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Letter
Construction of Smart Glutathione S-Transferase via Remote Optically-Controlled Supramolecular Switches. Yao Liu, Tiezheng Pan, Yu Fang, Ningning Ma, Shanpeng Qiao, Linlu Zhao, Ruidi Wang, Tingting Wang, Xiumei Li, Xiaojia Jiang, Fangzhong Shen, Quan Luo, and Junqiu Liu ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b02821 • Publication Date (Web): 15 Sep 2017 Downloaded from http://pubs.acs.org on September 15, 2017
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Construction of Smart Glutathione S-Transferase via Remote Optically-Controlled Supramolecular Switches. Yao Liu,† Tiezheng Pan,†, ‡ Yu Fang,† Ningning Ma,† Shanpeng Qiao,† Linlu Zhao,† Ruidi Wang,† Tingting Wang,† Xiumei Li,† Xiaojia Jiang,† Fangzhong Shen,† Quan Luo,*, † and Junqiu Liu† †
State Key Laboratory of Supramolecular Structure and Materials, Institute of Theoretical Chemistry, Jilin University, 2699 Qianjin Road, Changchun, 130012, China. ‡
School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi'an, 710072, China.
ABSTRACT: A supramolecular switch strategy that can reversibly “turn-on” and “turn-off” glutathione S-transferase (GST) is presented, which provides a proof-of-concept for a simple but efficient way to regulate the catalytic function of natural enzymes. This design is demonstrated by incorporating azobenzene/cyclodextrin-based supramolecular host-guest systems into the catalytic pocket of GST. The photoisomerization of trans and cis azobenzene leads to supramolecular complexation and dissociation of cyclodextrin, and thereby controls the enzymatic activity of GST by tuning substrate accessibility. This photo-switchable catalysis is reversible over multiple stimulus cycles. Furthermore, its capability is affected by the spatial size and binding affinity of different cyclodextrins, as well as the modification sites of azobenzene. The remote optical modulation method could offer great opportunities in the effort to create ‘smart’ catalysts. KEYWORDS: Azobenzene, cyclodextrin, glutathione S-transferase, switchable catalyst, supramolecular interaction. Enzymes are strictly regulated by sophisticated feedback loops and a variety of trigger-induced effects to control numerous biotransformations under physiological circumstances. Following Nature’s inspiration of enzyme regulation, the design of switchable man-made catalysts may bring precise control to biochemical reactions for biomedical applications. Typically, a variety of organocatalysts that contain stimuli-responsive functional groups such as azobenzene, spiropyrans, diarylethenes have been developed with switchable activity.1 Many ligands have also been utilized to regulate the catalytic activities of supramolecular catalysts based on ligand bindingdependent allosteric control.2 Recent advances have showed that the engineered allosteric proteins can serve as a promising scaffold for the development of proteinbased catalysts with both high efficiency and functional switches.3 These ‘smart’ catalysts exhibit excellent controllability and adaptivity through precise tuning of the active site to switch the chemo-, regio-, and stereoselectivities of reactant or product, which would expand the scope of applicable reactions for cellular physiology.1e,2g,3c The emerging field of switchable catalysts is growing rapidly toward the final goal of “bio-like” level of control over chemical reactions.4
Scheme 1. Design of light-responsive GST based on AzoCD supramolecular switches. The structures of GST, αCD, and β-CD obtained from the crystal data (PDB ID: 1Y6E, 3L2M, and 5E6Z); The structure of Azo-Maleimide built by GaussView and optimized by Gauusian 09; The open/closed states of GST were visualized in PyMOL.
