Temperature-Induced Switching of Enzyme Activity with Smart

Bioconjugate Chem. 2003, 14, 517−525

517

ARTICLES Temperature-Induced Switching of Enzyme Activity with Smart Polymer-Enzyme Conjugates Tsuyoshi Shimoboji,† Edmund Larenas,‡ Tim Fowler,‡ Allan S. Hoffman,*,† and Patrick S. Stayton*,† Department of Bioengineering, University of Washington, Seattle, Washington 98195, and Genencor International Inc., Palo Alto, California 94304. Received September 30, 2002; Revised Manuscript Received November 21, 2002

A method for thermally induced switching of enzyme activity has been developed, based on the sitedirected conjugation of end-reactive temperature-responsive polymers to a unique cysteine (Cys) residue positioned near the enzyme active site. The reversible temperature-induced collapse of N,Ndimethylacrylamide (DMA)/N-4-phenylazo-phenylacrylamide (AZAAm) copolymers (DMAAm) has been used as a molecular switch to control the catalytic activity of endoglucanase 12A (EG 12A). The polymer was conjugated to the EG 12A site-directed mutant N55C, directly adjacent to the cellulose binding cleft, and to the S25C mutant, where the conjugation site is more distant. The N55C conjugate displayed a larger activity shutoff efficiency in the collapsed polymer state than the S25C conjugate. Increasing the polymer molecular weight was also shown to increase the shutoff efficiency of the switch. Related to these effects of conjugation site and polymer size, the switching efficiency was found to be strongly dependent on substrate size. With a small substrate, o-nitrophenyl-β-D-cellobioside (ONPC), there was minimal blocking of enzyme activity when the polymer was in the expanded state. With a large substrate, hydroxyethyl cellulose (HEC), there was a large reduction of enzyme activity in the polymer expanded state, even with relatively small polymer chains, and a further reduction when the polymer was collapsed. Similar general trends for the interactive effects of conjugation site, polymer size, and substrate size were observed for immobilized conjugates. Kinetic studies demonstrated that the switching activity was due to the blocking of substrate association by the collapsed polymers. These investigations provide mechanistic insight that can be utilized to design molecular switches for a variety of stimuli-responsive polymer-protein conjugates.

INTRODUCTION

The ability to reversibly control protein and enzyme activities with external stimuli could provide new opportunities for the development of molecular diagnostics, bioprocesses, affinity separations, lab assays, BioMEMS, bioelectronics, and biosensor technologies. We have been developing a new approach to molecular switches that utilizes site-specific conjugation of stimuli-responsive polymers near the active sites of proteins. These stimuliresponsive polymers undergo a large change in size and physical properties in response to external stimuli such as temperature, pH, or specific wavelengths of light. We have previously demonstrated that the change in physical properties can be used as a temperature-, pH-, or lightdependent activity switch to control ligand capture and release (1-5). We report here the first investigation of these smart polymer-protein conjugates for thermalswitching of enzyme activity. * Corresponding authors. A.S.H.: address, Department of Bioengineering, Box 352255, University of Washington, Seattle, WA 98195; telephone, 206-543-9423; fax, 206-543-6124; email: [email protected]. P.S.S.: address, Department of Bioengineering, Box 351721, University of Washington, Seattle, WA 98195; telephone, 206-685-8148; fax: 206-685-8256; e-mail, [email protected]. † University of Washington. ‡ Genencor International Inc.

Several interesting strategies have been investigated for regulating enzyme activity by external signals. The first has been to conjugate small, responsive molecules near the active sites or binding sites of proteins. For example, binding-site tyrosine residues in streptavidin and antibodies have been chemically modified with nitro groups to introduce a pH-dependent biotin binding (6, 7). Another important example involved the introduction of photoresponsive groups near protein recognition sites (8-10). A second general strategy has been to genetically engineer a “responsiveness” into the protein, in fashion similar to nature’s strategy of allostery (11-13). The endoglucanase EG 12A from Trichoderma reesei preferentially hydrolyzes the internal β(1-4) linkages of amorphous cellulose (14). EG 12A is a single-domain cellulase that does not have a separate cellulose binding domain (15). EG 12A has been used in industrial textile processing, and the ability to finely regulate its activity could provide control over the degree of cellulose processing and fiber properties. Figure 1 shows the schematic design for constructing an enzyme that is thermally switched “on” and “off”. The smart polymer switch reversibly cycles between the expanded and collapsed states in response to temperature changes above and below the lower critical solution temperature (LCST).1 In this paper, we demonstrate the striking activity of the thermally responsive enzyme switch, as well as a char-

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518 Bioconjugate Chem., Vol. 14, No. 3, 2003

Figure 1. Molecular model of endoclucanase 12A and schematic model of the DMAAm-enzyme molecular switch. The red residues represent the catalytic glutamic acid side chains at the active site of EG 12A, the green residue is the Asn 55 position, the purple residue is the Ser 25 position, and the N-terminus is represented as the blue circle.

acterization of the importance of the polymer conjugation site, the molecular weight of the conjugated polymer, the size of the substrate, and the role of free polymer. Although the polymer we have designed for these studies is both light- and temperature-responsive, this paper focuses primarily on the use of temperature to switch the enzyme activity. EXPERIMENTAL PROCEDURES

