Proximity does not contribute to activity enhancement in the glucose oxidase-horseradish peroxidase cascade

Abstract

A proximity effect has been invoked to explain the enhanced activity of enzyme cascades on DNA scaffolds. Using the cascade reaction carried out by glucose oxidase and horseradish peroxidase as a model system, here we study the kinetics of the cascade reaction when the enzymes are free in solution, when they are conjugated to each other and when a competing enzyme is present. No proximity effect is found, which is in agreement with models predicting that the rapidly diffusing hydrogen peroxide intermediate is well mixed. We suggest that the reason for the activity enhancement of enzymes localized by DNA scaffolds is that the pH near the surface of the negatively charged DNA nanostructures is lower than that in the bulk solution, creating a more optimal pH environment for the anchored enzymes. Our findings challenge the notion of a proximity effect and provide new insights into the role of DNA scaffolds.

ARTICLE

Received 2 Aug 2016 | Accepted 14 Nov 2016 | Published 22 Dec 2016

A proximity effect has been invoked to explain the enhanced activity of enzyme cascades on DNA scaffolds. Using the cascade reaction carried out by glucose oxidase and horseradish peroxidase as a model system, here we study the kinetics of the cascade reaction when the enzymes are free in solution, when they are conjugated to each other and when a competing enzyme is present. No proximity effect is found, which is in agreement with models predicting that the rapidly diffusing hydrogen peroxide intermediate is well mixed. We suggest that the reason for the activity enhancement of enzymes localized by DNA scaffolds is that the pH near the surface of the negatively charged DNA nanostructures is lower than that in the bulk solution, creating a more optimal pH environment for the anchored enzymes. Our ndings challenge the notion of a proximity effect and provide new insights into the role of DNA scaffolds.

DOI: 10.1038/ncomms13982 OPEN

Proximity does not contribute to activity enhancement in the glucose oxidasehorseradish peroxidase cascade

Yifei Zhang1, Stanislav Tsitkov1 & Henry Hess1

1 Department of Biomedical Engineering, Columbia University, New York, New York 10027, USA. Correspondence and requests for materials should be addressed to H.H. (email: mailto:[email protected]

Web End [email protected] ).

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Enzyme cascades, which sequentially perform multiple enzymatic reactions, have a critical role in signal transduction and amplication in biological systems and also have a

wide range of potential applications in biotechnology15. In the past decade, researchers developed strategies to construct articial multienzyme systems6,7. In particular, the rapid development of DNA nanotechnology provides a programmable tool for the spatial organization of enzymes at specic sites8. When the components of an enzyme cascade were placed within nanometers on a DNA scaffold, a several-fold enhancement of the cascade throughput was observed812. These observations attracted a strong and growing interest in multienzyme catalysis, but the mechanism of the activity enhancement is still not clear13.

Glucose oxidase (GOx) and horseradish peroxidase (HRP) are the most frequently used enzyme pair to demonstrate the feasibility and advantages of multienzyme systems on DNA scaffolds. Mller and Niemeyer14 presented the earliest example of a GOxHRP complex by nucleic acid hybridization in 2008. Wilner et al.10 tethered GOx and HRP molecules to a DNA ribbon with a precisely dened distance between GOx and HRP pairs. By taking advantage of DNA origami, Fu et al.11 created an individual GOx and HRP pair on each DNA scaffold. A several-fold enhancement of the cascade throughput relative to the throughput obtained from equal numbers of free GOx and HRP molecules in solution was always observed, and a closer placement gave a higher overall activity. Several other enzyme cascades also showed enhanced activity when they are co-assembled with DNA nanostructures12,15,16. The activity enhancement was frequently attributed to facilitated transport, or so-called substrate channeling, where a shorter distance between the coupled enzymes leads to a faster transfer of a newly produced intermediate substrate molecule to the second enzyme.