Among the above-mentioned strategies to prepare intelligent catalysts, supramolecular manipulation is quite suitable to realize the “switch” due to its dynamically reversible nature.5 For instance, rotaxane architectures, cavitand/piperidinium complex and cucurbituril-based supramolecular complexes were constructed to reversibly turn on and off the enzyme activities.6 Inspired by the development of these supramolecular molecule-based
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switchable catalysts, the combination of supramolecular switches with natural enzymes has become a powerful method to achieve a higher level of enzyme regulation. Very recently, Brunsveld et al. pioneered the use of synthetic cucurbit[8]uril for supramolecular control of luciferase activity with a 20-fold fluctuation.7 Natural enzymes have been evolved to work with an astonishing velocity due to the synergistic effect between the catalytically reactive site and favorable substrate-binding pockets.8 In this regard, highly controlled and efficient catalysts could be designed by combining a supramolecular switch with enzyme molecules. Herein, a photo-switchable catalyst was constructed by using supramolecular host-guest interactions between cyclodextrins (CDs) and a maleimide-modified azobenzene derivative (Azo-MAM) to control the catalytic activity of GST (Scheme 1). In this design, the trans-Azo-MAMCD complexes were incorporated into the entrance of catalytic cavity of GST, which is a well-known detoxification enzyme with the ability to catalyze the xenobiotics into more hydrophilic and less toxic polypeptide derivatives at the expense of glutathione (GSH) (Figure 1A).9 The “click” reaction was performed to covalently attach the maleimide group of Azo-MAM to the thiol groups of the site-specifically engineered cysteines on protein surface. Due to the isomerization of trans-Azo-MAM under UV light (350 nm) irradiation, the Azo-MAM-CD complexes will dissociate to release CDs, which allows the substrate-binding site of GST to be accessible for GSH. Upon visible light (420 nm) irradiation, cis to trans isomerization of Azo-MAM lead to the reformation of the inclusion complex. This supramolecular complexation process is completely reversible and can provide a remotely controlled capability to directly manipulate the biological function of GST via light-driven transition between an original ‘closed’ state and a dissociated ‘open’ state. As shown in Figure S1, GST crystallizes as a dimer and two identical catalytic sites are located at the bottom of a intersubunit cleft, which contains several critical residues (e.g. W40, K44, N53, Q66, and D100) to stabilize the substrate GSH.10 In order to avoid unwanted reactions, all the cysteines of GST were replaced by Ser or His (C84S, C137S, C168H, C177S), then site-directed mutagenesis was performed to introduce two mutations (L117C and K124C) onto the outer edge of the GSH-binding cleft of GST for selective modification with Azo-MAM (Figure S2). 1H NMR spectroscopy and ESI-MS data have confirmed the successful synthesis of Azo-MAM (Figure S4 and S5), which displays an obvious conformational transition upon UV-vis light and similar dynamics with azobenzene (Figure S6). The bioconjugations between Azo-MAM and two mutated cysteine sites of GST were demonstrated by MALDI-TOF-MS (Figure S8). Furthermore, circular dichroism analysis showed that both mutations and chemical modifications have no significant influence on the secondary structure of GST (Figure S9), revealing its structural integrity to maintain high activity. In order to
Figure 1. A) The enzymatic reaction catalyzed by GST. B) Catalytic curves of the blank test without GST (black), the “closed” Azo-MAM-GST/L117C/K124C-α-CD (red) and the “open” Azo-MAM-GST/L117C/K124C-α-CD (blue). C) Absorption spectra of Azo-MAM-GST/L117C/K124C-α-CD under UV (blue) and visible (red) irradiation. The activity assay was conducted in 100 mM PBS buffer (pH=6.5), 2.98 µM Azo-MAM-GST/L117C/K124C-α-CD, 1 mM GSH and 1 o mM CDNB at 25 C.
achieve high “click” reaction efficiency, CD molecules were used to improve the water solubility of Azo-MAM. It is also intended that the formation of Azo-MAM-CD complexes could cooperatively and precisely prevent the substrate approaching the catalytic site of GST for reversible control of its enzymatic activity (Figure S2). The switchable activity of Azo-MAM-modified GST was evaluated by monitoring the UV absorbance of GSDNB (Δε=9.6 mM-1·cm-1) at 340 nm using α- and β-CD for functional regulation.11 Conditions for spectrophotometric activity assays were listed in supporting information. As shown in Figure 1B, Azo-MAM-GST/L117C/K124C shows very different dynamic curves in the presence of α-CD upon UV-vis irradiation. Its activity shifts from 182.1 ± 15.7 μmol·min-1·μmol-1 to 14.1±1.6 μmol·min-1·μmol-1 with a high blocking efficiency up to 92.2% (Table 1). The ‘on/off’ photoswitching was further proven to be correlated with a typical cis-to-trans isomerization of azobenzene via UV-