Materials. 4-Aminoazobenzene (Aldrich, Milwaukee, WI) was recrystallized from ethanol and water and dried in vacuo. N,N-Dimethyl acrylamide (DMA) (Fluka, Milwaukee, WI), diethyl ether, acryloyl chloride, triethylamine, and 2-mercaptoethanol (MEO) (Aldrich, Milwaukee, WI) were purified by distillation under reduced pressure. 2,2′-Azobisbutyronitrile (AIBN) (J. T. Baker, Phillipsburg, NJ) was recrystallized from methanol. Ethanol, dimethylformamide (DMF), tetrahydrofuran (THF), methylene chloride, potassium tert-butoxide, divinyl sulfone (DVS), and ethylenediamine tetraacetic acid (EDTA) (Aldrich, Milwaukee, WI) were used as received. Restric1 Abbreviations: DMA, N,N-dimethylacrylamide; AZAAm, N-4-phenylazo-phenylacrylamide; DMAAm, DMA-co-AZAAm copolymer; EG 12A, endoglucanase 12A; N55C, Asn55Cys sitedirected mutant; S25C, Ser25Cys site-directed mutant; Mn number-averaged molecular weight; ONPC, o-nitrophenyl-β-Dcellobioside; HEC, hydroxyethyl cellulose; LCST, lower critical solution temperature; MEO, 2-mercaptoethanol; AIBN, 2,2′azobisbutyronitrile; DMF, dimethylformamide; THF, tetrahydrofuran; DVS, divinyl sulfone; EDTA, ethylenediamine tetraacetic acid; TCEP, tris(2-carboxyethyl)-phosphine hydrochloride; HABA, 4′-hydroxyazobenzene-2-carboxylic acid; RBB-CMC remazol brilliant blue carboxymethylcellulose; endo H, endoglycosidase H; AS, ammonium sulfate; PHBAH, p-hydroxybenzoic acid hydrazide.

Shimoboji et al.

tion enzymes, XbaI and BglII, were purchased from New England Biolabs (Beverly, MA). QIA Prep Spin Plasmid Kit (QIAGEN, Santa Clarita, CA) and Quikchange SiteDirected Mutagenesis Kit (Stratagene, La Jolla, CA) were used as received. Butyl sepharose (Pharmacia, Piscataway, NJ), bis-tris propane, ammonium sulfate, o-nitrophenyl-β-D-cellobioside (ONPC) (Sigma, St. Louis, MO), hydroxyethyl cellulose (HEC) medium Viscosity (Fluka, Ronkonkome, NY), tris(2-carboxyethyl)-phosphine hydrochloride (TCEP), BCA protein assay kit, Magna Bind Streptavidin, EZ-Link sulfo-NHS-LC-LC-biotin, 4′-hydroxyazobenzene-2-carboxylic acid (HABA), streptavidin magnetic beads (Pierce, Rockford, IL), Ultrafree Biomax (Millipore, Bedford, MA), Precast gel (Novex, San Diego, CA), and Kaleidoscope MW marker (Biorad, Hercules, CA) were used as received. All other reagents were of analytical-grade. Synthesis of Azobenzene-Containing Monomer. An azobenzene-containing monomer, 4-phenylazophenylacrylamide (AZAAm), was synthesized as follows. 4-aminoazobenzene (0.2 mol) and triethylamine (0.26 mol) were dissolved in diethyl ether (200 mL). Acryloyl chloride (0.24 mol) dissolved in 80 mL of diethyl ether was added dropwise at 0 °C under nitrogen atmosphere with stirring. The reaction mixture was allowed to come to room temperature and stirred for 4 h. The reaction solution was filtered to remove triethylammonium chloride, washed with water, and evaporated. The product was recrystallized from ethanol-water mixture and dried in vacuo. The structures were confirmed by 1H NMR (d-DMSO): δ ) 5.7-6.7 (m, 3H), 7.4-7.8 (m, 5H), 7.8-8.2 (m, 4H). Synthesis of Temperature-Responsive Polymer. The temperature responsive polymer DMA-co-AZAAm copolymer (DMAAm) was synthesized with a hydroxyl terminus by free-radical polymerization in DMF at 60 °C for 20 h, using MEO as a chain transfer reagent and AIBN as an initiator (monomer concentration ) 2 mol/ L). The ratios of DMA and AZAAm were varied to change the percentage of incorporated azobenzene moiety in the polymer. To vary Mn, the monomer/MEO/AIBN molar ratios were varied from 100/0.25/0.05 to 100/16.0/3.2. The product was purified by precipitation into diethyl ether three times and dried in vacuo. The content of AZAAm incorporated in the copolymers was determined by 1H NMR (Spectrospin & Bruker, dpx200), comparing the ratio of aromatic and aliphatic hydrogens. The molecular weight (Mn) of the polymer was determined by gel permeation chromatography (GPC, Water, Styragel HR3 and 4) in THF, using polystyrene as a standard. The hydroxyl terminus of DMAAm was converted to a vinyl sulfone (VS) group for conjugation to a sulfhydryl group in the protein. DMAAm was dissolved in 20 mL of methylenechloride with 0.03 g of potassium tert-butoxide, and 100 µL of divinyl sulfone (DVS) (DVS/OH ) 10/1 molar ratio). The solution was stirred for 12 h at room temperature under nitrogen atmosphere. The polymer was precipitated and then washed in diethyl ether, and dried in vacuo. The LCST of the polymer was determined as the temperature at 10% of the maximum absorbance at 600 nm. The polymer concentration was 2 mg/mL in double-distilled water or 100 mM phosphate buffer (PB), pH 7.4. Site-Directed Mutagenesis of EG 12A. The EG 12A S25C and N55C site-directed mutants were constructed using the Quikchange (Stratagene, CA) method. A 20 ng sample of the plasmid was mixed with 125 ng of the mutant primers (sense and antisense). The synthesis product was digested by Dpn I and transformed into

