Poly(2‐oxazoline)s with a 2,2′‐Iminodiacetate End Group Inhibit and Stabilize Laccase

Abstract Poly(2‐oxazoline)s (POxs) with 2,2′‐iminodiacetate (IDA) end groups were investigated as inhibitors for laccase. The polymers with the IDA end groups are reversible, competitive inhibitors for this enzyme. The IC50 values were found to be in a range of 1–3 mm. Compared with IDA alone, the activity was increased by a factor of more than 30; thus indicating that attaching a polymer chain to an inhibitor can already improve the activity of the former. The enzyme activity drops to practically zero upon increasing the concentration of the most active telechelic inhibitor, IDA‐PEtOx30‐IDA (PEtOx: poly(2‐ethyl‐2‐oxazoline)), from 5 to 8 mm. This unusual behavior was investigated by means of dynamic light scattering, which showed specific aggregation above 5 mm. Furthermore, the laccase could be stabilized in the presence of POx‐IDA, upon addition at a concentration of 20 mm and higher. Whereas laccase becomes completely inactive at room temperature after one week, the stabilized laccase is fully active for at least a month in aqueous solution.


Introduction
The inhibition of enzymes is an important topic for controlling biocatalytic processes relevant in medicine, bioanalytics, and agriculture. Most enzyme inhibitors are small molecules that interactw ith enzymes in several ways. Improvingt he activity of such molecules is ag reat benefit because lower inhibitor concentrations will minimize side effects ande nvironmental pollution. Typically,s uch inhibitors are improved by chemical modification to increaseb inding to the active site of the respectivee nzyme and to increase specificity. [1,2] Another way to activate such inhibitors is to bind them to polymers or nanostructures to create multiple binding sites. [3,4] The modification of fullerene with an iminosugar,w hich is an inhibitor for Jack bean a-mannosidase, leads to 179 times higher activity of this inhibitor. [5] Bonduelle et al. have shown that aggregates of iminosugar-based glycopolypeptides form aggregates that increase the activity of the iminosugars as inhibitors for a-mannosidase by af actor of 30. [6] In both cases, the authors explained this improvement in activity by the multivalentb inding character.B inding inhibitors to the backbone of polymers can also create such as cenario. Such polymerbound inhibitors are often used to protect drugsf rom degradation in the body.F or example, serine protease inhibitors have been attached to poly(acrylic acid) andp olysaccharides to protect drugs such as insulin from proteolysis. [2,7,8] In addition to multivalent binding, the inhibitor can also be attached to the end group of an onaggregating hydrophilic polymer.I nt he case of ac ompetitive inhibitor,t his would lead to the situation depicted in Figure 1. According to this concept, the inhibitor could be activated by the factt hat the polymeric tail additionally blocks the active site of the enzyme. Also, the inhibitor can bind near the active site and would still be active due to its bulky tail. This might increaset he variability of ap otential enzyme inhibitor.O nt he contrary,t he polymer tail might hinder binding to the active site due to steric hindrance and it will also inducediffusion limitations.
In contrast to poly(ethylene glycol) (PEG), poly(2-oxazoline)s (POxs)i nteractw ith certaine nzymes,t os ome extent. For example,P Ox-enzyme conjugates based on horseradish peroxidase (HRP) and laccase are practically inactivei nw ater, [9] but are highly activated in organic solvents, similart ot he respective artificial enzymes. [10] Other enzymes are less affected by POx upon conjugation. [11] As shown by Saegusa et al.,c atalase Poly(2-oxazoline)s (POxs) with 2,2'-iminodiacetate (IDA) end groups were investigated as inhibitors forl accase. The polymers with the IDA end groups are reversible, competitive inhibitors for this enzyme. The IC 50 values were found to be in a range of 1-3 mm.C ompared with IDA alone, the activity was increased by af actor of more than 30;t hus indicating that attaching ap olymer chain to an inhibitorc an already improve the activity of the former.T he enzyme activity drops to practically zero upon increasingt he concentration of the most active telechelic inhibitor,I DA-PEtOx 30 -IDA (PEtOx:p oly(2-ethyl-2-oxazoline)), from 5t o8m m.T his unusual behaviorw as investigated by means of dynamic light scattering, which showed specific aggregation above 5mm.F