However, the mechanistic understanding of these experiments is still unsatisfactory. First, although the enzyme pairs were constructed by similar strategies, the activity enhancements vary a lot. For instance, Fu et al.11 co-assembled the GOxHRP pair on DNA origami and observed 15 times higher activity when the distance between two enzymes is 10 nm. In contrast, Xin et al.17 placed the GOx and HRP even closer using a similar strategy but found only a 1.5-fold activity enhancement. Second, a theoretical analysis of the GOxHRP cascade by Idan and Hess18,19 challenged the proximity channeling effect. They estimated the timescale when the intermediate ux from the upstream enzyme is equal to the ux from the bulk solution and pointed out that the facilitated transport owing to proximity is a temporary effect that only enhances cascade throughput until the concentration of intermediate substrate molecules in the solution becomes signicant. The duration of this normally very short-lived effect (on the millisecond timescale) can be extended by aggregating the enzyme pairs and/or introducing attractive interactions between the intermediate substrate and the scaffold. In brief, the aggregation provides more target enzymes in the vicinity of the rst enzyme, and the attractive interaction can increase the local concentration of intermediate and prevent its leaking to the bulk solution. Ultimately, the overall activity will be limited by the maximal reaction rate of the slower enzyme13,20. Proximity in itself should not inuence the maximal reaction rate of either enzyme; however, the reported activity enhancements appear to persist over the entire observation time which would indicate that the limiting maximal reaction rate has increased11,17,21. This contradiction between the theoretical analysis and experimental observations has not yet been resolved.

Here we examined the putative proximity effect for the GOxHRP cascade without relying on DNA nanotechnology.

We place GOx and HRP within 2 nm of each other using a crosslinker, show that the catalytic activity of both enzymes is retained and demonstrate that there is no enhancement in cascade throughput (either transiently or permanently) and no direct substrate transfer. As the proximity channeling is ruled out, the DNA scaffold must be responsible for the previously observed activity enhancement. We measure the pH dependence of the maximal reaction rate of GOx and HRP and nd that the observed activity enhancement can be explained by the lower pH near the DNA surface where the enzyme is located. These ndings challenge the traditional view of proximity channeling and provide new insights into the role of DNA scaffolds.

ResultsKinetics of GOx and HRP. GOx is a bi-substrate enzyme that oxidizes b-D-glucose with oxygen and produces glucono-d-lactone and hydrogen peroxide (H2O2; Fig. 1a). First we showed that the oxygen concentration is saturating for the duration of the experiment (1 h) by measuring the oxygen concentration directly with an oxygen meter in a sealed vessel containing 1 nM GOx in 1 mM glucose (Fig. 1b). The oxygen consumption rate is constant at 15 nM s 1 demonstrating that the reaction is not slowed by oxygen depletion or product inhibition. An

HRP-coupled colorimetric assay quantied the rate of formation of ABTS in the presence of 20 nM HRP and 2 mM 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)-diammonium salt (ABTS). Consumption of 54 mM oxygen in 1 h corresponded to the formation of 112 mM ABTS , proving that the stoichiometry of H2O2 to ABTS is 1:2. It also conrmed that the conventional assay using the GOx/HRP cascade is reliable as the high concentration of HRP (20 nM) ensures the rapid and complete conversion of H2O2 produced by 1 nM GOx. The colori-metric assay yielded a rate of H2O2 generation of15.40.1 nM s 1 (means.d.) in 1 mM glucose (the Michaelis Menten kinetics of glucose is shown in Supplementary Fig. 1).

The kinetics of HRP was determined by adding variable concentrations of H2O2 to 1 nM HRP and 2 mM ABTS (Fig. 1c).

HRP follows MichaelisMenten kinetics with a Km of HRP for H2O2 of 2.550.11 mM and a turnover number kcat of32.70.4 s 1 (means.e.). The concentration of the H2O2 stock solution has been determined by measuring the amount of

ABTS , which can be generated from a given H2O2 solution by HRP (Supplementary Figs 2 and 3).

