Single Molecule Studies of Allosteric Inhibition of Individual Enzyme

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Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

Single Molecule Studies of Allosteric Inhibition of Individual Enzyme on a DNA Origami Reactor Yan Xu, Yanjing Gao, Yingying Su, Lele Sun, FeiFei Xing, Chunhai Fan, and Di Li J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b02992 • Publication Date (Web): 09 Nov 2018 Downloaded from http://pubs.acs.org on November 10, 2018

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The Journal of Physical Chemistry Letters

Single Molecule Studies of Allosteric Inhibition of Individual Enzyme on a DNA Origami Reactor Yan Xu,†‡‖Yanjing Gao,‡¶Yingying Su,†§ Lele Sun, ‡¶Feifei Xing,§Chunhai Fan‡, and Di Li, *†‡

†School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, China ‡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

§Department of Chemistry, College of Science, Shanghai University, Shanghai 200444, China

‖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: Unraveling the conformational changes of enzymes together with inhibition kinetics during an enzymatic reaction has great potential in screening therapeutic candidates, however, remains challenging due to the transient nature of each intermediate step. We report our study on the non-competitive inhibition of horseradish peroxidase with single turnover resolution using single molecule fluorescence microscopy. By introducing DNA origami as an addressable nanoreactor, we observe the coexistence of nascent-formed fluorescent product on both catalytic and docking sites. We further propose a single-molecule kinetic model to reveal the interplay between products generation and non-competitive inhibition and find three distinct inhibitor releasing pathways. Moreover, kinetic isotope effect experiment indicates a strong correlation between catalytic and docking sites, suggesting an allosteric conformational change in noncompetitive inhibition. A memory effect is also observed. This work provides an in-depth understanding of the correlation between enzyme behavior and enzymatic conformational fluctuation,

substrate

conversion

and

product

releasing

pathway

and

kinetics.

TOC GRAPHICS

KEYWORDS. Enzyme inhibition, Kinetics, Single enzyme, Allosteric, Inhibition pathway, DNA origami


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Enzymes can accelerate the rate of chemical reactions with very high specificity1. Meanwhile, enzymes can also be inhibited by other molecules, for example, the end-product of a metabolic pathway. Therefore, enzymatic inhibition has been broadly regarded as a prevalence feedback for controlling cellular processes to maintain function efficiency and avoid internal extremes2-3. Particularly, non-competitive inhibitors that interact with an allosteric docking site distinct from catalytic site, are promising targets for new drug design because of alleviating undesirable sideeffects4-5. A comprehensive understanding of the inhibitor releasing pathways and kinetics could provide in-depth understandings of the correlation between enzyme conformations and catalytic activities6-8. However, owing to the transient nature of intermediate conformational states, it remains challenging for current microcopy tools to seize each intermediate state in an enzymatic reaction9. Moreover, recent studies in single-molecule enzymology indicated that the conformation of enzymes fluctuates during enzymatic catalysis, which further increases the complexity of inhibition kinetics10-12. Horseradish peroxidase (HRP), a 44 KDa monomeric metalloenzyme13, is a classical model for single-molecule enzymatic studies14-15. HRP catalyzes the oxidation of a nonfluorescent substrate Amplex Red into a fluorescent product resorufin in the presence of H2O2, which favors the observation with total internal reflection fluorescence microscope (TIRFM). Recent study indicated that HRP trapped in lipid vesicles could be non-competitively inhibited by resorufin16, suggesting the nascent-generated resorufin is an excellent probe to in situ study the conformational fluctuations of HRP. Indeed, very recent studies revealed HRP displays multiple intermediate conformational states with multiple product releasing pathways17-19. However, a comprehensive picture describing the pathway of products moving from catalytic sites to inhibition docking sites together allosteric kinetics is still unclear. TIRFM, broadly used in monitoring single


