Alleviated Inhibition of Single Enzyme in Confined and Crowded

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Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

Alleviated Inhibition of Single Enzyme in Confined and Crowded Environment Yanjing Gao, Xiaoguo Liu, Lele Sun, Yan Xu, Sichun Yang, Chunhai Fan, and Di Li J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b03736 • Publication Date (Web): 19 Dec 2018 Downloaded from http://pubs.acs.org on December 20, 2018

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The Journal of Physical Chemistry Letters

Alleviated Inhibition of Single Enzyme in Confined and Crowded Environment Yanjing Gao,† ‡ Xiaoguo Liu,¶ Lele Sun,† ‡ Yan Xu,† # Sichun Yang, Chunhai Fan† ¶ and Di Li*†§ ⊥

† Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation Facility, Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China § School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, China ¶ School of Chemistry and Chemical Engineering, and Institute of Molecular Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China ⊥

Center for Proteomics and Department of Nutrition, Case Western Reserve University, Cleveland, OH 4106, USA

# National Engineering Research Center for Nanotechnology, Shanghai 200241, China AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

ACS Paragon Plus Environment

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Abstract: Most proteins perform functions in intracellular milieu. The crowding, compartmentalized cytosol environment affects the protein structure, folding, conformational stability, substrate diffusion and substrate-enzyme binding. Moreover, enzymes are available at single or very low copy numbers in a cell, thus the conformation fluctuations of single enzyme in crowding environment could also greatly influence its kinetics. However, the crowding effect is poorly understood in kinetical aspect of enzymatic reactions. In the present study, individual horseradish peroxidase (HRP) is encapsulated in a liposome containing crowding reagents as mimics of viscous cytosol. The confined crowding environment possesses a profound influence on both catalytic activity and product inhibition of enzymes. By analysing the correlation between product generation and product inhibition, we find the allosteric noncompetitive inhibition of HRP is alleviated in the crowded and confined milieu. Small-angle X-ray scattering experiments provide straightforward proofs of structural changes of enzymes in crowding environments, which is responsible for the reduced enzyme activity and increased enzymesubstrate affinity. We expect this work may deepen the understanding of correlations between enzymatic conformations and activity performance in real cellular environments.

TOC GRAPHICS

KEYWORDS: Single enzyme, Enzyme activity, Allosteric inhibition, SAXS


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Enzymes usually perform their biological functions in volume (cytoplasm)- or surface (membrane)-confined environment.1 Particularly, cytosol is a highly crowded milieu composed of concentrated proteins and nucleic acids.2 The macromolecular crowding decreases the diffusive of substrate and product, limits the degree of freedom of enzyme conformation fluctuation, or shifts the equilibrium of protein–protein association and of protein–substrate interaction.2-5 Synthetic crowding reagents are commonly used to mimic cytosolic conditions to investigate crowding effect on enzymes.6-9 Various crowding effects including excluded volume effects, specific interactions between biomolecules and other solutes or solvents, physical and chemical heterogeneity in the reaction medium, were found to have great influence on various properties of proteins.10-15 For example, osmolytes and related molecules were found to be able to regulate the stabilities of protein-protein complex to organize the cellular interior.16 However, kinetical aspect of enzymes in crowding environment are largely ignored and poorly understood.17 Enzymatic kinetics are closely correlated with conformational fluctuations, while crowding can alter the kinetics by favoring certain protein conformations over others.12, 18-24 Therefore, the kinetics in crowding environments may be different from classical enzymatic assay operated in diluted buffer solutions. Moreover, reaction networks inside living cell involved numerous enzymes are always at small copy numbers, where the conformation fluctuations could thus be manifested and have potential physiological consequences.25-26 However, enzyme molecules are not synchronized with each other in an ensemble-averaged kinetic measurement. Recent advances in single molecule enzymology indicated that enzymes suffer a conformation fluctuation during enzymatic catalysis, which further complicates the dynamic interaction of crowders with enzymes.27-33 Furthermore, recent studies indicated the spatiotemporal activation