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vis spectroscopy, in which a gradual increase at 343 nm and a concomitant decrease at 430 nm were observed (Figure 1C and S10). In the absence of α-CD, however, only a slight activity change (from 136.05 to 154.84 μmol·min1 ·μmol-1) was detected for Azo-MAM-GST/L117C/K124C under the same condition (Figure S13F), revealing that photo-controlled dissociation and reformation of AzoMAM-CD complex directly involve in enzyme regulation. Furthermore, the switchable activity of the double modified GST was evaluated by using β-CD as a mediator. The corresponding blocking efficiency was found to reach > 90%, with the “open” activity of 183.5 ± 16.0 μmol·min1 ·μmol-1 and the “closed” activity of 15.9 ± 1.5 μmol·min1 ·μmol-1 respectively. A similar efficiency and “open/closed” activities were measured for the single modified GST (Azo-MAM-GST/K124C) via β-CD-based dissociation and complexation (Table 1). Notably, the substitution of β-CD with α-CD caused a much lower blocking effect (~45.0%) for Azo-MAM-GST/K124C in contrast to that of the other systems. The “open” and “closed” activities were measured to be 181.7 ± 10.3 μmol·min-1·μmol-1 and 100.0±2.1 μmol·min-1·μmol-1, respectively. This may be due to the spatial size of a single α-CD which is smaller than that of β-CD and is insufficient to completely block the catalytic pocket of GST. Also, the single modified GST without CDs as gating molecules has almost no effect on its activity upon UV-vis irradiation (from 153.94 to 163.92 μmol·min-1·μmol-1; Figure S13C). Taken together, both α-CD and β-CD displayed excellent enzymatic manipulations when precise incorporation of two Azo-MAM at position 117 and 124 for supramolecular complexation. Nevertheless, the question which CD would be the better one still need to be investigated in details, including the binding reversibility, kinetic and thermodynamic processes. The reversibility of remote photo-induced activity changes of GST variants was evaluated by multiple switching cycles under UV-vis light. Using α-CD as a supramolecular switch, Azo-MAM-GST/L117C/K124C can be controlled reversibly with only minor activity loss (Figure 2A). After 5 photoswitching cycles, α-CD still maintained a high blocking efficiency (80.9 ± 2.1%) towards the double modified GST and whose “open” and “closed” activities were 144.9 ± 11.2 and 27.5 ± 2.1 µmol·min-1·μmol-1 respectively (Table 1), which were also found to be relatively
Figure 2. The catalytic curves (A) and activities (B) of AzoMAM-GST/L117C/K124C over 5 photoswitching cycles (pH=6.5, [GST] = 3.43 µM, [GSH] = 1 mM, [CDNB] = 1 mM, o T = 25 C).
steady from the second to the fifth cycle (Figure 2B and Table S1). In the case of Azo-MAM-GST/K124C, similar controllability and recyclability were observed, even though a single α-CD cannot completely turn off the activity (Figure S13A Table S1). As for β-CD, however, both the single and double modified GST exhibited a significantly reduced controllability over several switching cycles, and the blocking efficiency of β-CD was measured to be 9.9 ± 7.2 % in the fifth cycle. This may be due to β-CD that contains a larger hydrophobic cavity than α-CD and can bind the cis-Azo with a relatively low Ka value (280 M1 ) to interfere with the recycled complexation process between trans-Azo and β-CD. On the other hand, the decreased blocking effect of β-CD could be attributed to the fact that its association constant is lower than that between α-CD and azobenzene,12 hence the β-CD complexation is not as efficient as the α-CD complexation to achieve robust switching capabilities for the modified GST.
Table 1. The blocking effect of CDs for single and double modified GST.
GST variants
Azo-MAMGST/K124C Azo-MAMGST/L117C/ K124C
Blocking molecule
Activity -1 -1 (μmol·min ·μmol )
Activity after 5 cycles -1 -1 (μmol·min ·μmol ) Open state
Closed state
Blocking efficiency after 5 cycles (%)
45.0 ± 1.8
170.1 ± 3.6
111.2 ± 2.4
34.6 ± 1.5
6.4 ± 3.6
17.5 ± 3.3
90.2 ± 0.8
54.2 ± 5.1
47.2 ± 4.4
12.9 ± 3.0
69.8 ± 2.5
182.1 ± 15.7
14.1 ± 1.6
92.2 ± 0.9
144.9 ± 11.2
27.5 ± 2.1
80.9 ± 2.1
20.4 ±2.0
183.5 ± 16.0
15.9 ± 1.5
91.3 ± 1.2
70.6 ± 9.5
63.6 ± 4.6
9.9 ± 7.2
62.1 ± 6.1
Open state
Closed state
α-CD
181.7 ± 10.3
100.0 ± 2.1
β-CD
179.7 ± 7.9
α-CD β-CD
Blocking efficiency (%)
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Figure 3. ITC analysis of the complexation of double modified GST (Azo-MAM-GST/L117C/K124C) with α-CD (A) and β-CD (B). [GST] = 10 µM.