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molecule reactions10, 20-21, however, suffers difficulties in following the single enzymatic reaction catalyzed by surface immobilized HRP, because CCD of TIRFM is unable to differentiate the source of fluorescence burst. Hence, it is unable to discriminate a nascent generate fluorescent product on immobilized HRP from a random touching of fluorescent product to the glass surface21. The key issue is a short of positioning system to locate HRP in TIRFM image, which enables the monitoring of long-time trajectory of fluorescent product generation events that occurred on HRP. In the present study, we introduce DNA origami as a single-enzyme reactor and study the noncompetitive inhibitor releasing pathway and kinetics of single HRP on origami. The addressable origami provides a “GPS” for TIRFM to directly capture the product generation and releasing event on single immobilized HRP. By analysis of the fluorescence trajectory, we confirm the coexistence of two distinct functional sites in one enzyme, i.e. catalytic site and docking site, and an allosteric conformational change upon inhibitor interacting with the two sites is also observed. Remarkably, we propose a single molecule inhibition model that reveals a thorough and quantitative view of enzymatic inhibition process and kinetics. DNA origami is a promising scaffold for the organization of biomolecules or nanoparticles because of its ability to site-specifically incorporate functional elements with high precise geometries22-29. In the present work, a triangular DNA origami baring two extended arm stands was used as scaffolds to immobilize individual HRP and a fluorophore, atto-488 (Fig.1A, Figure S1-3). Then, the triangular DNA origami carrying HRP and atto-488 was tethered to a glass coverslip via biotin-streptavidin interactions, and the substrate solution (10~200 nM Amplex Red, 1 μM H2O2) was flowed over the interface in a reaction chamber (Fig. 1A). The successful attachment of individual HRP on DNA origami was confirmed by AFM (Fig.1A, inset). The purpose of placing a fluorophore in close proximity to HRP is to set a “GPS” to locate the position


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of HRP in TIRFM images, thereby eliminates unwanted fluorescent bursts from random touching. In a typical assay, we first used 488 nm laser to excite atto-488 to locate HRP, and then switched to 561 nm to excite fluorescent product resorufin. Of note, the fluorescent product resorufin is negatively charged under neutral pH, thus the strong electrostatic repulsion between resorufin and DNA origami also prevents the random touching of diffusive resorufin on DNA origami. To further reduce random touching of fluorophore, we used liposome for comprehensive block of unmodified coverslip. Moreover, dissociated resorufin product molecules in solution are undetectable owing to their fast diffusion rate (Figure S4). Further control assays indicated only Amplex Red or H2O2 lead to no obvious fluorescent burst (Figure S5) in the absence of HRP. Furthermore, control experiments with O2 scavengers also excluded O2 induced production of resorufin (Figure S6).


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Figure 1. Single HRP immobilized on an addressable DNA origami nanoreactor. (A) Schematic illustration the immobilizing of single HRP on a surface immobilized triangle DNA origami. A fluorophore, atto-488, is rationally designed as “GPS” on the adjacent site of HRP to locate the position of HRP in TIRFM image. The fluorescent product generation event is followed by TIRFM. Inset: An AFM image of HRP loaded on triangle DNA origami. (B) PAINT image of triangle DNA origami loaded with both atto-488 and HRP. Atto-488 (red spots) and fluorescent product resorufin (green spots) were excited with 488 nm and 561 nm laser, respectively. (C) Amplified PAINT image and distance between atto-488 and resorufin obtained from the histogram distribution, which is close to the designed distance.


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Interestingly, the continuously generation of resorufin on HRP creates a necessary “blinking” for points accumulation for imaging in nanoscale topography (PAINT) to precisely locate atto-488 and resorufin (Fig. 1B)30-31. The distance between atto-488 and resorufin was well resolved from the distribution histogram (average distance ~72.5 nm), which is quite close to the calculated value between atto-488 and HRP from the number of nucleotide units (68.2 nm, Fig. 1C). Therefore, the co-localization of HRP and resorufin suggested that the fluorescence burst from nascent generated resorufin is exactly occurred on the immobilized HRP, which enables to follow the trajectory of fluorescent product generation event.