urthermore, the laccase could be stabilized in the presenceo fP Ox-IDA, upon addition at ac oncentration of 20 mm and higher.W hereasl accase becomes completely inactive at room temperature after one week, the stabilizedl accasei sf ully active for at least a monthi na queous solution. can be conjugated with poly(2-methyloxazoline) (PMOx) and poly(2-ethyl-2-oxazoline) (PEtOx) nearly completely retains its activity in water. [12] Another study reports on the conjugation of superoxide dismutase with amphiphilic POx-based block copolymers. [13] Here, the enzyme retained only 30 to 50 %o fi ts originala ctivity.M ero et al. showed that the conjugation of trypsin with PEtOx led to enzymes that showedh igh activity for small substrates, but ar educed activity for larger substrates. [14] Interestingly,e nzymes are fully active in POx-based networks. [15] There are studies that show the potential of POx derivatives as inhibitors. For example, human matrix metalloproteinases (MMPs), such as collagenase, are inhibited by telechelicP Ox terminated with N,N-dimethyldodecylammonium (DDA) as end groups for use in dental adhesives. [16] The two antibiotics ciprofloxacin and penicillin, whicha re both enzyme inhibitors, were shown to be active as end groups of POx. [17] POxw ith a2 ,2'iminodiacetate (IDA) end group was previously reported to diminish the activity of HRP as an entropically driven noncompetitive inhibitor. [18] Thisi sr emarkable because IDA is not an inhibitor of HRP. The interaction of these POx-IDA species with proteins is so strong that they form noncovalent, organosoluble conjugates with the latter. [19] Herein, we show how POx-IDAs inhibitt he enzyme laccase and even stabilizethis relatively fragile enzyme.

Results and Discussion
Laccase is an important, copper-based enzyme that is widely used in environmental bioremediation, [20] chemical synthesis, [21] biological bleaching, [22] and in biosensors for the detection of oxidizinga gents. [23,24] Typical inhibitors for this enzyme are several metal chelating ligands, such as diethylenetriaminepentaacetic acid, [25] dithiothreitol (DTT), [26] thioglycolic acid (TGA), [27] oxalic acid, [28] and citric acid. [28] These inhibitors diminish the activity of laccase in ac oncentration range of 5t o2 0mm.I DA barely inhibits laccase and shows 20 %i nhibition at 40 mm IDA. This weak inhibitor was attached to different POxs either at one end or at the two terminals. Different molecular weights and polymers (PMOxand PEtOx) were applied.
Initially,the binding reactionbetween laccase and PMOx-IDA was investigated by meanso fi sothermal titration calorimetry (ITC;F igure 2). In contrast to the interaction with HRP shown in ap reviouss tudy, [18] the reaction between laccase and PMOx 30 -IDA is an exothermic process. This indicates as trong binding affinity of the polymer to the enzyme. Only aw eak signal, andt hus, no bindingc ould be observed upon adding PMOx without the IDA end group to the enzyme;this indicates that the binding between laccase andP MOx 30 -IDA is driven by the IDA end group.A dditionally,t he titration curve allows the calculation of the binding constant (0.12 mm), presuming that PMOx-IDAa nd the enzymef orm a1:1 complex.
The inhibitory effect of POx-IDA was explored on the oxidation of [2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] diammonium salt (ABTS) by oxygen catalyzedb yl accase in the presence of various concentrations of the polymer.I tw as found that PMOx 30 -IDA inhibited more than 20 %o fthe laccase activity at ac oncentration of 1.25 mm.T his is a3 0times lower concentrationt han that of free IDA to achieve the same effect. Thus, conjugation of the polymerP MOx and IDA leads to a great activation of the latter;t his indicates that the general concept for polymerici nhibition, as proposed in Figure 1, seems to be valid for this system.
To study the type of inhibition caused by PMOx-IDA, the Michaelis-Menten parameters for the enzymer eactioni nt he presence and absence of CH 3 -PMOx 30 -IDA and IDA-PMOx 35 -IDA were determined. The Michaelis-Menten model was successfully used in ap reviouss tudy of thesep olymers as inhibitors for HRP,s howing noncompetitive inhibition. The kinetic experiments forl accase in this work were performed by varying the concentrations of both PMOx and ABTS. Calculation of the apparent parameters (V app max and K app m )w as realizedb yf itting of Michaelis-Mentenp lots( Figure 3). The concentration of PMOx-IDA was varied from 0t o5mm.