Smart Polymer−Enzyme Bioconjugates

Top10F′. The transformed cell was selected in agar plate with carbenicillin and grown in LB medium. The mutated plasmid was collected by mini-prep (QIAprep, QIAGEN). The following primers (Integrated DNA Technologies Inc., Coralville, IA) were used for the site-directed mutagenesis with the plasmid pGPTpyrG1 (16), including the EG 12A gene inserted between the unique XbaI and BglII cloning sites. The underlined bold sequences were the mutated portions:

1. S25C

5′-p-CAACCTTTGGGGAGCATGTGCCGGCTCTGGATTTGG-3′ 5′-p-CCAAATCCAGAGCCGGCACATGCTCCCCAAAGGTTG-3′

2. N55C

5′-p-GTGGTCCGGCGGCCAGTGCAACGTCAAGTCGTACC-3′ 5′-p-GGTACGACTTGACGTTGCACTGGCCGCCGGACCAC-3′

The site-directed mutations were confirmed bythe dyedeoxy sequencing method. Expression of EG 12A Mutants. Plasmid pGPTpyrG1 with the mutated EG 12A gene was transformed into A. niger for expression. In this plasmid, the EG 12A gene was placed under control of the strong glucoamilase (GAM) promoter for Aspergillus niger (16). The vector was transformed into strain dgr246p2. Transformants were selected on media lacking uridine and then screened for high remazol brilliant blue carboxymethylcellulose (RBB-CMC) activity in shake flask culture. The best producers in shake flasks were grown in 15 L fermentors. Purification and Characterization of the SiteDirected EG 12A Mutants. The fermentation broths were treated with Endoglycosidase H (Endo H) to inhibit heterogeneous glycosylation of the expressed EG 12A’s. EndoH (9 mg/mL) was added to the fermentation broth to a final dilution of 50 to 1 and incubated at 37 °C overnight. Ammonium sulfate (AS) was mixed to it to 0.5 M and centrifuged for 10 min. The pellet was discarded. A 20 mL sample of butyl sepharose beads in a drip column was equilibrated with 0.5 M AS in 50 mM bistris Propane, pH 5.5. A 15 mL of the enzyme solution (supernatant) was loaded and washed with 3 volumes of the equilibration solution. The EG 12A was eluted from the beads with 50 mM bis-tris propane pH 5.5. The fractions that had absorbance at 280 nm were collected. The buffer was exchanged to 50 mM sodium acetate buffer (AB), pH 5.5, and concentrated to 1 mg/mL concentration with an ultracentrifuge membrane (Ultrafree, MW cut off; 5 kDa). The site-directed mutations of S25C and N55C at the protein level were confirmed by peptide mapping method, in which the proteins were hydrolyzed by specific proteases and the fragments were analyzed by LC-MS. Conjugation of Temperature-Responsive Polymers to EG 12A Mutants. Conjugations of EG 12A mutants to DMAAm-VS with different Mn’s were carried out by reacting the vinyl sulfone group of the polymer with the sulfhydryl-containing cysteine side chains of the EG 12A mutants in 50 mM PB, pH 8.0. The cysteine residues in the EG 12A mutants form disulfide bonds during purification and storage. To reduce these disulfide bonds to sulfhydryl groups, TCEP at 10-fold molar excess to the mutant was added and incubated for 20 min at room temperature. A 50-fold molar excess of the polymer to the mutant was added and reacted overnight at 4 °C to obtain high conjugation efficiency. The buffer was

Bioconjugate Chem., Vol. 14, No. 3, 2003 519

exchanged to 50 mM AB, pH 5.5, at 4 °C using an ultrafiltration membrane (Ultrafree, Millipore) to stabilize the conjugate; then the conjugates were separated from the unconjugated mutants by thermally induced precipitation at 52 °C. The unconjugated mutants were retained in the supernatant. The precipitates were resuspended in 50 mM AB, pH 5.5. The thermally induced precipitation was repeated three times to remove the unconjugated mutants completely. The conversion of the conjugates was quantitated by the BCA protein assay method (Pierce). Immobilization of the Conjugates on Magnetic Beads. The conjugates were biotinylated by reacting with sulfo-NHS-LC-LC-biotin. The conjugate was dissolved in 100 mM sodium phosphate buffer, pH 7.4 at 4 °C, at a concentration of 200 µg/mL. A 10-fold molar excess of Sulfo-NHS-LC-LC-Biotin, dissolved in deionized water immediately prior to use, was added to the solution at room temperature. The solution was gently rotated at 4 °C for 5 h. The biotinylated conjugate was purified from unconjugated biotin by ultrafiltration using Biomax-5 (MW cut off: 5 kDa), followed by thermal precipitation at 52 °C. The average number of conjugated biotin molecules per EG 12A conjugate was determined using the HABA assay. In short, 0.5 mL of mixture of avidin (20 µM) and HABA (600 µM) was added to a quartz cuvette. Absorbance at 500 nm was measured on a UVvis spectroscope. To this solution, 50 µL of biotinylated conjugate solution was added. Absorbance at 500 nm was recorded 30 min after addition of the biotinylated protein solution. The concentration of biotin in the biotinylated protein solution was determined from the calibration curve of biotin standards. The biotinylated conjugates were immobilized on streptavidin magnetic beads. A 1 mg aliquot of magnetic beads was mixed with 20 µg of conjugate in 1 mL of 50 mM AB, pH5.5, containing 50 mM NaCl, 5 mM EDTA, and 0.2 wt % of BSA. The amount of the immobilized conjugate was determined by depletion of the conjugate from the supernatant. The immobilized conjugates were separated from the free polymer by washing the beads 10 times with 50 mM AB pH 5.5. Catalytic Activity Assay. The catalytic activity of the EG 12A conjugates was measured at 32 and 52 °C. The 32 °C temperature was chosen because it falls below the LCST transition, and 52 °C was chosen because it falls above the phase transition temperature of the polymer and EG 12A becomes unstable above 55 °C. To elucidate the mechanism of the stimuli-responses, the kinetic parameters Km and kcat of the conjugates were determined by Lineweaver-Burk plot assuming the MichaelisMenten equation. Two substrates with different sizes, ONPC and HEC, were chosen to investigate the effect of the size of the substrate on the switching activity. ONPC is hydrolyzed by EG 12A to generate a nitrophenol dye that is detected at 405 nm. The assay was conducted in 50 mM sodium acetate buffer pH 5.5. ONPC (2-10 mM) and the conjugate (100 nM) solutions were preincubated at 32 and 52 °C. The reaction was initiated by addition of ONPC into EG 12A solution in a total volume of 1 mL, and it was incubated at each temperature. Samples were taken at 8-10 different time points in order to produce reliable values for the initial velocity determinations. A 100 µL aliquot was taken from the reaction solution, and the reaction was terminated with the addition of 50 µL of 200 mM glycine buffer pH10. The absorbance at 405 nm was measured in a microtiter plate reader (Benchmark, BioRad). The concentration of nitrophenol was calculated using the extinction coefficient at 405 nm.


