Letter pubs.acs.org/macroletters
Polymer-Based Protein Engineering Enables Molecular Dissolution of Chymotrypsin in Acetonitrile Chad S. Cummings,†,‡ Hironobu Murata,† Krzysztof Matyjaszewski,†,§ and Alan J. Russell*,†,‡ †
Center for Polymer-based Protein Engineering, ICES, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, Pennsylvania 15213, United States ‡ Department of Biomedical Engineering, Scott Hall 4N201, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, Pennsylvania 15213, United States § Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States S Supporting Information *
ABSTRACT: While most effective in aqueous environments, enzymes are also able to catalyze reactions in essentially anhydrous organic media. Enzyme activity in organic solvents is limited as a result of inefficient substrate binding, lack of solubility, and inactivation by hydrophilic anhydrous solvents. With these facts in mind, atom transfer radical polymerization was used to synthesize chymotrypsinpoly(2-(dimethylamino)ethyl methacrylate) (CT-pDMAEMA) conjugates designed to be soluble and active in acetonitrile. CT-pDMAEMA solubility in organic solvents and the rate of CT-pDMAEMA-catalyzed transesterification in acetonitrile were examined at a range of water (0−15 M) and propanol (0.01−5 M) concentrations. The conjugates were soluble at the molecular scale in several organic solvents, exhibited good substrate binding with N-acetyl L-phenylalanine thiophenylester (KM as low as 17 mM), and had an activity (peak activity 330 μM/min/mg enzyme) many orders of magnitude higher than that of the insoluble native enzyme.
A
been used to increase the activity of lipase approximately 10fold in organic solvents.15 Still, even after modification, many enzymes often exist as aggregates in organic media, which allows only enzymes at the surface of the aggregate to catalyze reactions. If it were possible to molecularly dissolve enzymes in organic solvents, then one would expect dramatic increases in dissolved enzyme activity and the opportunity for tight substrate binding to enzymes even in organic media. Protein−polymer conjugates have proven to be useful for a variety of applications,16 but they are especially beneficial in nonaqueous media due to their increased activity and solubility compared to native enzymes in these solvents. We have previously examined the activity and stability response of polymer-based protein-engineered chymotrypsin to a variety of stressors (pH, temperature, protease degradation) in aqueous media.17,18 Specifically, chymotrypsin-poly(2-(dimethylamino)ethyl methacrylate) (CT-pDMAEMA) prepared by atom transfer radical polymerization (ATRP)19,20 showed pHdependent activity and increased stability to low pH conditions. Due to the amphiphilic nature of pDMAEMA and the high density polymer modification around the enzymatic core, we hypothesized that CT-pDMAEMA conjugates might also be soluble and active in organic solvents. In order to achieve the most stable protein−polymer conjugate with the largest
s nature’s nanocatalysts, enzymes are able to catalyze numerous reactions with high substrate specificity in aqueous environments under mild conditions. In addition, many enzymes show activity after lyophilization and suspension in organic media and ionic liquids.1−4 Nonaqueous enzymology has several potential benefits such as increased nonpolar substrate solubility, shifted thermodynamic equilibria, and reduction in water-dependent side reactions.5 If dissolved and active in organic solvents, enzymes can catalyze many reactions such as transesterification or aminolysis with impressive stereo-, regio-, or enantioselectivity. However, enzyme activity in organic media is often several orders of magnitude below that in aqueous conditions due mainly to poor substrate binding and the structural effects of the solvent.6 In nonpolar organic media, some enzymes remain structurally stable, as the solvation between nonpolar solvents and proteins favors a structured globular state.7 Conversely, activity in these solvents is largely reduced because enzymes do not have the required mobility for catalysis. In more polar organic solvents, such as acetonitrile, water molecules are pulled away from the enzyme, which favors the denatured protein state.8 Many different strategies, including complexation with other molecules,9,10 absorption to resins,11 and covalent coupling of polymers,12 have been used to increase enzymatic activity, solubility, and stability in hydrophilic organic solvents. Specifically, conjugation of a variety of enzymes with poly(oxazoline) polymers resulted in higher solubility13 and activity increases upward of 155,000fold.13,14 In addition, microemulsion-based organo-gels have © 2016 American Chemical Society
