeared and the plot of v against [S] 0 became sigmoidal. These results, which have not been previously reported, suggested that the enzyme could occur in two forms, one of which displays the usual hyperbolic dependence of v on [S] 0, and the other shows a sigmoidal dependence. So it appears that the purification process eliminated an isoform or a small percentage of the same enzyme that could be undergoing modification during the extraction process. The isoform or modified form would display a hyper-

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bolic dependence of v on [S] 0, that superimposes to sigmoidal dependence observed for the highly purified enzyme, would explain the kinetic results obtained and why the degree of latency increased during the purification process, an increase which has not been described in the bibliography. An intermediate plateau may appear on the v-[S] 0 plot if an allosteric enzyme which exhibits kinetic substrate cooperativity contains, as impurities, forms which have lost their cooperative properties, but which remain enzymatically active (37). On the other hand, for each purification step, the duration of the lag period showed a bell-shaped curve as a function of substrate concentration (Fig. 2). The existence of a lag phase in the expression of activity is a characteristic property of a hysteretic enzyme undergoing slow transition to another kinetically different form during catalysis (38). Thus it appears that the highly purified enzyme in the presence of substrate undergoes slow transition to a catalytically more active form, whereas the form eliminated during the purification process has lost the ability to suffer interconversion. The appearance of a lag-period in the kinetic curves of product accumulation, the tetrameric nature established for the enzyme (24) and the fact that lettuce

PPO does not undergo monomer-multimer interconversion (24) suggest that the kinetic behavior of PPO fulfills the theoretical analysis developed by Kurganov et al. (26) for the Monod et al. (25) model with slow transitions between the conformational states of the oligomeric enzyme. To develop this analysis, Kurganov et al. (26) assumed that the rate of achievement of equilibrium between the two conformational states of the oligomeric enzyme, called R and T, is slower than that of catalytic conversion of the substrate and the Frieden’s assumptions (38) used to account the phenomenon of hysteresis in a monomeric enzyme: (i) the complexes of R forms with the substrate (RS i) are at an equilibrium with free components (R 0) and the complexes of T forms with the substrate (TS i) are at an equilibrium with free components (T 0), (ii) the enzymatic transformation of the substrate to product is irreversible, (iii) the substrate concentration, [S], is maintained at a constant level, and (iv) there is no product inhibition. Thus in the absence of substrate there is a equilibrium between the free enzymatic forms, R 0 and T 0. In the presence of substrate and for a tetrameric protein, the mechanism would be described by the scheme:

A remarkable feature of kinetic cooperativity is that “slow” transition between enzyme forms may result in “slow” transients at the beginning of the reaction, before the steady state is reached. If the equilibrium between the two free enzyme forms is shifted toward the one that reacts more rapidly with the substrate, one will observe a lag phase before the steady state is reached. If alternatively this equilibrium is shifted toward the less reactive form, the enzymatic reaction will exhibit a burst. Furthermore, the Kurganov’s et al. (26) theoretical study predicts dependencies of ␶ on [S] 0 similar to those ones obtained experimentally for PPO (Fig. 2C) along with curves of product accumulation similar to those ones observed experimentally (Fig. 1) confirming the ability of this model to account the kinetic behavior of lettuce PPO. According to this mechanism, the positive

kinetic cooperativity observed in the steady state rate is due to the fact that the enzyme undergoes slow conformational changes toward a more active form induced by the binding of the substrate. In addition, the fitting by nonlinear regression the experimental data of v against [S] 0 (Fig. 2C) to Hill equation,

␯⫽

V max关S兴 0n H nH 共K H ⫹ 关S兴 0n H兲

a Hill coefficient (n H) of 3.88 was obtained. This value of the n H along with the tetrameric nature of the enzyme (24) support the validity of the mechanism proposed by Kurganov et al. (26) to account the positive cooperativity observed for highly purified PPO from lettuce.

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of allosteric enzymes, especially in their sensitivity to allosteric effectors and in the positive kinetic cooperativity toward allosteric ligands, so that hydrogen ions may be regarded as a particular type of allosteric effector. When we tested the diphenolase activity of the initial extract (step 1) at pH 4.5, a hyperbolic dependence of v on [S] 0 was obtained in contrast with the complex kinetic plot of v against [S] 0 observed for this activity at pH 6.5. This suggests that the activation by acid pH produced the immediate transition of the enzyme. This suggestion is in agreement with the above-mentioned involvement of conformational changes in the enzyme during activation (20, 21). Taking into account these data, we studied the effect of the pH on the cooperativity observed for the highly purified enzyme (step 4). To do so, the diphenolase activity was assayed at different pH values varying the substrate concentration. The steady-state rates obtained at the different pH values are shown in Fig. 3A. The experimental data of v against [S] 0 at each pH were fitted by nonlinear regression to Hill equation. The values of the n H are shown in Table 1. When the diphenolase activity was assayed at pH 4.5, the enzyme followed a hyperbolic dependence of v on [S] 0 (Fig. 3A) according to the Michaelis–Menten