The Michaelis-Menten plots reveal that the increase in PMOx 30 -IDA concentrationi ncreases the Michaelis constant, K m , from 0.026 mm of the native enzyme to 0.5 mm at 5mm of PMOx 30 -IDA, whereas no significant change in the maximum oxidation rate, V max ,o ccurs. This is typical for ac ompetitive inhibition mechanism as am ajor mechanism for the singly functionalized PMOx. The Lineweaver-Burk plots (Figure3B) clearly confirmed that the competitive inhibition mechanism given in Figures 1a nd 3c an describe the inhibition of the laccase by As observed in Figure 4, IDA-PMOx-IDA concentrations of 1.25 and2 .5 mm afford competitive inhibition that leads to increaseda pparent K m values, whereas V max is not affected. Fur-ther increasing the polymer concentration to 5mm affords a lower apparent V max value and ah igher apparent K m value. This could indicate ad ifferent inhibitionm echanism.R osenfeld and Sultatos reported that the kinetics of the inhibition, in some cases, changed with concentration, which suggested that the  inhibitorc ould be binding to as econdary binding site outside the active side of the enzyme. This can lead to apparent activation of the enzyme and in other cases to further inhibition. [29] The inhibition of laccase in the presence of one-sided and telechelic PMOx and PEtOx was taken furtheru pt o8m m POx to determine IC 50 values and the inhibition constant.The inhibition curves were fitted with Equation (1 < ), which describes a competitive enzymei nhibition: in which V max and K m are the kinetic parameters of the free enzyme,a nd K i is the competitive inhibition constant. The inhibition constants K i and the IC 50 values were determined after fitting the inhibition values to the competitive mechanism. Figure 5s hows ag raphic representation of four examples for PMOx and PEtOx with one-side-andt elechelic-terminated IDA.
In contrast to typical inhibition curves, the inhibitor does not work up to ac oncentration of > 0.5 mm in all cases. This can be explained by the fact that POx withouta ne nd group can activate laccase at low concentrations ( Figure 6). The activation effect is more pronouncedf or PMOx than that for PEtOx.
To eliminate this effect, the inhibition curvesw ere fitted with ac oncentration of 0.5 mm as as tartingv alue. As observed in Figure 5, the curvesa re well fitted to the competitive mechanism. The inhibition constants K i are in the range of 0.04 to 0.15 mm.C loser inspection of the inhibitionc urvesr eveals that, in most cases, the curve does not fit the inhibition rates at inhibitor concentrations of 7mm and higher. This is probably due to ad ifferenti nhibition mechanism at higher concentrations, whichh as been reported for low-molecular-weight, competitive inhibitors. [30] In the case of IDA-PEtOx 30 -IDA, the activity can be inhibited by more than 99 %, which makes this polymer ad ead-end inhibitor.T he results of K i and IC 50 values  for all polymers, calculated from the respective curves, are presented in Table 1.
As observed from the IC 50 values in Ta ble 1, all polymers are inhibitors for laccase and are generally more active than that of IDA alone. The IC 50 values are in ar ange of 1.3 to 3.9 mm. Thus, the activation factor for IDA attached to POx is between 30 and 60. Furthermore, it can be seen that the telechelic polymers are up to two times more active than the one-side-terminated analogues.T his could be interpreted as an effect caused by multiple binding at the protein. This is in contrast to the results found for HRP,f or which telechelic termination had no furthereffect on the inhibition potency. [18] Moreover,t he dependence of activity on the molecular weighto ft he polymers wasi nvestigated with one-and twoside-terminated IDA polymers with two different molecular weights. The telechelic macromolecules with high molecular weighta re also up to two times more active than that of the smaller ones because the larger polymer tail would result in a strongerb locking of the actives ite. Additionally,u pon comparing the inhibition of PMOx and PEtOx derivatives with similar lengths, the IC 50 and K i valuesa re almost identical,t hat is, the hydrophilicity of the polymers is not the major driving force, which is most likely to be the end group in combination with the bulky tail. The respective polymers without specific end groups show aw eak inhibition of the enzymea th igher concentrations. In contrast, one-sided POx-IDA with lower molecular weights are more than two times more active inhibitors than that of the respective higherm olecular weight polymers. This is possibly because the affinity to the active side of the enzymei sh igher for the low-molecular-weightp olymers due to lower steric hindrance.
As observed in Figure 7, the IC 50 curves observed with DMP as substrate look similar to those found with ABTSa ss ubstrate. K i and IC 50 values for the two polymers were calculated from the respective curvesa nd are listed in Ta ble 2. These data show that the inhibition of the polymers for laccase with DMP as as ubstrate is stronger,a ss uggested by the tenfold lower K i values. This is expected for competitive inhibition because DMP has al ower affinity to the actives ite of the enzymet han that of ABTS (K mABTS = 0.026, K mDMP = 0.037).