Figure 2. Single-turnover analysis of single HRP catalytic reaction. (A) A typical fluorescence trajectory of an immobilized HRP in the presence of 100 nM Amplex Red and 1 μM H2O2. Right inset: Distribution histogram of intensities of on-time in the trajectory. (B) A segment of fluorescence trajectory in (A) (purple region). (C) The percentage of two on-levels intensity increased with elevated concentration of substrate.


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We thereby used the nascent generated resorufin as an in-situ probe to explore the enzymatic reaction of single HRP. The fluorescence trajectory was recorded from a fluorescent spot of resorufin in Fig. 1B that contains stochastic on-off signals (Fig. 2A). A control experiment indicated that the burst frequencies are independent of laser intensity; thereby excluded the possibility of photo-induced bursts (Figure S7). Therefore, we attributed the digital nature of this trajectory to nascent product generation turnovers (detailed discussions, see Supplementary Information). Surprisingly, the fluorescent bursts revealed two types of on-level intensities (Fig. 2B), and the two on-level intensities were statically in a 2-fold ratio (as shown in the distribution histogram in Fig. 2A, inset). However, HRP is a monomeric metalloenzyme with one catalytic site32; it is obscure to understand why two product molecules exist simultaneously on single enzyme. Moreover, we found that appearance frequency of the 2-fold on-level increased with substrate (Amplex Red) concentrations (Fig. 2C). To understand the simultaneous coexistence of two product molecules on the same enzyme, we performed bulk kinetic measurements of HRP enzymatic reaction in the presence of various concentrations of resorufin, and the obtained Eadie-Hofstee plots suggested resorufin is a noncompetitive inhibitor for HRP (Figure S8)33. It is now clear that the two types of on-level with approximately 2-fold intensities difference imply the coexistence of two functional domains in HRP, i.e. docking site and catalytic site. As the reaction proceeds, the concentration of resorufin increases, which explains the increasing percentage of two on-levels of intensity with increased substrate in Fig. 2C. To further confirm this, we also performed a control experiment with an enzymatic reaction of an HRP mimicking DNAzyme. Upon stacking with hemin, a G-quadruplex DNAzyme reveals an HRP-mimicking catalytic activity34. Structural analysis suggests that the HRP-mimicking DNAzyme possesses only one catalytic site in the hemin-binding pocket35. Of


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note, in the present study we employed a G-quadruplex sequence with as adjacent adenine at 3’ end that significantly improves the activity of DNAzymes at acidic pH34. Accordingly, we observed a consistent height of intensity in the DNAzyme-catalyzed intensity-time trajectory (Figure S9), indicating the two types of on-level intensity in Fig. 2A was indeed originated from two different domains of HRP.


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Figure 3. Multiple product releasing pathways and kinetics. (A) A fragment of fluorescence trajectory of an individual HRP catalysis. (B) Averaged catalytic kinetics of HRP. -1 (upper panel) and -1 (bottom panel) were mean values from >50 trajectories on the same concentration of substrate. Solid lines were fitted with equations (1) and (2), the subsequent parameters were: k1=2.09 s-1, k-1=0.25 s-1, k2= 0.46 s-1, k3=4.53 s-1, k4= 6.28 s-1, k5= 2.26 s-1. Note here the values of the kinetic parameters were statistical averaged from >30 trajectories. (C)


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Heterogeneous reactivities in catalysis. -1 (upper panel) and -1 (bottom panel) were classified into three subpopulations, which were hidden in ensemble averaging on the same concentration of substrate. -1 (upper panel) and -1 of three distinguished types also revealed the dependence of substrate concentration. Type Ⅰ (red): k1=1.55 s-1, k-1=0.48 s-1, k2= 0.67 s-1, k3=2.02 s-1, k4= 2.24 s-1, k5= 1.01 s-1. Type Ⅱ (blue): k1=1.51 s-1, k-1=0.51 s-1, k2= 0.41 s1,

k3= 0.67 s-1, k4= 1.81 s-1, k5= 0.93 s-1. Type Ⅲ (green): k1=1.44 s-1, k-1=0.54 s-1, k2= 0.55 s-1, k3=

k5=1.0 s-1, k4= 0.07 s-1. Solid lines were simulations of equation (1) and (2). (D) Schematic chart for HRP catalytic kinetic mechanism. S: Amplex Red; P: resorufin.