FIG. 2. Effect of substrate concentration on the diphenolase activity at different purification steps: (A) Step 2. Extract concentrated with ammonium sulfate [11 ␮g/ml] (B) Step 3. High molecular weigh fraction obtained by chromatography on Sephacryl HR S-200 [2.5 ␮g/ml] (C) Step 4. Fraction eluted at 150 mM ClK by chromatography on DEAE [0.3 ␮g/ml]. The closed circles represent the steady-state rates and the open ones represent the lag period duration. Enzyme activity was measured in 50 mM sodium phosphate buffer, pH 6.5. In (C) the experimental data of v against [S]0 are fitted to the Hill equation by nonlinear regression.

It is important to point at that this positive cooperativity induced by the substrate has never been described for any PPO or tyrosinase. A cooperative behavior in the steady-state kinetic of diphenolase activity of PPO has previously been described for grape (31, 32) and broad bean (33) enzymes. In both cases the cooperativity was pH-induced, and the enzyme displayed negative cooperativity. Effect of pH Changes in the concentration of hydrogen ions usually result in modifications in the regulatory properties

FIG. 3. Effect of substrate concentration on the diphenolase activity at different pH values. (A) Effect on the steady-state rate. Experimental data were fitted to the Hill equation. (B) Effect on the duration of the lag period. The reaction medium contained 0.15 ␮g/ml of the enzyme eluted at 150 mM ClK by chromatography on DEAE (step 4) in 50 mM sodium acetate [(F) pH 4.5, (‚) pH 5, (␶) pH 5.5] or sodium phosphate buffer [(䊐) pH 5.5, (E) pH 6.5 and (}) pH 7.3].

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Kinetics Parameters Which Characterize the 4tBC Oxidation by PPO at Different pH Values pH

nH

K H (mM)

V max (␮M/min)

4.5 a 5.0 a 5.5 a 5.5 b 6.5 b 7.3 b

1 1.80 1.98 2.83 3.88 3.5

1.19 1.23 3.84 5.06 5.49 3.90

147.50 165.12 145.65 154.92 110.79 110.59

a b

hyperbolic dependence of v versus [S] 0 and there is no lag over the whole substrate concentration range. The progressive decrement of K H with increasing [H ⫹] suggests that the conformational transition, induced by the substrate and favored by the [H ⫹] would be toward an enzyme form with higher affinity for the substrate. Effect of SDS

Assayed in 50 mM sodium acetate buffer. Assayed in 50 mM sodium phosphate buffer.

equation, i.e., the value of the n H was 1 (Table 1). However, when the activity was measured at higher pH values, the enzyme displayed positive kinetic substrate cooperativity. These results demonstrate that positive cooperativity, induced by substrate, was dependent on pH of the assay, reaching its maximal degree at pH 6.5. As can be seen in Table 1, the degree of positive cooperativity decreased as [H ⫹] increased until it disappeared at pH 4.5. Table 1 also shows that when the n H value decreased, the enzyme affinity for the substrate increased, i.e., the Hill constant (K H) value increased. Regarding the effect of pH on the V max, it appears that this latter had a tendency to augment as [H ⫹] increased. Figure 3B shows the variation of lag period with the substrate concentration at different pH values. As can be seen, the widest ␶ v [S] 0 profile together with the maximum ␶ value was obtained at pH 6.5. At this pH value the cooperativity is the highest. When the cooperativity decreased, there was a displacement in the ␶ v [S] 0 profile toward lower substrate concentrations. At the same time, there was a drop of the ␶ maximum values and a reduction in the substrate concentration range where the lag period appear. So, at pH 4.5, where there was no cooperativity, the reaction rate immediately reached its steady-state value and there was no lag over the whole substrate concentration range. These results confirm that the appearance and disappearance of the lag period are concomitant with the appearance and disappearance respectively of cooperativity. Therefore, these data are consistent with the model developed by Kurganov et al. (26). These results suggest that the slow isomerization, induced by the substrate, could be modulated by a sensitive pK, so upon protonation of a strategic ionizable group the protein undergoes the conformational transition. Thus, the substrate would induce the enzyme transition to a catalytically more active form and the [H ⫹] increment would favor this isomerization, so at pH 4.5 practically all the enzyme would be in the more active form and as a result the enzyme displays a