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profiles of the same signaling proteins (for example, kinase) result in different gene activation patterns and diverse physiological responses.34-35 Therefore, the kinetical aspect of single enzyme in cytosol environment could greatly affect the kinetics of signaling pathway.11, 18 In the present study, we investigate the enzymatic kinetics of a single allosteric enzyme, horseradish peroxidase (HRP), inside a liposome containing crowding reagents as mimics of cytosol.36 By analyzing the correlation between the active state of enzyme and its inhibition state, we found the allosteric inhibition of HRP by its product is alleviated in the crowded and confined liposome environment. Specially, small-angle X-ray scattering (SAXS) results revealed a more compact HRP conformation with its catalytic site exposes slightly outward in crowded environments. This study establishes a direct connection between enzymatic kinetics and crowding effect that correlates to structural fluctuations. This work represents an advance in crowding effects and opens an unexplored field of influential of environmental factors on enzymatic kinetics. Liposomes have been used extensively as nanocontainers for enzymes in diverse biomedical applications.37 Herein, HRP, an intensively studied allosteric enzyme,36,38,39 was encapsulated in a liposome with a diameter of 100 nm (Figure S1). To mimic viscous cytosol environment, various concentrations of PEG or Ficoll were introduced as crowding reagents to coencapsulated with HRP. The as-prepared liposome was then tethered on a supported lipid bilayer on glass slide via biotin-streptavidin interactions (Figure 1A, and Figure S2). The number of HRP molecules in one liposome was estimated from photobleaching experiments (Figure S3). Under optimized conditions, the encapsulation of HRP was governed by Poisson distribution, an average occupancy was calculated to be 0.73 molecules per HRP-PBS containing liposome and 0.74


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molecules per HRP-PEG containing liposome, suggesting most liposomes trapped single enzyme.

Figure 1. Catalytic reaction of single HRP in macromolecule crowder containing liposomes. (A) Schematic illustration of the strategy of studying enzymatic kinetics of single HRP in crowded liposomes. PEG molecules were introduced as crowding reagents to mimic the viscous cytosol environment. (B, C) The presence of fluorescent spots indicated enzymatic reactions of individual HRP-PBS-containing liposomes (B) and HRP-PEG-containing liposomes (C). (D) The time trace of fluorescence intensity from individual liposomes. Following an initial rapid rise, the fluorescence intensity reached a plateau within a few seconds, and the fluorescence intensity fluctuation is inevitable.

Particularly, HRP catalyzes the oxidation of a nonfluorescent substrate Amplex Red (10acetyl-3,7-dihydroxyphenoxazine) into a fluorescent product resorufin (7-hydroxy-3Hphenoxazin-3-one) in the presence of H2O2. Amplex Red is an uncharged molecule that can passively diffuse across the lipid bilayer walls, while the negatively fluorescent product resorufin cannot cross the phospholipid bilayer at neutral pH, leading to their accumulation in the


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liposome.39 Moreover, H2O2 can also freely diffuse through phospholipid bilayer. Therefore, product generation by individual enzyme could be determined by monitoring the increase of fluorescence upon the addition of Amplex Red and H2O2 (Figure 1B and C).39-40 Surprisingly, instead of continuous increasing, the fluorescence intensity of the HRP-containing liposome reaches a plateau (Figure1D). This phenonium was previously explained as enzymatic product resorufin is a noncompetitive inhibitor of HRP.39 Thereby, the allosteric site of HRP becomes increasingly occupied, gradually reduces the enzymatic reaction rate to a value close to zero, resulting in a plateau in fluorescence intensity.41-42 In order to further confirm the product inhibition, we also performed a control enzymatic experiment with an HRP-mimicking DNAzyme.43-45 In contrast to HRP containing liposomes, the fluorescence intensity of DNAzyme-containing liposomes kept increasing and did not achieve a saturation plateau even after a long enough time interval (1h) (Figure S4). Since crystal structure of DNAzyme indicates only a catalytic site, this control experiment further verifies the plateau in HRP experiment is a result of product inhibition. Of note, possible contribution from photooxidation of substrates and photobleaching of resorufin were ruled out by control experiments (Figure S5 and S6).46 The initial reaction rate (V0), i.e. initial slope of the fluorescence time traces in Figure 1D, thus represents the rate of product generation, showing a clear dependence on the concentrations of substrates and encapsulated enzymes (Figure S7). The plateau fluorescence intensity (Imax) in Figure 1D corresponds to the number of product molecules that fully inhibit the enzyme.34 These two unrelated parameters (V0 and Imax), however, were found to be evidently correlated with each other in each enzyme and over long times, indicating the two structurally distinct sites, active site and allosteric inhibitory site,47 are fluctuating in a coordinated fashion.39 A kinetic model describing the allosteric inhibition was derived as39:


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P(t)=(2E0kc/kI)1/2tanh[(0.5E0kckI)1/2t]

(1)

where P(t) is the time dependence of product concentration within a liposome, E0 is the total number of enzyme molecules per liposome; kc represents the catalytic rate of enzyme, which could be presented by initial reaction rate; kI was the product binding rate at the inhibitory site, which could be presented by the plateau level (Imax). Inspired by this, we aimed to probe the allosteric inhibition, or allosteric structural fluctuations of HRP in crowded and confined liposome by analyzing the Pearson correlation between lg(V0) and Imax. Briefly, PEG molecules of various molecular weights (PEG200, PEG2000, PEG6000) with a mass fraction of 10% were coencapsulated with HRP in liposomes to mimic crowding environments. Time traces of fluorescence intensity from individual liposomes were collected (Figure 2A), and the initial slope and plateau intensity of each time traces were plotted in Figure 2B. Interestingly, the initial rates (V0) in crowding environments were retarded compared with in buffer solution (Figure 2B, top panel). We counted the fluorescence time races of randomly selected 400 HRP-loading liposomes containing either PBS buffer or PEG crowders (PEG200, 2000, 6000) and extracted the reaction time (τ, defined as the time where fluorescence intensity keeps increasing before reaching plateau). The statistic distribution in Figure 2C also indicated a prolonged reaction time in the presence of PEG. We next theoretically calculated the diffusion coefficient (DS) and diffusion velocity constant (k) of Amplex Red in this system and found that substrate diffusion is not the rate limiting step for the confined enzymatic reaction (for details, see Part 4 in supporting information). Therefore, the slowed initial reaction rate (V0) and prolonged enzymatic reaction time (τ) were attributed to a reduced enzyme activity in the confined crowding environment instead of decreased substrate diffusion. In addition, the plateau intensities (Imax) in crowding environments increased together with a broader distribution (Figure


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2B, right panel). Although the emission wavelength of resorufin showed a red shift in different crowding solutions (Figure S8), its fluorescence reveal highly consistent quantum yield (Figure S9), possible different photons emitted in different environments was thereby excluded.48 Furthermore, the solution pH was not altered by crowding agents (Figure S9), thus, the red shifted plateau intensities indicated more products are generated in crowding liposomes. As the reaction rate was retarded, reasonable explanation for the improved product generation could be alleviated product inhibition. To evaluate the correlation between product generation and product inhibition in different environments, we calculated the Pearson correlation between initial reaction rate (lg(V0)) and plateau intensity (Imax). The Pearson correlation coefficients in PBS, PEG200, PEG2000 and PEG6000 were calculated to be 0.342, 0.244, 0.190 and 0.032, respectively, with 2-tailed significant tests, 95% confidence intervals in SPSS software (Figure 2D).49-50 Pearson correlation coefficients between 0.36 and 0.67 indicates a moderate correlation, between 0.21 and 0.35 indicates a weak correlation, while a value below 0.20 suggests an extremely weak correlation or no correlation. The present Pearson correlation analysis indeed indicated a correlation between the structural fluctuations of active and allosteric site of HRP in buffer solution, while the correlation is weakened in PEG environments. To further evaluate the crowding effect on enzymatic inhibition, we also introduced another macromolecule crowder Ficoll to replace PEG and analyzed the enzymatic behavior of single HRP in Ficoll containing confined liposome (Figure S10).8 The obtained Pearson correlation coefficient of HRP in Ficoll containing confined liposome was calculated to be 0.019, even lower than in PEG 6000 (Figure 2D). This further confirmed that the inhibition of HRP was alleviated in the crowding liposomes.