Shimoboji et al. Table 1. DMAAm Polymer Physical Properties polymer

MEOa (mol %)

AZAAma (mol %)

Mwb

Mnb

Mw/Mnb

yield (%)

M-10 M-6 M-3

0.25 1.0 2.0

5.5 8.0 9.0

14900 9100 5700

9600 5700 3200

1.55 1.59 1.79

31.6 48.5 74.1

a

Molar ratio to total monomer in feed. b Determined by GPC.

Figure 2. The DMAAm LCST behavior under UV vs vis photoirradiation. The relative absorbance was measured at 600 nm for 2 mg/mL DMAAm (10 kDa) in 50 mM sodium acetate buffer, pH5.5. The heating rate was 0.5 °C/min. The DMAAm solutions were photoirradiated with UV and vis light for 10 min and 3 h, respectively, before the LCST measurements.

HEC is a soluble cellulose derivative that is hydrolyzed by EG 12A to generate hydroxyethyl glucose. This product was subsequently quantitated by reaction with p-hydroxybenzoic acid hydrazide (PHBAH). PHBAH reacts with glucose to produce glucosazone, which is detected at 405 nm. In brief, the conjugate (100 nM) and HEC (Cellulose WP-40, Fluka-BioChemika) (0.25-2 w/v %) solutions in 50 mM sodium acetate buffer, pH 5.5, were preincubated in a microtube at each temperature. The reaction was initiated by addition of HEC into EG 12A solution in a total volume of 1 mL, and it was incubated at 32 and 52 °C. Samples were taken at 8-10 different time points in order to produce reliable values for the initial velocity determinations. A 100 µL aliquot was taken from the reaction solution, and the reaction was stopped by addition of 50 µL of 2% NaOH solution. A 20 µL sample of the reaction solution was mixed with 400 µL of PHBAH solution (1 w/v % in 0.3 N NaOH) in microtubes. The top of the cap of the microtube was punctured with syringe needle, and the tube was placed in boiling water for 10 min (for steam treatment). The solution was cooled, and 200 µL of the solution was placed into microtiter wells. The absorbance at 405 nm was measured. The glucose concentration was calculated from the standard curve made by a series of concentration of glucose. RESULTS

Synthesis and Characterization of Azobenzene and DMA Copolymers. The DMAAm copolymers display dual temperature- and photo-responsive phase behavior (17, 18). The DMAAm copolymers exhibit a shift toward higher LCST values under vis illumination (Figure 2). The monomer ratios were selected so that the maximal difference in polymer expansion and collapse between the vis and UV states occurred in the range of 40-45 °C, where the enzyme exhibits an optimal balance between thermal stability and activity. For these studies focused on the development of a thermo-sensitive enzyme switch, three different DMAAm polymers with different Mn’s were synthesized in order to investigate the role of polymer size in the thermal switching activities for the polymer-EG 12A conjugates. The physical properties of the copolymers utilized in this study are summarized in Table 1. The thermally induced phase transition behaviors of the DMAAm polymers at three Mn’s (10, 6, 3 kDa) are shown in Figure 3. The LCST values fall between 37 and 43 °C. To create a thiol-reactive end group for conjugating to the genetically engineered cysteine on the

Figure 3. The LCST behavior of DMAAm as a function of polymer Mn. The relative absorbance at 600 nm was measured for 10, 6, and 3 kDa polymer solutions (2 mg/mL) in 50 mM sodium acetate, pH 5.5. The heating rate was 0.5 °C/min.