PPO activation by SDS is a well-known phenomenon, it has been suggested that it is due to a limited conformational change (14) as well as it could be equivalent to the physiological role fulfilled by lipids in vivo (19). Besides, the complex kinetic plot of v against [S] 0 observed for the latent enzyme didn’t appear when the initial extract (step 1) activity was assayed in presence of 0.7 mM SDS. Taking the above considerations into account, we decided to study the effect of SDS on the observed cooperativity for the highly purified enzyme (step 4). To investigate this effect, the enzyme activity was assayed varying the substrate concentration in presence of different SDS concentrations. Figure 4A shows the plot of v against [S] 0 obtained for each SDS concentration and Table 2 shows the corresponding kinetic parameters.

FIG. 4. Effect of substrate concentration on the diphenolase activity in presence of different SDS concentrations. (A) Effect on the steady-state rate. Experimental data were fitted to the Hill equation. (B) Effect on the duration of the lag period. The reaction medium contained 0.15 ␮g/ml of the enzyme eluted at 150 mM ClK by chromatography on DEAE (step 4) in 50 mM sodium phosphate buffer, pH 6.5, and different SDS concentrations: (F) 0.7 mM, (■) 0.35 mM, (Œ) 0.17 mM and (}) without SDS.

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Kinetics Parameters Which Characterize the 4tBC Oxidation in the Presence of Different SDS Concentrations a [SDS] (mM)

nH

K H (mM)

V max (␮M/min)

0 0.17 0.34 0.70

3.88 1.97 1.62 1

5.49 2.72 1.55 1.13

110.79 179.32 195.05 244.18

a

Assayed in 50 mM sodium phosphate buffer (pH 6.5).

When the diphenolase activity was assayed in presence of 0.17 mM SDS, a sigmoidal dependence of v on [S] 0 was obtained, although the n H value was half-one obtained in absence of detergent. In presence of 0.35 mM SDS, the n H value was even lower and the addition of 0.7 mM SDS results in the disappearance of sigmoidicity of the plot of v against [S] 0. So the degree of positive cooperativity decreased as SDS concentration increased. This disappearance of the cooperativity resulting from the presence of SDS suggests that SDS also favors the enzyme transition, which is in accordance with the above-mentioned idea that the presence of SDS provokes a conformational change in the enzyme (14). In this way, in presence of 0.7 mM SDS, practically all the enzyme would be in the more active form and for this reason the enzyme displays a hyperbolic dependence of v versus [S] 0 and there is no lag over the whole substrate concentration range. In addition, the enzyme affinity for the substrate increased with increasing concentration of SDS. The same happened with variations of V max with SDS concentration. It is therefore acceptable to consider that the conformational transition, favored by SDS, would be toward an enzyme form more active and with higher affinity for the substrate. As shown in Fig. 4B, in which the variation of lag period with the substrate concentration at different SDS concentrations are presented, there was a displacement in the ␶ v [S] 0 profile toward lower substrate concentrations as the cooperativity decreased, a drop of the ␶ maximum values and a reduction in the substrate concentration range where the lag period appear. So in presence of 0.7 mM SDS, where there was no cooperativity, the reaction rate immediately reached its steady-state value over the whole substrate concentration range. This again confirms that the appearance and disappearance of the lag period are concomitant with the appearance and disappearance of cooperativity, respectively. In summary, in the present study we describe the existence of two PPO forms, which present different kinetic characteristics. One of them, in an activated state, displays the usual hyperbolic dependence of v on [S] 0. The other one, in latent state, undergoes slow transition to a catalytically more active form in the

presence of substrate and so displays sigmoidal kinetics. This form was purified and its kinetic characterized. The presence of the activated form masks the cooperative behavior of the latent form and this could perhaps explain why this cooperativity has not been previously described in the literature, whereas the latency and subsequent activation (by low pH or SDS) of many plant PPOs is well-known. The elucidation of this cooperative kinetic behavior may help to clarify the activation mechanism of the enzyme in vivo, since, as we have also observed, this cooperativity is dependent on physiological modulators (such as the pH) and SDS detergent, which would play an equivalent role to the physiological one fulfilled by lipids in vivo (19). These results might throw some light on the mechanisms which regulate the enzyme activity in vivo and support the assigned role of PPO in plant defense since the positive cooperativity observed would enable the plant to trigger an immediate defense response in case of injury. ACKNOWLEDGMENTS This work was partially supported by a grants from the DGICYT (Spain), Project PB 98-0385. Soledad Chazarra has a fellowship from the Asociacio´n Cultural Caja Murcia.

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