Interestingly,the inhibitor IDA-EtOx 30 -IDA can practically fully inhibit laccase at ac oncentration of 7-8 mm.T his is unusual, although not unique, for competitive inhibitors. To explore if this effect might derive from certain superstructures formed at higher concentrations, dynamic light scattering (DLS) experiments werep erformed in the respective assay buffer solution at concentrations of 3a nd 8mm,r espectively.O ur hypothesis was that the POx-IDA might form aggregates at higher concentrations, which would then act as multivalent inhibitors. The latterare known sometimes to activate the attached inhibitor structures by up to 179 times. [5] Such an effect might explain as eemingly increasing activity of POx-IDA at higher concentrations. As observed in the intensity plots shown in Figure 8, solutions at both concentrationss how ap eak at 2.5 nm, which can be attributed to the single polymer chains, and ap eak at 250 nm, which mostl ikely originates from unspecific aggregates.T he only difference is ap eak at 28 nm,   which only occurs at higher concentration. This peak could be an aggregate, which might indeed be responsible for the full inhibition of laccase. The intensity of this peak is very low,r esulting in less than 0.01 %o fa ll molecules in the number plot of the DLS curve (Figure 8, right). Thus, it seems unlikely that this aggregate is responsible for the highera ctivity.
To investigate if the POx-IDA inhibitors for laccase are reversible, laccase solutions that contained 20 mm of IDA-PMOX 35 -IDA and IDA-PEtOx 30 -IDA were prepared. Then, different volumes of these solutionsw ere added to the ABTS assay and the resultinga ctivity was compared with that found for al accase solution without polymer.
As observed in Figure 9, the relative enzymea ctivity increases with greater dilutiona nd reaches its original native activity at ac oncentration of 0.5 mm polymer in the assay solution. This is in agreement with the inhibition curves shown in Figure 5, which confirms that the inhibition of laccase with the polymers described herein is fully reversible and that the activity is fully preserved after one week of storage.
Laccase is ar ather fragile enzyme, which quickly loses its activity during storage, particularly in aqueous solution.Several stabilizers were used fort his enzyme,b ut,s of ar,o nly immobilization and covalent crosslinking led to greatlyi mprove storage stability. [31][32][33][34] To investigate if POx-IDA was not only inhibiting, but also stabilizing laccase, solutions( 100 mm acetate buffer,p H5,2 .2 10 À3 mg mL À1 )o ft he enzymec ontaining various POx at different concentrationsw erep repared and stored at room temperature for up to 28 days. The concentration of POx was set to 5, 10, and 20 mm in the incubated solutions. The activity was measured after differenti ntervals of storage at room temperature by adding 25 mLo ft he stock solution to 0.975 mL laccase assay,w hichr esulted in aP Ox concentration 0.5 mm. As observed in Figure 10, laccase in water becomes practically inactive after 18 days of storage. POx withouta ne nd group already stabilizes laccase. The presenceo f2 0mm PMOx and PEtOx resulted in ar etention of 20 %o fa ctivity after 28 days of storage. Upon adding 5mm IDA-PMOx 35 -IDA and IDA-PEtOx 30 -IDA, the activity after 28 days was still about6 0%, showingt he role of the IDA endg roups. If the polymer concentrationw as increased from 5t o1 0mm,a ni ncrease in stabilityw as observed. Laccase in the presence of 10 mm IDA-  Thus, POx-IDAs stabilizet he enzyme in its deactivated state. In contrast to laccase, HRP is not stabilized by POx-IDAs ( Figure 11). We hypothesize that this is due to ad ifferent inhibition mechanism. As shown previously,P Ox-IDA is an oncompetitivei nhibitor for HRP.T his could be expected because the stabilitym echanismsa re not universal and must be explored for each protein separately. [35,36]

Conclusion
We showedt hat POxs with IDA end groups werec ompetitive inhibitors for laccase and act as stabilizers for this enzyme. This supports the concept illustrated in Figure1 that inhibitors bound to hydrophilic polymers as end groups are initially activated and the bulky polymeric tail additionally blocks the active site of an enzyme. The behavior of POx-IDA towards lac-case is essentially the oppositeo fthat towards HRP.P Ox-IDA are noncompetitive inhibitors for HRP and do not stabilize this enzyme. Thus, polymers with enzyme inhibitors as end groups are av ersatile and interesting way to bring to functions to these relevant drugs.