EG 12A enzymes, the hydroxyl termini of all the polymers were converted to a vinyl sulfone moiety by reaction with divinyl sulfone. Construction and Characterization of the SiteDirected EG 12A Mutants. Figure 1 shows a 3-D schematic structure of EG 12A, with the site-directed mutation positions constructed for this study displayed in relation to the catalytic side chains at the active site. The criteria for selection of the Asn 55 and Ser 25 positions was that they be solvent exposed and on the outer edge of the concave cellulose binding cleft. The Ser 25 position was chosen to be more distant from the binding site cleft than Asn 55. The site-directed mutants were successfully constructed as demonstrated by DNA sequencing, and the mutant proteins were subsequently produced in an established A. niger expression system (16). The purities of the S25C and N55C mutants were determined to be 88.4% and 94.0%, respectively, by SDSPAGE gel analysis. The N55C mutant displayed a small change in Km of 33.9 versus 19.5 mM for wild-type with the model substrate, o-nitrophenyl β-D-cellobioside (ONPC) and no significant change in kcat (13.6 vs 12.8 s-1). S25C exhibited a Km of 20.8 mM and a kcat of 14.5 s-1. The two mutants thus exhibited kinetic values that were largely unperturbed from the wild-type enzyme, and the subsequent smart polymer conjugates were normalized to the activity of the unconjugated mutants. Both mutants were conjugated to the DMAAm-VS in the presence of TCEP to reduce the interprotein dimers that formed during purification. The polymer-enzyme conjugates were separated from the unconjugated mutants by repeated thermally induced precipitation at 52 °C. Table 2 shows the overall yields of the conjugates. Control experiments showed that the EG 12A enzyme was not precipitated in physical mixtures of enzyme and polymer, demonstrating that only conjugated enzyme was purified by this technique. The S25C conjugates exhibited higher yields than the N55C conjugates, suggesting that the 25 position is more accessible and reactive than the 55 position.



Table 2. Conversion Efficiencies of the EG 12A-DMAAm Conjugations conjugate

total yield (µg)

%

S25C-DMAAm (10 kDa) S25C-DMAAm (6 kDa) S25C-DMAAm (3 kDa) N55C-DMAAm (10 kDa) N55C-DMAAm (6 kDa) N55C-DMAAm (3 kDa) WT + DMAAm (10 kDa)

198 292 167 141 148 144 0

23.5 34.6 19.8 19.4 20.3 19.8 0.0

Figure 5. Characterization of HEC enzyme activities for free DMAAm-EG 12A conjugates in solution at as function of polymer size and conjugation site. The activities were measured for 100 nM conjugates with free polymer using HEC (20 mg/ mL) as a substrate and normalized to the activity of N55C or S25C EG 12A controls as 100% at each temperature. EG 12AM10, 10 kDa DMAAm-EG12A conjugate; EG 12A-M6, 6 kDa DMAAm-EG 12A conjugate; EG 12A-M3, 3 kDa DMAAmEG 12A conjugate; and EG 12A + M10, physical mixture of enzyme plus 10 kDa DMAAm.

Figure 4. Characterization of ONPC enzyme activities for free DMAAm-EG 12A conjugates in solution at as function of polymer size and conjugation site. The enzyme activities were measured for 100 nM conjugates using ONPC (8mM) as a substrate and normalized to the activity of N55C or S25C EG12A controls as 100% at each temperature. EG 12A-M10, 10 kDa DMAAm-EG12A conjugate; EG 12A-M6, 6 kDa DMAAm-EG 12A conjugate; EG 12A-M3, 3 kDa DMAAmEG 12A conjugate; and EG 12A + M10, physical mixture of enzyme plus 10 kDa DMAAm.

Temperature-Responsive Properties of the Free EG 12A Conjugates in Solution. Switching Activities as a Function of Polymer Size and Conjugation Site. The EG 12A conjugates were first characterized in solution with free polymer present. Figure 4 shows the comparison of the thermo-responsive activity changes of the N55C and S25C conjugates toward the small molecule substrate ONPC at 32 °C below the LCST and at 52 °C above the LCST. The activity of the conjugates was normalized to that of unconjugated N55C or S25C at each temperature. The physical mixtures of the mutants and the polymers were used as controls. While the physical mixtures did not show switching effects, both the N55C and S25C conjugates exhibited a sharp and reversible shutoff of the enzyme activities at 52 °C. In particular, the N55C-DMAAm of Mn 10 kDa displayed a nearly complete switching of the enzyme activity at 52 °C. As the Mn of the conjugated polymer was decreased from 10 to 6 to 3 kDa, there was a concomitant decrease in

switch efficiency (Figure 4). For the S25C conjugates, a small but significant activity was retained, even with the 10 kDa DMAAm, when the conjugation site was moved to this more distant position relative to the active site carboxylate residues. Taken together, these results demonstrate that both the polymer Mn and the position of conjugation are critical for determining the magnitude of the switching effect. Switching Activities as a Function of Substrate Size. The effect of substrate size on the switching activity was investigated by using the large HEC as a substrate. Figure 5 shows the results of the thermo-responsive switching activity measurements using HEC as a substrate for the N55C and S25C conjugates. At 32 °C, the enzyme activities were much lower than the unconjugated mutant EG 12A controls, and the physical mixtures of unconjugated enzyme and free polymer, while they were largely unchanged for the small molecule ONPC substrate. This suggests that the steric hindrance of the expanded, conjugated polymer interfered with the binding of the larger HEC to EG 12A. When the temperature was increased to 52 °C, the activity of the N55C and S25C conjugates toward HEC was shut off. This effect was observed not only with the 10 kDa N55C-DMAAm conjugate, but also with the 6 kDa polymer. This finding was in contrast to the incomplete shutoff of the smaller ONPC activity with the 6 kDa DMAAm polymer. When the conjugation site was moved to the more distant S25C position, the expanded polymer again blocked activity to a significant level at all three polymer sizes. The same


Shimoboji et al. Table 4. Quantities of Immobilized Conjugates and WT EG 12A Control conjugate

immobilized quantity (µg/mg beads)