Considering the superiority of α-CD to regulate the catalytic activity of Azo-MAM-GST/L117C/K124C, the catalytic and binding properties were quantitatively analyzed for better understanding this association process. First, Lineweaver-Burk plots were obtained to calculate the kinetic parameters by varying the concentration of one substrate while keeping the other substrate at a fixed concentration. Comparison of the kcat/Km ratio showed 4.9-fold and 4.0-fold differences between open and closed states of the double modified GST for CDNB and GSH, where the increased kcat and the decreased Km after UV irradiation represent more opportunity for substrates to rebind to the open enzyme (Figure S14 and Table S2). Then, the thermodynamic parameters of the interaction mechanisms of GST variants with CDs were characterized by isothermal titration calorimetry (ITC) (Figure 3). As shown in table 2, the association constant (Ka) between Azo-MAM-GST/L117C/K124C and α-CD was determined to
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be 2.69×105 M-1 in 1:2 complexation, indicating that excess α-CDs can simultaneously include two trans-Azo-MAM moieties on GST surface. This Ka value is much higher than that between α-CD and azobenzene (~103 M-1) estimated by 1H-NMR and ITC.13 It is suggested that hydrogen bonding interactions may occur between the hydroxyl groups of α-CDs and the polar residues of GST variant (e.g. R41, K112, T116, F120, and Q206). The large negative enthalpy change (∆H) (-1.91 ± 0.21×105 J·mol-1) and entropy change (∆S) (-535.29 J·mol-1·K-1) also confirmed a significantly favorable binding and a decreased translational freedom of Azo-MAM-GST/L117C/K124C and α-CDs upon complexation. By contrast, the study of ∆H and ∆S of βCD with the double modified GST showed that the inclusion complexation is enthalpy-driven process (∆H: -2.33 ± 0.29×104 J·mol-1) with minor positive entropy (∆S: 20.81 J·mol-1·K-1) of interaction. The entropic gain may arise from the relatively loose binding between Azo-MAMGST/L117C/K124C and a larger β-CD cavity, which allows for more translational and conformational freedoms than that of α-CD inclusion complex. Therefore, the switching capability of β-CD to reversibly manipulate the catalytic activity of GST variant cannot reach the level of α-CD. On the other hand, the 1:1 stoichiometry of β-CD inclusion complex also revealed that there was only one β-CD anchored to the double modified GST (Table 2, Figure S15A and S15B). This may be due to the steric hindrance between two β-CDs when they bind to the double modified GST simultaneously. Similar differences in thermodynamic parameters were obtained for the inclusion complexation of the single modified GST by α-CD and β-CD (Table 2). In addition, the substrate-binding ability of “open” GST variants towards GSH was demonstrated by ITC experiments (Figure S15C and S15D). It is worth to note that the association constant Ka decreases by 31.8% with the increase of modification sites on GST surface, which provides an explanation for why the “open” activity of the single modified GST (170.1 ± 3.6 µmol·min-1·μmol-1) is a little higher than that of the double modified GST (144.9 ± -1 -1 11.2 µmol·min ·μmol ) after the second switching cycle (Figure 2 and Table S1). In conclusion, supramolecular switches have been demonstrated to be an efficient way to reversibly control the catalytic activity of natural GST. The Azo-MAMmodified GST exhibits excellent controllability and recyclability with the blocking efficiency up to 80.9 % after five complete switching cycles when α-CD was used as a
Table 2. The thermodynamic parameters of the inclusion complexation of GST variants and CDs. GST Variants Azo-MAMGST/L117C/K124C Azo-MAMGST/K124C
Blocking Molecule
Association -1 Constant (M )
α-CD
2.69 ± 0.66×10
β-CD α-CD
1.45 ± 0.11×10
β-CD
9.41 ± 2.06×10
Binding Stoichiometry (CD : GST)
ΔH -1 （J·mol ）
ΔS -1 -1 (J·mol ·K )
ΔG -1 (KJ·mol )
-535.29
-31.29
4
20.81
-29.45
5
-548.24
-29.35
4
59.87
-28.32
5
1.81 ± 0.16
-1.91 ± 0.21×10
1.46 ± 0.32×10
5
0.98 ± 0.09
-2.33 ± 0.29×10
5
0.90 ± 0.08
-1.93 ± 0.21×10
0.79 ± 0.06
-1.05 ± 0.11×10
4
5
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mediator to block the substrate-binding pocket of GST through remote optically-controlled host-guest complexation. Study on the catalytic and binding properties of GST variants has confirmed that the positions and numbers of chemical modification sites, the binding strength of α-CD to trans/cis-Azo-MAM, and the kinetics/thermodynamics of complexation process are crucial for the blocking effect of enzyme activity as compared with that of β-CD. This strategy is simple, but powerful for enzymatic regulation at the single-molecule level. It is intended that the construction of “smart” catalysts could realize precise spatiotemporal manipulation of biochemical reactions for providing a conceptual approach to explore the synergistic function of natural enzymes without sophisticated activations or regulations.
AUTHOR INFORMATION Corresponding Author * E-Mail:
[email protected] Notes The authors declare no competing financial interest.
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Supporting Information Experimental details including chemical synthesis, structure characterization, spectroscopy analysis, SDS-PAGE, enzyme engineering, MALDI-MS, circular dichroism, activity assays and ITC experiments. The Supporting Information is available free of charge on the ACS Publications website.
ACKNOWLEDGMENT This work was supported by the Natural Science Foundation of China (nos. 21474038, 21004028, 21234004, 21420102007, 21574056, and 91527302), the Chang Jiang Scholars Program of China, the Science Development Program of Jilin Province (nos. 20160520005JH and 20140101047JC). We also acknowledge Professor Quanshun Li for providing us with the enzyme to test the general utility of supramolecular strategy for enzyme regulation.
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