Having confirmed the coexistence of catalytic site and docking site, we analyze the fluorescence trajectory to probe more information about the product releasing pathways and kinetics36-37. As discussed above, each sudden intensity increase represents a product formation on HRP, each intensity decreases marks product dissociation from HRP, and each off-on cycle corresponds to a single turnover of a catalytic formation of a product and its subsequent dissociation from single enzyme, therefore the trajectory carries rich dynamic information38. We extracted the duration time from the catalytic trajectories (Figure S10), then defined τoff as the waiting time before product formation, and τon as the waiting time before product dissociation after its formation (Fig. 3A). devotes averaging. To focus on the understanding of non-competitive inhibition by resorufin, we omitted the contribution from H2O2 by adding large excess of H2O2. Clearly, the single-turnover trajectory in Fig. 3A separated the enzymatic reaction into two parts (Fig. 3B), i.e. the substrate-enzyme binding (reaction i; E+SES) and the enzymatic conversion process (reaction ii; ESEP) that occurred in τoff, and the product releasing process (reaction iii, EPE+P) that occurred in τon. Considering


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the coexistence of catalytic site and docking site, the product releasing thereby involves another pathway, i.e. the nascent product moves to the nearby docking site with the aid of a next substrate binding (reaction iv’, E·P+S→E·S·P), follows a next enzymatic conversion (reaction iv, E·S·P→E·P·P), and then product dissociates from enzyme (reaction v; E·P·P→E+2P). The enzyme in off-state then experiences a conformational switch to its original state (reaction vi). We hypothesize that protein structural change induced by the second substrate binding and product moving to docking site in step iv’ is relatively fast compared with other steps, thereby the dynamic contribution of step iv’ was ignored. Since HRP contains only one catalytic site, the enzymatic reaction could follow a first-order single-molecule kinetic model. -1 and -1 was derived as：

〈𝜏𝑜𝑓𝑓〉 ―1 =

〈𝜏𝑜𝑛〉 ―1 =

1 ∞

∫0 𝜏𝑔𝑜𝑓𝑓(𝜏)𝑑𝜏 1 ∞

∫0 𝜏𝑔𝑜𝑛(𝜏)𝑑𝜏

=

=

{

𝑘2[𝑆]

(1)

[𝑆] + 𝑘2/𝑘1 + 𝐾

1 𝑘3 ― 𝑘5

}

1 + (𝑘3 + 𝑘4[𝑆])2 𝑘5

(2)

where ցoff(τ) and ցon(τ) correspond to the probability density function of τoff and τon, respectively, [S] is the concentration of substrate, k1, k2, k3, k4, k5 and k6 are the rate constants corresponding to each reaction step in Fig. 3D, K is the dissociation constant of substrate (k-1/k1) (detailed derivations, see Supplementary Information). The derived Eq. (1) and (2) suggested a correlation between single molecule product formation and dissociation rates with substrate concentration [S]. Surprisingly, the product dissociation rate -1 is also a [S]-dependent function, indicating that substrate also contributes to product