Free GOx and HRP cascade. In order to characterize the GOxHRP cascade reaction if both enzymes are free in solution, we measured the ABTS concentration as function of time for different ratios of HRP to GOx in the presence of 1 mM glucose (Fig. 2). According to the measured kinetics of HRP, the maximum reaction velocities of 1, 2 and 20 nM HRP are 33, 65 and 650 nM s 1, respectively, and are all greater than the reaction velocity of 1 nM GOx (15.4 nM s 1). There is a clear transient stage at the beginning in the case of 1 nM GOx with 1 nM HRP (Fig. 2a,b). Increasing the amount of HRP gradually shortens the transient time, leading to slightly higher concentrations of ABTS at equal time points. The transient stage is almost undetectable when a 20-fold excess of HRP was used. The increase in reaction velocity of HRP causes a change in the product formation curve (Fig. 2b), which resembles the changes observed when GOx and HRP are placed on DNA scaffolds9,10,17.

All of these reactions converge to a state in which the H2O2

consumption rate is equal to the reaction velocity of 1 nM GOx in 1 mM glucose (15.4 nM s 1). The fact that the nal activity converges to an identical value demonstrates that the only

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13982 ARTICLE

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Figure 1 | Individual kinetics of GOx and HRP. (a) Schematic representation of GOx/HRP cascade. (b) Oxygen concentration in a sealed vessel containing 1 nM GOx and 1 mM glucose in PBS (red), and the concentration of ABTS in PBS containing 1 nM GOx, 20 nM HRP, 1 mM glucose and 2 mM ABTS (blue)

as function of time, error bars represent the s.d. of the three replicates. (c) HRP follows MichaelisMenten kinetics; the activity of HRP was measured in PBS containing 1 nM HRP, 2 mM ABTS and different concentration of H2O2.

potential benet of an improved transport is a shortening of the initial transient stage if the intrinsic kinetics of the involved enzymes do not change. This conclusion had been drawn in the literature decades ago when biochemists were rst dening the concept of substrate channeling2225.

The time course of ABTS production was modelled with a system of ordinary differential equations using the kinetic parameters measured individually for GOx and HRP (Supplementary Fig. 4). This model assumes that on the timescale of the experiment the produced H2O2 is well mixed.

A detailed justication for this fact is given in our previous work19. For illustration, consider that, for a solution containing 1 nM HRP and 1 nM GOx, the average distance between the enzyme pairs is approximately 1 mm. Therefore, the H2O2 generated by GOx can diffuse from a GOx molecule to a HRP molecule in 0.6 ms (the diffusion coefcient of H2O2 is 1.71 10 9 m2 s 1 at 25 C (ref.

26)). As it takes about 10 s to mix the enzyme and substrate solution before the data acquisition commences, all simulation curves were shifted by 10 s. As shown in Fig. 2, the model ts the experimental results. The slight decrease in the nal activity may be due to deactivation of GOx or product inhibition by the accumulating gluconolactone, but neither deactivation nor inhibition signicantly inuences the activity. Figure 2c shows that, in the cascade reaction, it takes 500 s to establish the steady state when 1 nM HRP is used. Increasing the concentration of HRP can shorten the transient time owing to a more rapid conversion of H2O2. As the maximum reaction velocity of the

HRP (Vmax is 33 nM s 1 for 1 nM HRP) always exceeds the production of H2O2 by GOx (15 nM s 1 for 1 nM GOx), the

H2O2 concentration converges to a constant value so that the actual reaction velocity of HRP equals 15 nM s 1 (Supplementary

Fig. 5).

We then carried out the reaction at lower concentrations of HRP, where the reaction rates are limited by the activity of HRP (Fig. 3). Reducing the HRP concentration from 0.4 to0.2 nM now slows the rate of ABTS production by half. The H2O2 concentration quickly exceeds the Km of HRP and continues to rise throughout the experiment (Supplementary Fig. 6), making the reaction rate of HRP approach its maximum value. The model also describes the process well despite the slight deactivation of HRP in the nal stage.