Experimental Section
Instruments: 1 HNMR spectra were recorded in CDCl 3 by using a Nanobay AVANCE-III HD-400 spectrometer with a5mm BBFOsmart probe from Bruker BioSpin GmbH operating at 400 MHz, and on a DD2-500 spectrometer with a5mm triple resonance H(C,X) probe from Agilent Te chnologies operating at 500 MHz. UV/Vis spectroscopy was performed on an Analytik Jena Specord 210 spectrophotometer with ad ouble-beam photometer to monitor the enzyme activity.S ize-exclusion chromatography (SEC) was performed on a Viscotek GPCMax instrument equipped with ar efractive index (RI) detector (tempered to 55 8C) by using aT osoh TSKgel GMHHR-M (5.0 mmp ores, 2 + 1 precolumn) column set. As an eluent, saline N,N-dimethylformamide (DMF + LiBr,2 0mmol) was used at 60 8Ca taflow rate of 0.70 mL min À1 .C alibration was performed   with polystyrene standards (Viscotek). ITC was performed on aM i-croCal VP-ITC instrument that measured heat evolved or absorbed in liquid samples as ar esult of mixing precise amounts of reactants. DLS measurements were performed on aM alvern Zetasizer Nano S( ZEN 1600) instrument in aqueous buffer at 25 8Ca nd polymer concentrations of 3a nd 8mm.A ll polymerizations were performed by using am icrowave-assisted synthesizer from CEM with avertically focused IR sensor.
Materials:A ll chemicals and solvents were purchased from Acros, Merck, Fluka, and Sigma Aldrich. HRP (EC 1.11.1.7) and laccase from T. versicolor were purchased from Sigma Aldrich. DMP was purchased from Acros. ABTS was purchased from Sigma-Aldrich.
Synthesis of POx-IDA:T he syntheses of the polymers terminated with IDA were performed according to procedures reported in the literature. The composition of the polymers was calculated from 1 HNMR spectra in CDCl 3 . [18] Analytical data for the resulting polymers are given in Ta ble 3.
Laccase assay with ABTS substrate:T he activity of pure laccase from T. versicolor was determined according to aM ajcherczyk modified assay with 0.5 mm ABTS as ac olor-generating substrate in 100 mm acetate buffer at pH 4.5. [37] Coloration was monitored at aw avelength of 420 nm at 25 8Cb yu sing as pectrophotometer (Analytik Jena AG, Jena Germany). Different concentrations of POx (in the range from 0.5 to 8mm)w ere dissolved in ABTS solution (900 mL). Then, laccase (100 mL, 0.05 mg mL À1 ,a bout 0.8 mm)w as mixed with the aqueous, buffered ABTS polymer mixture and the increase in absorbance was measured for 5min. The molar extinction coefficient of oxidized ABTS is 36.6 m À1 cm À1 .
Laccase assay with DMP substrate:T he laccase activity was determined according to am ethod reported by Paszczyń ski et al. by using 2.8 mm DMP substrate in 100 mm acetate buffer pH 4.5. [38] The reaction mixture was prepared analogously to that for the ABTS assay and the increase in absorbance was photometrically determined at awavelength of 468 nm for 5min. The molar extinction coefficient of oxidized DMP is 49.6 mm À1 cm À1 .
Storage stability of laccase:T he stability of the enzyme was tested by incubating 1mLo ft he enzyme (2.2 10 À3 mg mL À1 )a nd polymer at different concentrations (5, 10, 20 mm)f or 28 days in acetate buffer at pH 4.5. The activity of the incubated enzyme was then determined at different time points as follows:t he polymer enzyme solution (25 mL) was added to the ABTS assay solution (1 mL) at 25 8C. The activity was compared with the initial activity of laccase at the beginning of the measurement.
Storage stability of HRP:T he stability of HRP was tested by incubating the enzyme (1 mL, 1.25 10 À3 mg mL À1 )and polymer at concentrations of 10 and 20 mm,f or 20 days in 0.2 m phosphate/0.1 m citrate buffer at pH 5. The activity of the incubated enzyme was then determined at different time points as follows:t he polymer enzyme solution (25 mL) was mixed with the ABTS buffer solution (1425 mL, 0.2 m phosphate/0.1 citrate buffer at pH 5a nd 5mm of ABTS) then hydrogen peroxide solution (50 mL, 0.3 wt %) was added and the increase in absorbance was photometrically determined at 25 8Ca taw avelength of 405 nm. The activity was compared with the initial activity of HRP at the beginning of the measurement.