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releasing. We thus preformed the single molecule enzymatic reaction under various concentrations of Amplex Red, and then applied eq. (1) and (2) to fit the experimental data (Fig.3B, averaged kinetic plots of substrate concentration vs. reaction rate). The single enzyme product formation rate (Eq. 1) predicts variable saturation level and initial slops with increasing [S], if different HRP molecules reveal heterogeneous binding affinity (K) and catalytic activity towards resorufin (k2). The heterogeneous substrate-dependent product formation rate was confirmed by in the upper panel of Fig. 3B. In addition, Eq. (2) predicts three types of product releasing pathways if HRP has heterogeneous activities between two product dissociation pathways. (Ⅰ) if k5k3, -1 decreases with increasing [S], the indirect dissociation pathway is dominant, which means the product gives priority to move to the docking site before dissociation. As previously described in Fig.3D, in this pathway, the secondary substrate be adsorbed in the active site of the enzyme synchronous with the migration of previously formed product to allosteric site; (Ⅲ) if k5=k3, -1 is constant at any [S]. We counted the different dissociation curve for the same HRP over various concentrations of resorufin, and indeed observed three entirely different [S] dependence with subpopulations (Fig. 3C): 51% of HRP molecules are type-Ⅰ, 22% are type-Ⅱ and 27% are type-Ⅲ. The single molecule analysis unraveled the masked heterogeneous in product releasing pathways and dynamic. Surprisingly, single enzymatic reveals a large proportion of substrate-assist dissociation pathway, which is in stark contrast with traditional understanding on ensemble level. Now, it is clear that the catalytic product resoufin is a non-competitive inhibitor of HRP, and resorufin possesses heterogeneous releasing kinetics and pathways. We then wonder if there is an


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allosteric conformational change between the two product releasing pathways, i.e. from either catalytic site or docking site. Correlated structural changes are the core of allosteric interactions. Previous computational results have suggested correlated structural motion in non-competitive inhibition8; however, experimental support on the allosteric conformational change is still rare because of buried molecularly level information at ensemble average. The established single molecule measurement in the present work provides a powerful means to probe dynamic information of each enzymatic step, which enables to focus in the non-competitive inhibition step (ⅳ) to correlate the dynamic fluctuation (k4) with local structural changes. Catalytical cycle of HRP with substrate cause sequential oxidation-reduction reaction within heme pocket. However, oxygen intermediates produced in active center of HRP existed in very shot life time, it is beyond our scope of present study for direct observing their presences. Wellestablished crystallography study indicates that the heme pocket of HRP features histidine (His42) and arginine (Arg38) amino acid residues that located on the distal side of heme39-40. Moreover, upon both H2O2 and Amplex Red bind to HRP, molecular dynamics simulations suggest that proton transfer between H2O2 and heme ligand is energetically favorable if mediated by water molecule in the active site41-42. Therefore, proton transfer from His42 to water is the rate-limiting step of the enzymatic conversion reaction (ESEP)43-44. We thus performed solvent kinetic isotope effects (SKIE) experiment to explore the proton transferring process in step ii and iv, respectively45-46. Briefly, we carried out a control single molecule experiments in D2O diluted buffer with other experimental conditions remained unchanged (Figure S11). The extracted fluorescence trajectory of each HRP catalytic event in D2O diluted buffer was shown in Fig. 4A and B. The enzymatic dynamic in D2O is in stark contrast with in H2O. (1) The dwell time of product generation, i.e. τoff, in D2O is different. The average


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value of off-time in D2O ( ~ 11.290 s) is considerably larger than that in H2O ( ~ 3.190 s), wherever the average value of on-time remained unchanged ( ~ 0.098 s, ~ 0.100 s). The slower product generation rate in D2O was attributed to higher stability of O-D bond over O-H bond, which slows the rate of proton transfer and prolongs τoff. (2) Fitting the enzymes-averaged data in D2O (Fig. 4C and D) with eq. 1 and 2, we obtained k2, D2O=0.22 s-1, k4, D2O=8.71 s-1, which are different from k2, H2O=0.46 s-1, k4, H2O=6.28 s-1. For the enzymatic conversion in the catalytic site (step ii), k2, H2O /k 2, D2O =2.09, suggesting a primary isotope effect, indicating H2O involves in the catalyze reaction. While for the enzymatic conversion in step iv, k4, H2O / k4, D2O =0.72, indicating a secondary isotope effect where H2O is not involved. Thus, HRP reveals distinctively different states in enzymatic conversion occurred in step ii and iv. We speculated that the different states imply an allosteric conformational change. Upon nascent generated products moving to the docking site, HRP experiences a conformational change, therefore the local environment around catalytic site is changed. As a result, H2O could not enter the catalytic pocket and involve in the second round of catalytic conversion (step iv) (Fig. 4 E). As far as we know, this is the first experimental support of the allosteric conformation change in noncompetitive inhibition.