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Figure 2. Fittings and analysis of fluorescent time traces from individual liposomes (A) Repetitive fittings of time traces of three randomly selected fluorescent spots. (B) Scatters distribution of lg(V0) and Imax from each fluorescent time trace. Top panel: distributions of lg(V0), right panel: distributions of Imax. Red and blue color indicated the data obtained from HRP-PBS containing liposomes and HRP-PEG containing liposomes, respectively. Distributions of reaction time (τ) in HRP-PBS containing liposomes and HRP-crowder containing liposomes (crowders are varying molecular weight for PEG with a mass fraction of 10%: PEG200, PEG2000, PEG6000). The reaction time were calculated from the time traces in 400 liposomes. (D) The Pearson correlation between lg(V0) and Imax in various crowder-containing liposomes.

We then asked what is the behind mechanism of the alleviated product inhibition in the crowding liposomes. We first verified that resorufin is still a noncompetitive inhibitor of HRP in crowding environment (containing different molecules (PEG200, PEG2000, PEG6000 and Ficoll) with a mass fraction of 10%) through the parallel Eadie-Hofstee plots (Figure 3A, Figure S11 and S12a).51 From bulk enzymology experiments, we found the Michaelis-Menten constant


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(KM) decreased in the PEG-containing bulk solution, indicating the affinity of Amplex Red towards HRP is increased in crowding environment. In addition, kcat and the enzymatic activity (kcat/KM) also declined in the PEG containing bulk solution with increase of PEG viscosity (Table 1 and Table S2). The reduced enzyme activity could be responsible for the lowered initial reaction rate in crowder contaning liposomes. We thus proposed that, the enzyme activity was strongly dependent on crowding environment regardless of in homogenous solutions or confined liposomes. We next investigated if the retarded inhibition in crowding environments also existed in bulk solutions, as we observed at single molecule level in liposomes. We pre-mixed different concentrations (0 µM, 5 µM and 10 µM) of resorufin inhibitor with HRP and performed the enzymatic assay in PBS and PEG-containing bulk solutions, respectively. Interestingly, florescent intensities of catalytic generated resorufin are all decreased (Igenerated=Itotal-Ipre-mixed. Igenerated, Itotal and Ipre-mixed presents the intensity of resorufin generated from catalytic reaction, the total intensity recorded by fluorescence spectrophotometer and the intensity of pre-mixed resorufin, respectively) (Figure 3B). However, at the end of the enzymatic reaction, there was no significant difference of the florescence intensity of resorufin over different PEG molecules (Figure 3C and Figure S12b). These results indicated that although HRP possesses different initial reaction rate under various crowding conditions (Figure S13), equal quantity of product is generated at the end of reaction regardless of solution viscosity. We hypothesized that, in homogenous bulk solutions, once product generated, it could diffuse away from HRP surface, thus the activity of HRP would then be less-effectively inhibited. Therefore, after a long enough time interval, equal substrate is converted to an equivalent product regardless of various reaction environments.

While

in

confined

and

crowded

liposomes,

generated

resorufin

is


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compartmentalized in the liposomes, thereby the accumulated product resorufin is confined on the surface of HRP and could not diffuse away. As a result, the alleviated product inhibition is a synergy of both confined and crowded environments.

Figure 3. Allosteric inhibition of HRP in crowder containing bulk solutions. (A) Eadie-Hofstee plots of enzyme activity kinetic measurements in different reaction buffer. Each initial rate (V0) was obtained under different concentrations of H2O2. Each line was obtained with pre-mixed resorufin (red line: 0 μM, blue line: 5 μM, yellow line: 10 μM). (B) Histograms of the florescent intensity of resorufin generated from enzymatic reaction in different reaction buffer. NR presented the normalized florescent intensity of resorufin. (C) Box plots of the final florescent intensity of resorufin generated from enzymatic reaction with various concentrations of resorufin pre-mixed (upper: 0 μM, middle: 5 μM, bottom: 10 μM). NR presented the normalized florescent intensity of resorufin. Colors indicated enzymatic reaction performed in different buffer. P >> 0.05 of t test indicated no significant difference of the generated resorufin under various buffer conditions, independent with the concentration of the pre-mixed resorufin.