N55C-DMAAm (10 kDa) N55C-DMAAm (6 kDa) N55C-DMAAm (3 kDa) S25C-DMAAm (10 kDa) S25C-DMAAm (6 kDa) S25C-DMAAm (3 kDa) WT

18.8 19.3 19.5 19.0 19.5 19.8 19.8

Figure 6. Characterization of ONPC switching activities for immobilized EG12A conjugates as a function of polymer molecular weight and conjugation site. The activities were measured using 8 mM ONPC as a substrate at 32 and 52 °C in 50 mM sodium acetate buffer, pH5.5, and normalized to the activity of WT EG 12A. DMAAm-EG12A conjugates of the following are shown for the N55C and S25C conjugates: 10 kDa Mn, M-10; 6 kDa Mn, M-6; and 3 kDa Mn, M-3. Table 3. Biotinylation Results for DMAAm-EG 12A Conjugates conjugate

number of biotins (#/EG 12A conjugate)

N55C-DMAAm (10 kDa) N55C-DMAAm (6 kDa) N55C-DMAAm (3 kDa) S25C-DMAAm (10 kDa) S25C-DMAAm (6 kDa) S25C-DMAAm (3 kDa)

1.4 1.4 1.5 1.3 1.4 1.5

general trend was still found of a more complete shutoff effect as polymer Mn was increased, although the absolute magnitude of the off-state activities were consistently higher at the corresponding polymer sizes than for the N55C conjugates. Temperature-Responsive Activity Changes of Immobilized DMAAm-EG 12A Conjugates. Immobilized enzymes are used in many applications and the switching properties were thus also investigated when the conjugates were bound to beads and free polymer was removed. The conjugates were biotinylated and immobilized onto streptavidin magnetic beads, followed by intensive washing to remove the free polymer chains. The degree of biotinylation was well controlled near a stoichiometry of one (Table 3). The quantities of the conjugates and the control WT EG 12A immobilized on the SA magnetic beads were controlled to equivalent levels (Table 4). A comparison of the catalytic activities of the immobilized and purified N55C and S25C conjugates toward ONPC and HEC at 32 and 52 °C is shown in Figures 6 and 7. The general trend of switching activity as a function of conjugation position, of polymer size, and of substrate size followed those of the conjugates in

Figure 7. Characterization of HEC switching activities for immobilized EG12A conjugates as a function of polymer molecular weight and conjugation site. The activities were measured using 8 mM ONPC as a substrate at 32 and 52 °C in 50 mM sodium acetate buffer, pH5.5, and normalized to the activity of WT EG 12A. DMAAm-EG12A conjugates of the following are shown for the N55C and S25C conjugates: 10 kDa Mn, M-10; 6 kDa Mn, M-6; and 3 kDa Mn, M-3.

solution. The magnitude of the shutoff effects were generally good, although there was a significant residual activity in all samples at 52 °C, including with the 10 kDa conjugates. This residual activity was approximately 12% for the N55C-10 kDa DMAAm conjugate. To investigate the effect of the free polymer on the switching activity of the immobilized conjugates, free DMAAm was added back to the beads at defined stoichiometries. The effect of free polymer addition on the activity of the immobilized conjugates at 52 °C is shown in Figure 8. The addition of free DMAAm polymer enhanced the shutoff effect for both S25C and N55C conjugates, with complete shutoff of enzyme activity for the N55C conjugates when 100-200 times excess of the free polymer was added. These results suggest that the conjugated polymer directs the aggregation of the free polymer to the conjugation site near the enzyme active site, where the higher effective size of the aggregate leads to more effective blocking of the substrate from the active site.


Figure 8. Effect of free polymer addition to immobilized EG 12A activities remaining above the LCST of the DMAAm. DMAAm of 10 kDa Mn was added to the immobilized N55C and S25C conjugates at the defined concentrations noted on the bar graph. The activities were measured for 100 nM conjugates using ONPC (8 mM) as a substrate at 52 °C in 50 mM sodium acetate buffer, pH 5.5, and normalized to the activity of immobilized WT EG 12A at each temperature.

Figure 9. The complete activity profiles for the DMAAmN55C EG 12A conjugate as a dual function of temperature and UV or Vis light irradiation. Initial velocities were determined for the 100 nM N55C-DMAAm (11 kDa) conjugate and the N55C control using 8 mM ONPC as the substrate in 50 mM sodium acetate buffer, pH5.5. The samples were irradiated with either the UV or vis light for 10 min and 3 h, respectively, before the activity determinations.

Dual Photo- and Temperature Switching Activity Profiles. The light-responsive enzyme switching has been described in a recent communication (18). The dual relationship between temperature and UV or vis irradiation on the activities of the N55C control and the N55CDMAAm (10 kDa) conjugate are shown in Figure 9 with ONPC as substrate. The activity of the N55C increased with increasing temperature, and UV or vis irradiation did not influence the enzyme activity. The temperature response of the polymer switch generally follows the shift in LCST behavior under UV versus vis photoirradiation (Figure 2), confirming the action of the polymer as a switch between the expanded and collapsed states. The thermal cycling properties and reversibility of the DMAAm polymer switch are demonstrated in Figure 10. As the product accumulation profile demonstrates, the response time to turn on or off the enzyme activity was faster than the scale of seconds it took to switch between the two


Figure 10. Reversibility of the DMAAm-N55C EG 12A enzyme switch. The total product (o-nitrophenol) accumulation was measured for 100 nM conjugate using ONPC (8 mM) as a substrate in 50 mM sodium acetate buffer pH 5.5. The reaction vessel was transferred between 32 and 52 °C water baths with small aliquots removed to determine product accumulation as a function of time.