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Figure 4. Allosteric conformational change in non-competitive inhibition reveled by solvent kinetic isotope effects (SKIE) experiment. (A) A segment of intensity-time trajectory of HRP catalysis at 100 nM Amplex Red and 1 μM H2O2 in D2O diluted buffer. (B) A magnified fluorescence trajectory showing 2-fold of on-time intensity. (C, D) Histogram of distribution of off-time (C) and on-time (D) in H2O and D2O. (E) The role of water is changed in allosteric regulation.


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Previous single-molecule enzymatic studies indicated that enzymatic reactions reveal dynamic disorder, and memory effect exists between adjacent pair of turnovers because of conformational fluctuation of proteins47-48. The inherent structural fluctuation of enzymes, however, hinds that the memory effect may also exist in inhibition reactions. The ability to distinguish multiple inhibition pathways and dynamics motivated us to explore the dynamic disorder of inhibition reactions. As mentioned above, the product releasing and inhibition occur in τon, we thereby determined the time dependence of turnover rate (number of on-off circle per second) from each single-turnover fluorescence trajectory (Fig. 5A). The observed large temporal variations suggested a dynamic fluctuation of catalytic activities of single HRP. To further clarify interaction of products and enzyme, the conditional probability distribution (p(x,y)) of on-times was calculated. For pairs of on-times (x and y) separated by several numbers of turnovers, p(x,y) could represents mutual influence between on-times. When two on-times are independent, or in the absence of dynamic disorder, p(x,y)= p(x) p(y). For pairs of on-times of adjacent turnovers, a clear diagonal line was present (Fig. 5C), indicating the previous reaction has an impact on the following reaction. Meanwhile, for pairs of on-times separated by 10 turnovers, the distribution was independent. The disparity between Fig. 5C and 5D indicates an apparent evidence of dynamic disorder due to the conformational motion during catalysis. Especially, when the substrate concentration was saturated, substrate-assisted releasing pathway was dominant in τon reaction (k5 being rate-limiting), therefore, dynamic disorder might arise from the slowing fluctuations in inhibition dynamics during products releasing, further validating the conformational fluctuations in allosteric inhibition.


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Figure 5. Memory effect in single-molecule HRP catalytic kinetics. (A) Trajectory of rates of turnovers for single-molecule HRP at 200 nM Amplex Red and 1 M H2O2. Data points were calculated every 20 turnovers. (B) Autocorrelation analysis of τon (up) and τoff (bottom) which derived from the same trajectory. Solid lines were exponential fits with decay constants of moff =1.5±0.05 and mon=1.01±0.07 turnovers. (C) The 2D conditional histogram for on-times of two adjacent turnovers, which is derived from the trajectories of 10 HRP molecules. A subtle diagonal feature is observed. (D) The 2D conditional histogram for two on-times separated by 10 turnovers for the HRP molecules in (C). The diagonal feature vanishes because the two on-times become independent of each other at the 10-turnover separation. The scale of x and y are normalized from 0 to 1 s. We then extract the sequence of individual τon and τoff from each single-turnover fluorescence trajectory and interrogated the correlation between adjacent turnover with auto correlation functions (C(𝜏) =

[∆𝜏(0)∆𝜏(𝑚)]