The results above demonstrate that the diffusion of the intermediate substrate H2O2 is not the rate-determining step even in the initial stage of the free GOxHRP cascade. Small molecules such as H2O2 with a diffusion coefcient on the order of 10 9 m2 s 1 diffuse so fast into the bulk solution that no channeling effect can be expected for such a cascade system. In addition, although the rate of product formation converges to a certain value, the concentration of the intermediate substrate may continue to increase if the downstream enzyme is rate limiting. Increasing the rate of the non-limiting step can only shorten the transient stage (which may appear to be enhanced throughput), whereas improving the activity of the rate-limiting enzyme can signicantly and permanently enhance the throughput.

GOxHRP conjugate. In order to place GOx and HRP in close proximity to each other, we covalently conjugate GOx and HRP with a small molecular linker, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), with a spacer length of 8.3 . As the free cysteine residues of the GOx are hard to access, we grafted more sulfhydryl groups from the lysine residues via N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) modication and subsequent dithiothreitol thiolation (Fig. 4a). HRP was puried by size exclusion chromatography prior to modication (Supplementary Figs 7 and 8). After the conjugation, the maximal distance between HRP and GOx is2.3 nm if the exible linker is fully stretched. The conjugate was separated by size exclusion chromatography on a HiLoad 16/600 Superdex 200 prep grade column (Supplementary Figs 9 and 10). The molar ratio of GOx and HRP in the conjugate is 0.94: 1, as determined from its absorbance at 280 and 403 nm (Supplementary Fig. 11). SDSPAGE shows that the conjugate is a mixture of multimers with a different stoichiometry of GOx to HRP and that all HRP was conjugated with GOx (Fig. 4b). The activity of GOx was determined as14.1 nM s 1 for the amount of conjugate containing 1 nM HRP in 1 mM glucose. The Vmax and Km of the conjugated

HRP were almost the same as those of free HRP, indicating that its activity as well as its afnity for H2O2 had not been affected by the conjugation (Fig. 4c). The specic activities

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Figure 2 | Free GOx and HRP cascade when GOx is the limiting enzyme. (a) Concentration of ABTS in the presence of 1 nM GOx and 1, 2 or 20 nM HRP and (b) the initial period of panel (a). (c,e,g), Activity of GOxHRP cascade with respect to the consumption of H2O2 in the presence of 1, 2 and 20 nM HRP and (d,f,h) the initial periods of panels (c,e,g). All assays were independently replicated three times.

of GOx and HRP in the conjugate were not signicantly different from their original value, respectively (Fig. 4d,e). We then carried out the cascade reaction with this conjugate (Fig. 4f). The catalytic process of the GOxHRP conjugate can also be described by our model, without requiring additional terms for a substrate channeling effect. The steady state is reached after 500 s, and the nal overall activity was limited by

the slower enzyme, GOx (Fig. 4g). It clearly demonstrates that simply linking two enzymes together or placing them close to each other has no benets on the overall activity, that is, proximity is not the reason for the activity enhancement for the GOxHRP cascade or other multienzyme system involving small-molecular intermediates with high diffusion coefcients.

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Figure 3 | Free GOx and HRP cascade when HRP is the limiting enzyme. (a) The time courses of ABTS production and the simulation curves in the presence of 1 nM GOx and 0.2 nM or 0.4 nM HRP. (b) Activity of the GOxHRP cascade with respect to the consumption of H2O2. The assays were independently replicated twice.

Testing for substrate channeling with catalase (CAT) competition. To further conrm that the proximity does not result in substrate channeling, we introduce varying concentrations of CAT to the GOxHRP conjugate catalysed reaction (Fig. 5a). CAT consumes H2O2 in the bulk solution and diverts it away from HRP but should not affect the direct channeling of H2O2 from GOx to HRP. As shown in Fig. 5b, the addition of CAT can completely suppress the formation of ABTS , indicating that all the H2O2 enters the bulk solution and is accessible to the CAT. The time course of ABTS can also be well described by our model augmented with a term describing the CAT-induced sequestering of H2O2 (Supplementary Figs 1215). If a second term describing the direct channeling of a fraction of the GOx output is introduced, a t to the data in Fig. 5b leads to the conclusion (with 95% condence) that the directly channeled fraction is o0.5% (see Supplementary

Information for details).