Received: February 17, 2016 Accepted: March 28, 2016 Published: March 30, 2016 493
DOI: 10.1021/acsmacrolett.6b00137 ACS Macro Lett. 2016, 5, 493−497
Letter
ACS Macro Letters potential to increase solvation in organic solvents, we chose to examine the solubility and activity in acetonitrile of the largest CT-pDMAEMA conjugate that was prepared. This conjugate consisted of a chymotrypsin core surrounded by 12 pDMAEMA polymer chains each with an average molar mass of 23.1 kDa. The hydrodynamic diameter (Dh) of the conjugate in aqueous media was ∼34 nm, and the overall molar mass of the bioconjugate was 305 kDa. Prior to examining activity of the CT-pDMAEMA conjugates in anhydrous environments, we determined the effect of polymer conjugation on solubility of the conjugates in acetonitrile and a variety of other solvents. The hydrodynamic diameter values of CT-pDMAEMA conjugates were measured using dynamic light scattering (DLS) in acetonitrile/water/ propanol mixtures (Figure 1). Propanol and water were
Table 1. Solubility of Chymotrypsin and CT-pDMAEMA in Organic Mediaa sample native CT CTpDMAEMA
turbidity (Abs at 500 nm)
Dh (nm)
acetonitrile acetonitrile
0.122 0.004
510 ± 260 29 ± 7
dichloromethane chloroform tetrahydrofuran acetone
0.011 0.050 0.184 0.332
solvent
21.1 211.7 270.0 546.6
± ± ± ±
2.0 60.3 131.7 204.0
a
Comparative solubility of native chymotrypsin (0.02 mg/mL) and CT-pDMAEMA (0.24 mg conjugate/mL, 0.02 mg enzyme/mL) was determined by measuring turbidity and the number-average hydrodynamic diameter (Dh) of chymotrypsin particulate suspensions in organic media.
Following confirmation that CT-pDMAEMA was highly soluble at the molecular scale in acetonitrile, we next sought to examine the kinetics of chymotrypsin-catalyzed transesterification and hydrolysis of N-acetyl L-phenylalanine thiophenylester (APTE).22 In the model reaction (Scheme 1) N-acetyl Lphenylalanine propyl ester (APPE) was produced by the CTpDMAEMA-catalyzed transesterification of APTE with 1propanol in acetonitrile (AN). CT-pDMAEMA also catalyzed the hydrolysis of APTE, resulting in N-acetyl phenylalanine (AP). As a result of both the TE and hydrolysis reaction, thiophenol was liberated then subsequently detected and quantified with colorimetric analysis using 4,4′-dithiodipyridine (DTDP).23 Using this model reaction, we looked at the effect of both water and propanol concentrations on the rate of reaction (kcat) and substrate affinity (KM). Since thiophenol is a byproduct of both the transesterification and hydrolysis reactions, we used DTDP to quantify product formation. Thus, the resulting apparent kcat and KM values corresponded to the total rate of substrate consumption. We were very surprised to observe water-like KM values for the acetonitrile-soluble chymotrypsin. The kcat and KM values of enzymes in organic solvents are often many orders of magnitude lower and higher, respectively, than in aqueous buffer.24 The molecular solubility of CT-pDMAEMA conjugates enabled tighter binding of the APTE substrate, resulting in sharply lower KM values. When insoluble and in aggregate form, native enzyme does not have the same opportunity for tight substrate binding. Not surprisingly, the total turnover number (kcat) for CT-pDMAEMA-catalyzed degradation of APTE increased with increasing water content (Table 2). In addition, native chymotrypsin displayed no transesterification activity at equivalent enzyme concentrations even when increasing water content (SI Table 1). When increasing native chymotrypsin concentration 10-fold (from 0.02 mg/mL to 0.2 mg/mL), the activity of native chymotrypsin (1.3 ± 0.1 μM/ min/mg CT) was still 2 orders of magnitude below CTpDMAEMA activity (170 ± 20 μM/min/mg CT). As water content increased, the increase in CT-pDMAEMA activity was likely due to more structural flexibility. Activity was measured at water concentrations up to the APTE solubility limit. Previously, when insoluble chymotrypsin transesterification and hydrolysis activities were measured with increasing water concentrations in organic solvents, the enzyme exhibited a bellshaped activity curve.25 Conversely, as the water content increased, CT-pDMAEMA conjugates simply increased activity in acetonitrile (Table 2), although the catalytic efficiency did
Figure 1. Dependence of CT-pDMAEMA conjugate hydrodynamic diameter (Dh) on (a) water content with fixed propanol concentration (500 mM) and (b) propanol content with fixed water concentration (1000 mM). CT-pDMAEMA was dissolved in acetonitrile at a concentration of 0.24 mg/mL, and Dh was determined using dynamic light scattering at 25 °C.