⟨∆𝜏2⟩

, τ is either τoff or τon, m is the turnover index number in the sequence


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and Δτ(m)=τ(m) - , where < > denotes averaging), to quantitatively evaluate the extent of τoff and τon reactions affecting activity fluctuation as well as memory time of τon reactions. According to this function, when dynamic disorder is absent, C(0)= 1 and C(τ)= 0 (m>0). However, as depicted in Fig. 5B, C(τ) ≥ 0 and C(τ) could be well fitted with single exponential decay with the initial amplitude (m=1), indicating dynamic disorder was indeed present. The fitted decay constants were calculated as moff = 1.50±0.05 turnovers and mon=1.05 ±0.07 turnovers. With an average turnover time of 1.50 s for this trajectory, correlation times for τoff and τon reactions were ~2.25 s and 1.51 s, respectively. The two correlation times quantitatively reflect the timescale fluctuation of k1 and k5, which were attributed to the conformational fluctuation kinetics leading to rate-limiting in τoff and τon reactions. The Amplex Red assay has been widely employed in recent years as an example to demonstrate the catalysis mechanism of HRP. Although the catalysis of HPR with H2O2 has been extensively studied, there are still debates on the electron transfer mechanism between the substrates and the enzymes49, i.e. the product is generated through a two-electron-oxidation process or through a radical reaction after a one-electron-oxidation. The only difference lies in whether there exists an enzyme independent dismutation reaction to form fluorescent resorufin50. However, our work and other work from Lu’s group51-52independently found that resorufin molecules are delivered from HRP one by one, which are unlikely due to the combination of two radicals. We are not in position to prove one way or another in our current work as it is beyond the scope of present work. However, we speculate the coexistence of docking sites and catalytic sites may result in an inner-enzyme electron transfer through proteins backbones that lead to two-electron transfer from a single Amplex Red to HRP.


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In conclusion, we presented a single molecule study of enzymatic product releasing dynamic in non-competitive enzymatic inhibition reaction. We introduced addressable DNA origami as nanoreactors to precisely position single HRP, although, the confinement of HRP causes reduced enzyme activities due to increased structure rigidity of hybridized dsDNA arm with DNA origami.53 DNA origami provided a direct guidance which enabled real time monitor the nascent fluorogenic product on individual enzyme with single turnover resolution with TIRFM. We observed a simultaneous appearance of two fluorogenic products arising from catalytic and docking site of the same enzyme, respectively. By examining the single enzymatic reaction in real time at single turnover resolution, we illuminated heterogeneous intermediate conformation states and quantitatively identified various product releasing pathways. Moreover, this work also disclosed direct evidences of an allosteric conformational correlation between two protein functional sites in non-competitive inhibitions. Although further structural experiment is necessary, the present work draws a generalized picture about the non-competitive inhibition process from the generation of enzymatic product, moving to docks sits and allosterically inhibiting the enzymatic activity, to finally dissociating from enzymes in various pathways. We expect the insightful information may bring further understanding of enzymatic reaction and shed light on artificial regulating enzyme activities, for example, new drug discovery. Materials and methods Oligonucleotides. Sequences of the oligonucleotides used in this work are available in the Supplementary Information. Modification of HRP with ss-DNA. LC-SPDP was used to crosslink HRP with thiol-modified ss-DNA53. Briefly, 100 μL of 2 μM HRP solution was first reacted with a 20-fold excess of LC-