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Table 1. Michaelis-Menten constants of HRP in different media. KM (μM) kcat0 (s-1) kcat1 (s-1) kcat2 (s-1) kcat0/KM (μM-1 s-1) PBS 7.83±0.53 178.6 94.0 45.4 22.8 PEG 200 4.43±0.68 68.2 42.6 21.2 15.4 PEG 2000 4.48±0.23 42.6 27.6 17.0 9.5 PEG 6000 4.22±0.31 27.0 21.0 11.6 6.4 # KM is the Michaelis-Menten constant. kcat0, kcat0, kcat0 are the catalytic number of HRP when the concentration of pre-mixed resorufin are 0 μM, 5 μM, 10 μM, respectively.

Now, we are aware that the confined crowding environment possesses a profound influence on both catalytic activity and product inhibition of enzymes. We then wondered if this new-found crowding effect is correlated with the structural changes of enzymes in crowding environments. We thereby performed synchrotron small-angle X-ray scattering (SAXS) measurements to investigate the global configuration of HRP in different crowding environments.52-54 The scattering profiles of HRP with gradient concentrations (2.5 mg/mL, 5 mg/mL, 10 mg/mL) are well overlapped with each other, indicating the absence of aggregation, interparticle interference and radiation damage (Figure S14). A higher HRP concentration (10 mg/mL) resulted in stronger signal-to-noise ratios, especially in the high-q range, which was selected as the optimized condition to conduct the following experiments (HRP in PBS, PEG200, PEG2000 and PEG6000) (Figure 4A). We performed Guinier analysis of the scattering profiles at the low q-range to determine the radius of gyration (Rg) for HRP molecules, calculated the pair distance distribution function P(r), and the excluded volume of hydrated particle solution (Porod volume (VP)) (Table 2 and Table S1).55 The P(r) function of HRP in PBS, PEG2000 and PEG6000 exhibited typical Gaussian distributions, indicating a compact globular structure (Figure 4B). The Rg of HRP was 23.4 ± 0.5 Å in PBS buffer, close to theoretical value of HRP (Rg = 22.0 Å). The Rg, maximum


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particle dimension (Dmax), and Vp of HRP in PEG2000 and PEG6000 decreased with the increase of PEG molecular weight, which suggested that HRP was compressed into a more compact state by surrounding crowders. It should be noted that, for PEG200, a significantly larger slope at lowq range was observed (Figure 4A), as well as the greater relevant parameters (Table 2). These suggested an obvious overall structural change of HRP monomers. Ab initial structure determination by DAMMIN showed a globular particle with a tail (Figure S15), most likely that some PEG molecules adhere to one end of HRP, since PEG200 is a widely used surface modifier56. Our data (Table 1) captured the possible attachments of PEG200 had little affection on the catalytic abilities and conformation of HPR. However, larger PEG molecules served exclusively as crowding agents. The optimized pdb model fits of PBS, PEG2000 and PEG6000 well agreed with experimental profile, with χ2 of 1.36, 3.14 and 2.05 (Figure 4C). The separate presentation of HRP ribbon models as reconstructed using CRYSOL and their superposition envelopes provided a clear structural comparison (Figure 4D and E, Figure S16), suggesting certain crowding environment (PEG6000) force HRP to exhibit a more compact structure and expose its heme site, which may be responsible for the reduced enzyme activity and increased enzyme-substrate affinity. Since SAXS detection could only interrogate the molecular conformation in nanoscale, the changes of allosteric inhibition site of resorufin to HRP could not be resolved.


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Figure 4. SAXS measurements for HRP in different conditions. SAXS measurements for HRP in different conditions. (A) Double-logarithmic representation of the averaged scattering profiles of HRP (10 mg/mL) in different media. For clarity, plots were shifted along the I(q) axis. The slopes at low-q range were -846.0 ± 41.2 (PBS), -1262.2 ± 39.1 (PEG 200), -611.5 ± 50.0 (PEG 2000) and -471.1 ± 47.3 (PEG 6000), respectively. (B) The pair distance distribution P(r) functions for HRP in different solutions, based on the analysis of experimental SAXS data by applying DAMMIF program. (C) The optimized model fits of HRP by applying Normal Mode refinements in various solutions. The fitting lines (yellow solid lines) were the theoretical curves calculated from the crystal structure of HRP. (D) The ribbon models of HRP as reconstructed using CRYSOL by applying Normal Mode refinements. Ribbon models of HRP in PBS and PEG6000 was shown in tan and blue, respectively. Heme sites were marked with red circles. (E) Overlapping ribbon models of HRP in PBS and PEG6000.