included in the measurement media to mimic the solutions used in the activity experiments. Propanol was a necessary substrate in the transesterification reaction, and water content has been shown to have a large effect on enzyme activity in nonaqueous solvents.21 For each of the organic media conditions, the hydrodynamic diameter of CT-pDMAEMA was equivalent to that in aqueous conditions, indicating that CT-pDMAEMA readily dissolves at the molecular scale in acetonitrile/propanol/water mixtures. Native chymotrypsin under the same conditions formed aggregates (Table 1), as expected since the native enzyme is insoluble in acetonitrile. Even at concentrations ∼10 times higher than native chymotrypsin, CT-pDMAEMA conjugates still did not form aggregates in acetonitrile (SI Figure 1). CTpDMAEMA conjugates were also molecularly soluble in dichloromethane and more soluble than native chymotrypsin in chloroform, tetrahydrofuran, or acetone (Table 1). 494
DOI: 10.1021/acsmacrolett.6b00137 ACS Macro Lett. 2016, 5, 493−497
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ACS Macro Letters
Scheme 1. Chymotrypsin-pDMAEMA-Catalyzed Transesterification of N-Acetyl L-Phenylalanine Thiophenylester (APTE) and 1-Propanol and Subsequent Reaction of Thiophenol with 4,4′-Dithiopyridine (DTDP) to Quantify Product Formation
Table 2. Effect of Water Concentration on Apparent Michaelis−Menten Parameters for CT-pDMAEMACatalyzed Transesterification and Hydrolysis of APTEa water (mM)
propanol (mM)
500 750 1000 1250 1500 2000 5000 10000 15000
500 500 500 500 500 500 500 500 500
kcat (min−1) 3.7 8.6 16 18 21 43 46 68 74
± ± ± ± ± ± ± ± ±
0.1 0.3 0.5 0.5 0.6 1.4 0.8 1.4 0.6
KM (mM) 17 20 22 20 22 40 50 93 99
± ± ± ± ± ± ± ± ±
1 2 2 2 2 2 1 3 1
Table 3. Effect of Propanol Concentration on Apparent Michaelis−Menten Parameters for CT-pDMAEMACatalyzed Transesterification and Hydrolysis of APTEa
kcat/KM (mM−1 min−1)
water (mM)
propanol (mM)
± ± ± ± ± ± ± ± ±
1000 1000 1000 1000 1000 1000 1000 1000 1000
10 25 50 100 200 500 1000 2500 5000
0.21 0.43 0.74 0.86 0.95 1.1 0.92 0.73 0.74
0.03 0.04 0.07 0.08 0.1 0.07 0.03 0.03 0.01
a
kcat (min−1) 11 16 22 28 37 16 13 2.3 0.8
± ± ± ± ± ± ± ± ±
0.3 0.4 0.4 0.4 0.4 0.5 0.6 0.1 0.03
KM (mM) 24 25 25 32 38 22 36 31 32
± ± ± ± ± ± ± ± ±
2 1 1 2 0.4 2 4 4 2
kcat/KM (mM−1 min−1) 0.47 0.64 0.89 0.87 0.97 0.74 0.36 0.07 0.03
± ± ± ± ± ± ± ± ±
0.04 0.05 0.05 0.05 0.05 0.07 0.04 0.01 0.002
a
Michaelis−Menten parameters were calculated using DTDP to quantify product formation over time at different water concentrations in acetonitrile at 30 °C. Substrate (APTE) concentration was varied from 0 to 100 mM. DTDP reacts with both the hydrolysis and transesterification products; thus, these values describe the combined rate. Initial velocity plots are available in the Supporting Information.
Michaelis−Menten parameters were calculated using DTDP to quantify product formation over time at different propanol concentrations in acetonitrile at 30 °C. Substrate (APTE) concentration was varied from 0 to 100 mM. DTDP reacts with both the hydrolysis and transesterification products; thus, these values describe the combined rate. Initial velocity plots are available in the Supporting Information.