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SPDP in PBS buffer (pH 8.0) for 2 h, excess LC-SPDP was removed by ultrafiltration with 30 kD cutoff filters (Amicon). Next, LC-SPDP modified HRP was conjugated to 10-fold excess of thiolmodified ss-DNA. The reaction mixture was incubated in PBS buffer (pH 7.4) for 8 h under room temperature. Excess DNA was removed by ultrafiltration with 30 kD cutoff filters. Assembly of HRP-DNA conjugate on triangle DNA origami. Triangle DNA origami with extended DNA stands for hybridization with HRP were prepared in 1×TAE-Mg2+ buffer (40 mM Tris, 20 mM Acetic acid, 2 mM EDTA and 12.5 mM Magnesium Acetate, pH 8.0). The assembly of HRP and DNA origami were achieved by DNA hybridization. In detail, HRP-DNA conjugates were mixed with DNA origami in 1 × TAE-Mg2+ buffer with a molar ration of 5:1. The solution mixture was cooled from 37°C to 4°C with the following gradient: 37°C for 5 min, 36-10°C, 0.2 °C /min, 4°C for storage. AFM imaging. The imaging of HRP linked DNA was character by MultiMode 8 AFM with NanoScope V Controller (Bruker, Inc) in fluid under Peak force mode. Before imaging, 3 μL sample was stored on a freshly cleaved mica surface and left to absorb to the surface for 3 min. Single-molecule enzymatic reaction. The enzymatic reaction of single HRP on origami nanoreactor was monitored by a commercial total internal reflection fluorescence microscopy (Nstorm, Nikon). The laser was activated and focused on the sample for 10 min to remove autoactivated fluorescence before taking movies. The movies were analyzed using a home-written program to extract the fluorescence intensity trajectories from localized fluorescence spots individually across the entire movie. Single-molecule enzyme catalysis experiments was carried out in a flow cell that formed by double-sided tapes sandwiched between a quartz slide and a streptavidin-functioned borosilicate coverslip54. Then, 100 μL of 100 pM HRP-origami


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conjugates or DNAzyme linked origami was dropped on the coverslip, and incubated for 20 min. The coverslip was then rinsed for 3 min with 1  TAE-Mg2+ buffer to remove unbound HRPorigami conjugates. Two holes were drilled on the quartz slide to connect polyethylene tubing and a syringe pump for continuous solution flow at rate of 10 μL/min during recording. Super-resolution PAINT imaging of DNA origami and products. Upon addition of Amplex Red and H2O2, the enzymatic reaction of HRP generates product constantly, which results in fluorescence flashes. PAINT was then performed through recording the nascent product. Continuous wave circularly polarized 488 and 561 nm laser beam were focused onto an area of 50 × 50 μm2 of the sample to excite atto-488 and resorufin, respectively. Videos of fluorescent products flashing and imaging of the fluorescence labelled origami was real-time monitored and analyzed through a software developed by Jungmann et al. to exact the position of resorufin and atto-488.55 Data availability. The data supporting the main findings of this study are available within the main article and its Supplementary Information (Supplementary Figs 1-11) and Supplementary Tables 1-2) or from the authors upon request ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: ………….. Experimental section, all nucleic acid sequences, data availability and theoretical derivation. AUTHOR INFORMATION


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ORCID †Di Li: 0000-0003-1674-0110 Chunhai Fan: 0000-0002-7171-7338 Author Contributions Y. X, Y. G. and Y. S. contributed equally to this work. Notes The authors declare no competing financial interests. ACKNOWLEDGMENT This work was supported by the National Basic Research Program of China (2013CB932803, 2013CB933800), National Key R&D Program of China (2016YFA0201200, 2016YFA0400900), NSFC (21675166, 21390414, 21473236, 31371015, 21329501), Key Research Program of Frontier Sciences, CAS (QYZDJ-SSW-SLH031) and Shanghai Rising-Star Program (17QB1402900). REFERENCES (1) Benkovic, S.J.; Hammes, G.G.; Hammes-Schiffer, S. Free-energy landscape of enzyme catalysis. Biochem. 2008, 47, 3317-3321. (2) Goldstein, A. The mechanism of enzyme-inhibitor-substrate reactions. J. Gen. Physiol. 1944, 27, 529-580. (3) Gorris, H.H.; Rissin, D.M.; Walt, D.R. Stochastic inhibitor release and binding from single-enzyme molecules. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 17680-17685. (4) Kleinboelting, S.; Ramos-Espiritu, L.; Buck, H.; Colis, L.; van den Heuvel, J.; Glickman, J.F.; Levin, L. R.; Buck, J.; Steegborn, C. Bithionol potently inhibits human soluble adenylyl cyclase through binding to the allosteric activator site. J. Bio. Chem. 2016, 291, 9776-9784.


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Figure 1



Figure 2


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Figure 3



Figure 4


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Figure 5



TOC figure


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Single Molecule Studies of Allosteric Inhibition of Individual Enzyme

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