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Table 2. SAXS measurements of the structural parameters of HRP under different conditions Rg Dmax Porod volume PBS 23.4±0.5 69.4 68863 PEG 200 34.7±0.3 138.0 78807 PEG 2000 22.8±0.6 67.8 62889 PEG 6000 21.8±0.7 66.5 61012

Enzyme functions are closely related to structures. Previous work indicated that aromatic substrate can enter into the amino acid residues formed substrate-binding pocket and be oxidized at the exposed heme edge.38 The conformation of the access channel with a flanking entry point, narrow and deep inside the globular HRP, determines the accessibility of substrates to the active site. Thus, a more compact structure would reduce the substrate entrance ability.57 However, the exposed heme site in crowding environment can facilitate the binding probability of substrate to HRP, which explains the decreased KM. In addition, a hydrophobic surrounding of active site will have a positive impaction of the affinity of HRP towards aromatic substrates.22, 57-58 In the present study, the exposed heme site and the hydrophobic surrounding predominated increases enzymesubstrate affinity in crowding environment. In summary, macromolecular crowding has been found to influence enzyme folding, enzymesubstrate interaction. In the present study, we demonstrated the first example of single enzyme in crowding and confined liposome as a mimic of viscous cytosol and exploited the largely ignored kinetical aspect of enzymes in crowding environment. By analyzing the Pearson correlation coefficients of product generation and product inhibition, we found the noncompetitive enzymatic inhibition is alleviated owing to the synergy contribution from both confined and crowded environmental factors. Specially, the current SAXS experiments provided straightforward proofs of structural changes of enzymes in crowding environments. Along with


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rapid development of DNA nanotechnology59,60, the merging of enzymes and DNA nanostructure may make it convenient and time saving to obtain the kinetical behavior of single enzymes. These findings are also closely related to understanding how enzymes work in cytosol environment. It will be interesting to see how the kinetical fluctuates of single enzyme in cytosol affect the universal dynamical output of signal pathways. Materials and methods Materials Horseradish peroxidase (HRP,EC 1.11.1.7), Poly(ethylene glycol) (MW: 200, 2000, 6000, 10000), Streptavidin (lyophilized powder), Ficoll (MW: 400 kD), Hemin, Amplex Red, Resorufin, hydrogen peroxide (30% solution), 1×PBS buffer (135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4, pH 7.4) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and prepared with deionized water. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(capbiotinyl)

(Biotin-PE),

1,2-

dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Alexa Fluor 647 NHS Ester (Alexa647) was purchased from Molecular Probes (Eugene, OR, USA). DNA strands were synthesized by Sangon Biotech Co. (China). Preparation of liposomes. To prepare small unilaminar liposomes (SULs). DOPC and BiotinPE were mixed with an optimized mass ratio of 100:1.6 in chloroform in a 5 mL glass vial. Fluorescent lipids (NBD-PE) were added with desired molar ratio. Then chloroform was thoroughly dried under a stream of N2 and stored in vacuum for 3 h. SULs, used for preparing supported lipid bilayers (SLB), were made by hydrating the lipid film with 1×PBS buffer to