Figure 11. Thermal stability of the EG 12A enzyme switch. The temperature was cycled between 32 and 52 °C for 2 min each. The activities were measured for 100 nM N55C or the conjugates with free polymer using ONPC (8 mM) as a substrate at 32 °C after incubation for 20 min in 50 mM sodium acetate buffer pH5.5.

temperature baths. There was some instability of the EG 12A to repeated thermal cyclings as shown in Figure 11, but the DMAAm conjugation did not significantly destabilize the enzyme further. The activities of both N55C and the conjugates decreased with increasing cycle number in equivalent fashion. Kinetic Analysis of the Switching Mechanism. Michaelis-Menton analysis of the catalytic activity data was conducted to provide mechanistic insight into the polymer switching activity. The effects of temperature changes under UV or vis photoirradiation on the Michaelis-Menton kinetic parameters kcat and Km for N55C and N55C-DMAAm (10 kDa) conjugates are shown in Table 5. These parameters were determined for both the small molecule ONPC substrate and the larger HEC substrate. The Km values of the conjugates were increased substantially compared to those of unconjugated N55C at 52 °C compared to 32 °C. The kcat values of the conjugates remained only slightly reduced in comparison to the N55C control. These results suggest that the shutoff effect is primarily due to the large decrease in substrate affinity upon polymer collapse above the LCST. The


Shimoboji et al.

Table 5. Michaelis-Menton Analysis of Switching Mechanism ONPC Km [mM] 32 °C 52 °C 32 °C 52 °C

HEC kcat [s-1]

N55C 1.9 13.6 N55C-DMAAm 20.7 2.06 1140 4.10 15.8 33.9

Km [m/mL]

kcat [s-1]

14.4 29.2

4.7 12.7

190 9660

4.5 9.3

switching is thus connected to the steric blocking of the active site by the collapsed DMAAm polymer when it is attached close to the active site. The activity of the DMAAm conjugates toward HEC is reduced at 32 °C, where the polymer exists in the expanded state. This reduced activity is also due to a large increase in Km. The decrease in the affinity of the large HEC substrate, even when the DMAAm polymer is in the expanded state, is again consistent with a simple steric blocking mechanism, which is enhanced by the large size of the HEC substrate. DISCUSSION

A new approach has been developed for controlling enzyme activity using smart polymer switches that serve both as antennae and as actuators. We have previously shown that smart polymer collapse and expansion in response to small changes in temperature or pH can be used to control biotinylated-ligand binding to streptavidin (1-4). A similar general approach for controlling enzyme activity was investigated in this work, with a new family of dual temperature- and photo-responsive polymers. These polymers reversibly cycle between an expanded state, hydrated state, and a collapsed, hydrophobic state above and below their characteristic phase transition LCST. This LCST varies under UV versus vis irradiation, which provides the ability to use temperature and/or light to regulate polymer expansion and collapse. The EG12A enzyme has a distinctly different binding site compared to the previously studied streptavidin-biotin system. The binding site is a relatively broad and concave groove that is more open and accessible to the conjugated polymer chain than the nearly completely buried binding site of streptavidin. This likely plays an important role in the improved on and off reversibility of the enzyme switch compared to streptavidin, as well as the considerably faster substrate off-rate compared to that of biotin. An important conclusion that can be drawn from these initial studies is that the polymer switch is largely acting by blocking substrate access when the chain is in the collapsed state, i.e., the off-state. In the expanded state below the LCST, i.e., the on-state, the hydrated polymer chain did not significantly alter the small substrate access as evidenced by the catalytic activities and Km values. The key corollary is that the switch can be optimized by the interplay of conjugation site, polymer size, and substrate size. With a polymer of a given Mn, the efficiency of the off-state was reduced when the conjugation site was moved away from the active site. Consistent with this steric mechanistic model, the efficiency could be improved at the distant conjugation site by increasing the polymer chain length. These two design parameters of polymer Mn and conjugation site also trade off with respect to substrate size. In contrast to the small molecule ONPC substrate, we found significant blocking of the large HEC substrate by the expanded polymer

chain with even the 3 kDa DMAAm. Taken together, these findings suggest that the polymer switch approach should allow optimization for a given substrate through an appropriate choice of polymer size and conjugation site. While it is clear that the blocking of substrate access is underlying the switching effects, the origins of this blocking mechanism could be different for the conjugates free in solution versus in the immobilized state on beads. From an applications standpoint, both are interesting as there are assay and microfluidic technologies where either or both of the systems might be useful. The immobilized conjugates were purified from free polymer and were found to possess strong switching activity. With these isolated conjugates, the mechanism should be close to the schematic model of polymer collapse and expansion in Figure 1. The remaining activity could be abrogated by the addition of free polymer, suggesting that larger polymer chains and/or a closer conjugation site could optimize the enzyme switch for the surface-immobilized enzymes. On the other hand, the free conjugates in solution could be forming colloidal aggregates above the LCST, and the substrate blocking effects could be due to transport limitations to the active sites of the aggregated enzymes. These experiments were conducted at low concentrations ca. 100 nM, where there were no detectable changes in optical density (i.e., clouding) above the LCST and no aggregates could be pelleted. The time response in going from the collapsed off-state to the expanded on-state is at least as fast as seconds, consistent with very small aggregates that can dis-aggregate quickly. Finally, it should be noted that there is still a strong positional dependence of the conjugation site on the switching activity that follows the same trend as that of the immobilized system. Therefore the orientation of the enzyme active site relative to the polymer must be connected to the poor accessibility of substrate, which would only be possible in very small aggregates or isolated conjugates. Further experimentation with dynamic light scattering techniques will be necessary to determine the nature of the aggregates formed at these concentrations. ACKNOWLEDGMENT