decrease at the highest water concentrations tested. It is likely that the dense pDMAEMA shell, generated using “graftingfrom” ATRP to surround the enzyme, protected the chymotrypsin from denaturation at the sufficiently high water concentrations. CT-pDMAEMA had a bell-shaped curve for activity, while APTE substrate affinity decreased with increasing propanol concentration (Table 3). Substrate affinity plausibly decreased as a result of the partitioning of the substrate in the polymer chains attached to surface of the enzyme. Combined with observation of increased D h at higher propanol concentrations, it is likely that the reduced activity was due to unfolding and denaturation of the CT-pDMAEMA conjugate above 500 mM propanol. In order to determine the mechanism of the reaction, further analysis was carried out with a large concentration range of propanol. Kinetic constants were determined at 10 mM, 25 mM, and 50 mM propanol (Table 3). As can be seen from the double reciprocal plot with different concentrations of propanol (Figure S4), it became clear that CT-pDMAEMA catalyzed the
reaction through a sequential mechanism. This mechanism indicates that both APTE and propanol must bind to the enzyme prior to product formation.26 The sequential mechanism is in contrast to the “ping-pong” mechanism where the enzyme releases product prior to the binding of the second substrate. We then decided to determine the effect of water and propanol concentration on the individual rates of CTpDMAEMA-catalyzed transesterification and hydrolysis. Using high pressure liquid chromatography (HPLC), the initial rates of APPE (transesterification reaction) and AP (hydrolysis reaction) product formation were separately quantified. The initial rates of the reactions were quantified using 500−1500 mM water and 500−5000 mM propanol with a constant substrate (APTE) concentration of 20 mM (Figure 2). As water content increased, the rate of both transesterification and hydrolysis increased. Conversely, as propanol concentration was increased, the activity of both reactions 495
DOI: 10.1021/acsmacrolett.6b00137 ACS Macro Lett. 2016, 5, 493−497
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ACS Macro Letters
concentrations in acetonitrile. The CT-pDMAEMA conjugate had a water-like KM that resembled that of the native enzyme. The molecular dissolution of an active enzyme in an organic solvent used in organic syntheses represents a significant step toward application of nonaqueous enzymology.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.6b00137. Experimental details, synthesis protocol for APTE and APPE, initial rate vs substrate plots for CT-pDMAEMAcatalyzed transesterification at different water and propanol concentrations, plot of CT-pDMAEMA solubility in acetonitrile, and comparative activity of chymotrypsin and CT-pDMAEMA in acetonitrile (PDF)
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Figure 2. Dependence of CT-pDMAEMA-catalyzed transesterification and hydrolysis initial rates on (a) water content (fixed propanol concentration-500 mM) and (b) propanol concentration (water content-1000 mM). Transesterification rate (open circles), hydrolysis (open triangles, left y-axis), ratio of transesterification/hydrolysis (closed squares, right y-axis). Rates of CT-pDMAEMA (0.7 μM) catalyzed transesterification and hydrolysis in acetonitrile at 30 °C were calculated by measuring product formation over time using RPHPLC.
AUTHOR INFORMATION
Corresponding Author
* E-mail:
[email protected]. Author Contributions
The manuscript was written through contribution of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge support from CIT and MCS Seed funding at Carnegie Mellon University.
decreased. Since it was possible to separately quantify product formation from each reaction, the ratio of transesterification to hydrolysis was calculated (Figure 2). At each concentration, the rate of formation of transesterification products was higher than that of hydrolysis. However, at water concentrations above 1000 mM, CT-pDMAEMA-catalyzed hydrolysis rates increased more quickly than CT-pDMAEMA-catalyzed transesterification. Thus, when varying water content, we concluded the optimum water concentration was 1000 mM. Finally, we examined the effect of propanol concentration on enzyme activity. Using the optimal water content (1000 mM), we found that a propanol concentration of 2500 mM resulted in the highest transesterification to hydrolysis ratio, although the highest activity for transesterification was at 1000 mM propanol. Consistent with results from the calorimetric assay, higher concentrations of propanol caused reduced enzyme activity. The transesterification: hydrolysis ratio is important to consider when determining which specific reaction conditions are most advantageous for biocatalysis. Even though high concentrations of water may yield the highest overall activity, much of the product results from the hydrolysis reaction, which necessitates an extra step after catalysis to remove unwanted side products. In conclusion, we synthesized a CT-pDMAEMA conjugate that showed dramatically increased activity and molecular dissolution in acetonitrile. While other strategies have yielded high enzyme activity, those approaches did not enjoy the benefits of molecular dissolution of chymotrypsin in acetonitrile. Rates for CT-pDMAEMA-catalyzed transesterification and hydrolysis were proportional to water concentration and inversely proportional to propanol concentration. Native CT had no detectable activity at equivalent enzyme and substrate
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REFERENCES
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