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achieve a final lipid concentration of 5 mg/mL, then agitated for half an hour with a magnetic stirrer and extruded repetitively through a 100 nm polycarbonate (PC) membrane. The average diameter of SULs was measured by dynamic light scattering (DLS, Malvern, Nano-ZS90). To prepare enzyme containing liposomes, 1 µM HRP solution (prepared in 1×PBS, or 10% w/w PEG 200 (PEG2000, PEG6000, Ficoll)) was used to hydrate the lipid film. Untrapped enzymes were removed by three times of dialysis (12 h/per time) through cellulose ester dialysis membranes under 4 °C with 1×PBS (pH 7.4) as dialysate. Tethering of enzyme-containing liposomes on SLB. Supported lipid bilayers was formed by incubating 50 µL of SUL solution to a homemade sample chamber for 30 min, excess unfused SULs were removed by thoroughly rinsing with deionized water. HRP-containing liposomes were tethered on SLB via biotin-streptavidin interaction (Figure S2). The strategies of tethering DNAzyme-containing liposomes, HRP-PEG-containing liposomes and HRP-Ficoll-containing liposomes on SLB were the same as described above. TIRFM imaging of single enzyme reaction in liposomes. TIRFM measurements were conducted on a commercial total internal reflection fluorescence microscopy (TIRFM, N-storm, Nikon) with a high numerical aperture oil immersion 100× objective lens (NA 1.49) and electron multiplying charge-coupled devices (EMCCD) camera (Andor, iXon 3). The temporal florescence signal of single enzyme reaction from immobilized liposome was obtained under 561 nm laser irradiation after introducing 50 µL substrate mixture of Amplex Red (10 µM) and H2O2 (100 µM) immediately to the sample chamber. The acquired videos were analyzed using a homewritten program, which extracted the fluorescence intensity trajectories from localized fluorescence spots individually across the entire video.


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Bulk enzymology experiments. HRP activity was measured by following the H2O2-dependent oxidation

of

Amplex

Red

using

the

fluorescence

spectrophotometer

(Cary-100

spectrophotometer, ex: 532 nm, em: 585 nm) at room temperature. The reaction mixtures containing 0.5 nM HRP, 100 µM Amplex Red and various concentrations of H2O2 were prepared immediately in a quartz cuvette and placed into the spectrophotometer to monitor the timeresolved fluorescence immediately. For inhibition experiments, HRP should be pre-incubated with resorufin (0 µM, 5 µM, 10 µM) for 5 min at room temperature. The reaction buffer was 1×PBS, PEG 200 10% (w/w), PEG 2000 10% (w/w), PEG 6000 10% (w/w) or Ficoll 10% (w/w), respectively. The concentration of resorufin was determined by calibrating the fluorescence intensity with resorufin standard curves. SAXS measurements. Small-angle X-ray scattering (SAXS) detection was performed on beamline BL19U2 of the National Center for Protein Science Shanghai (NCPSS) at Shanghai Synchrotron Radiation Facility (SSRF)3. The sample-to-detector distance was 2125.0 mm for the current measurements, it was set such that the detecting range of momentum transfer q (q = 4πsinθ/λ, where 2θ is the scattering angle) of the SAXS experiments was 0.01 - 0.5 Å-1. 100 µL of HRP solutions with increasing gradient concentrations (2.5 mg/ml, 5 mg/ml, 10 mg/ml) were prepared in 1×PBS buffer. Each sample was exposed to X-rays while flowing through the 1.5 mm-diameter quartz capillary using the automated sample loading system, and the exposure time was set at 1 s. 20 frames were recorded of each sample and buffer to obtain the optimized signalto-noise ratios. The same test was carried out for HRP (10 mg/mL) dispersed in various solutions (PEG 200 10% w/w, PEG 2000 10% w/w, PEG 6000 10% w/w). BioXTAS RAW 1.2.1, ATSAS 2.7.2 package, Crysol, ab initio shape determination program DAMMIF were used for data processing and model reconstruction.


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ASSOCIATED CONTENT Supporting Information. Experimental section, all nucleic acid sequences, data availability, and theoretical derivation. AUTHOR INFORMATION ORCID Sichun Yang: 0000-0002-1726-0576 Chunhai Fan: 0000-0002-7171-7338 Di Li: 0000-0003-1674-0110

Author Contributions Y. G. and X. L. contributed equally to this work. Notes The authors declare no competing financial interests. ACKNOWLEDGMENTS This work was supported by National Key R&D Program of China (2016YFA0201200, 2016YFA0400900), NSFC (21675166, 21603262, 21390414, 21329501), Key Research Program of Frontier Sciences, CAS (QYZDJ-SSW-SLH031) and Shanghai Rising-Star Program (17QB1402900). The authors thank the staffs from BL19U2 beamline at Shanghai Synchrotron Radiation Facility (SSRF), for assistance during data collection.


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Alleviated Inhibition of Single Enzyme in Confined and Crowded

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