We would like to thank Dr. J. Milton Harris of Shearwater Polymers for his advice on the addition of the polymer end hydroxyl groups to vinyl sulfone groups of DVS. We also wish to thank Dr. R. To for helping the construction of the EG 12A mutants and Dr. David Hyre for the molecular model of EG 12A. The NIH (Grant No.53771), UW Office of Technology Transfer, the Washington Research Foundation, and the Washington Technology Center are gratefully acknowledged for their support of this project. LITERATURE CITED (1) Stayton, P. S., Shimoboji, T., Long, C., Chilkoti, A., Chen, G., Harris, J. M., and Hoffman, A. S. (1995) Control of protein-ligand recognition using a stimuli-responsive polymer. Nature 378, 472-474. (2) Bulmus, E. V., Ding, Z., Long, C. J., Stayton, P. S., and Hoffman, A. S. (2000) A pH- and temperature-sensitive copolymer-streptavidin site-specific conjugate for pH-controlled binding and triggered release of biotin. Bioconjugate Chem. 11, 78-83. (3) Ding, Z. L., Long, C., Hayashi, Y., Bulmus, E. V., Hoffman, A. S., and Stayton, P. S. (1999) Temperature control of biotin

Smart Polymer−Enzyme Bioconjugates binding and release with a streptavidin-poly(N-isopropylacrylamide) site-specific conjugate. Bioconjugate Chem. 10, 395400. (4) Ding, Z., Fong, R. B., Long, C. J., Hoffman, A. S., and Stayton, P. S. (2001) Size-dependent control of the binding of biotinylated proteins to streptavidin using a polymer shield. Nature 411, 59-62. (5) Shimoboji, T., Ding, Z. L., Stayton, P. S., and Hoffman, A. S. (2002) Photoswitching of ligand association with a photoresponsive polymer-protein conjugate. Bioconjugate Chem. 13, 915-919. (6) Morag, E., Bayer, E. A., and Wilchek, M. (1996b) Immobilized nitro-avidin and nitro-streptavidin as reusable affinity matrixes for application in avidin-biotin technology. Anal. Biochem. 243, 257-263. (7) Tawfik, D. S., Chap, R., Eshhar, Z., and Green, B. S. (1994) pH on-off switching of antibody-hapten binding by sitespecific chemical modification of tyrosine. Protein Eng. 7, 431-434. (8) Willner, I., Shai, R., and Riklin, A. (1991) Photoregulation of papain activity through anchoring photochromic azo groups to the enzyme backbone. J. Am. Chem. Soc., 113, 3321-3325. (9) Bieth, J., Wasserman, N., Vratsanos, S. M., and Erlanger, B. F. (1970) Photoregulation of biological activity by photochromic reagents, IV. A model for diurnal variation of enzymic activity. Proc. Natl. Acad. Sci. U.S.A. 66, 850-854. (10) Namba, K., and Suzuki, S. (1975) Photocontrol of enzyme activity with a photochromic spiropyran compound-modification of R-amilase with spiropyran compound. Chem. Lett. 9, 947-950. (11) Posey, K. L., and Gimble FS. (2002) Insertion of a reversible redox switch into a rare-cutting DNA endonuclease. Biochemistry 41, 2184-2190.

Bioconjugate Chem., Vol. 14, No. 3, 2003 525 (12) Seetharaman, S., Zivarts, M., Sudarsan, N., and Breaker, R. R. (2001) Immobilized RNA switches for the analysis of complex chemical and biological mixtures. Nat. Biotechnol. 19, 336-341. (13) Marvin, J. S., Corcoran, E. E., Hattangadi, N. A., Zhang, J. V., Gere, S. A., and Hellinga, H. W. (1997) The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors. Proc. Natl. Acad. Sci. U.S.A. 94, 4366-4371. (14) Hakansson, U., Fagerstam, L., Pettersson, G., and Andersson, L. (1978) Purification and characterization of a low molecular weight 1,4-beta-glucan glucanohydrolase from the cellulolytic fungus Tricoderma viride QM 9414. Biochim. Biophys. Acta 524, 385-392. (15) Ward, M., Wu, S., Darberman, J., Weiss, G., Larenas, E., Bower, B., Rey, M., Clarkson, K., and Bott, R. (1993) Cloning, sequence and preliminary structural analysis of a small, high pI endoglucanase (EG III) from Tricoderma reesei. Found. Biotech. Ind. Ferment. Res. 8, 153-158. (16) Sandgren, M., Shaw, A., Ropp, T. H., Wu, S., Bott, R., Cameron, A. D., Stahlberg, J., Mitchinson, C., and Jones, T. A. (2001) The X-ray crystal structure of the Trichoderma reesei family 12 endoglucanase 3, Cel12A, at 1.9 A resolution. J. Mol. Biol. 308, 295-310. (17) Kroger, R., Menzel, H., and Hallensleben, M. L. (1994) Light controlled solubility change of polymers: copolymers of N,N-dimethylacrylamide and 4-phenylazophenyl acrylate. Macromol. Chem. Phys. 195, 2291-2298. (18) Shimoboji, T., Larenas, E., Fowler, T., Stayton, P. S., and Hoffman, A. S. (2002) Photoresponsive polymer-enzyme switches. Proc. Natl. Acad. Sci. U.S.A. 99, 16592-16596.

BC025615V

Temperature-Induced Switching of Enzyme Activity with Smart

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