pH dependence as a potential source of activity enhancement. If the proximity effect is discarded, other explanations for the strongly enhanced throughput of the GOxHRP cascade on DNA scaffolds are needed. We checked that the activity of GOx and HRP is not altered by the buffer used for DNA origami (Supplementary Fig. 16). Idan and Hess18,27 suggested that the aggregation of multiple enzyme pairs on a large scaffold can lead to throughput enhancement. The colocalization of enzymes does ensure the fast consumption of intermediate and prevent it escaping28, but the work presented by Fu et al.11 clearly showed that enhancement occurs even if there are individual enzyme pairs on each scaffold and the enhancement effect is permanent. Lin and Wheeldon pointed out that attractive interactions between a DNA scaffold and the substrate can facilitate the transport of the substrate to the enzyme29,30, and the model by Idan and Hess18 suggested that the effect could be long-lasting. However, DNA does not attract the negatively charged substrate ABTS31 and the throughput enhancement is caused by facilitated transport and should disappear as the concentration of the intermediate substrate builds up in the reaction vessel.

Recently, signicant enhancement of throughput for the GOxHRP cascade was reported in giant DNA structures. Linko et al.32 encapsulated GOx and HRP in DNA origami tubes with dimensions of 33 27 60 nm3. The activity of

GOx origami and HRP origami increased about threefold and vefold, respectively. Zhao et al.21 anchored different enzymes in DNA nanocages (54 27 27 nm3) and found that many

encapsulated enzymes showed much higher activities than their

free form. Specically, the kcat for the HRP increased from

32 to 290 s 1 after encapsulation, and other enzymes such as GOx, malate dehydrogenase and lactate dehydrogenase all showed increased activities. This suggests that the DNA itself was the major reason for the increase in the enzymatic activities. Zhao and colleagues suggested that the enhancement is because the hydration layer attracted by negatively charged DNA can stabilize the enzymes located around the highly ordered water molecules. However, the ordered water layer only contributes within a few angstroms33,34, and the stabilization of the enzyme is not directly related to a higher activity.

We believe that the many negative charges on large DNA structures, and in particular on DNA origami, are very important for the activity of conjugated enzymes. Similar to polyelectrolyte lms35, the pH close to the surface of a DNA nanoplate is much lower than that in the bulk solution because the protons are attracted to the negatively charged interface, as illustrated in Fig. 6a. The relatively lower local pH will signicantly shift the pH-activity prole of anchored enzymes, as demonstrated by Goldstein and colleagues 50 years ago. They immobilized trypsin on a water-insoluble polyanionic carrier and found that the pH-activity prole of immobilized trypsin shifted toward more alkaline pH values by 12.5 pH units, and this effect is highly depended on the ionic strength of the bulk solution36. A similar effect was also observed for a polyanionic derivative of chymotrypsin and papsin37,38. For the DNA origami, the surface pH is related to the bulk pH and the surface electrical potential c by equation (1) (ref. 39):

pHs pHb

FC2:3RT 1 where pHs is the pH at the surface, pHb is the pH in the bulk solution, F is the Faraday constant, R is the universal gas constant, and T is the temperature.

Although the exact electric surface potential of DNA origami has not yet been reported, we can estimate its magnitude from similar situations. Knudsen et al.40 measured the surface potential of a single-stranded DNA-grafted brush polymer by atomic force microscopy to be 130 mV. Wong and Melosh41

estimated the electrostatic potential at a DNA-hybridized surface. For a DNA-grafting density of 3 1013 cm 2, the surface

potential is also about 130 mV. The density of negative

charges on DNA origami is signicantly higher, although a theoretical description of the resulting surface potential is not straightforward41. We calculated the pH distribution near the surface of DNA origami in a pH 7.5 solution with 10 mM monovalent salt concentration by solving the Poisson

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Boltzmann equation for an innite sheet of uniform charge density of 36 nm 2 (from ref. 21):

r2C

2FC0e sinh

2

In equation (2), C0 denotes bulk salt concentration, and e is the product of the vacuum permittivity constant and dielectric constant of water (see Supplementary Information for details). The pH prole is shown in Fig. 6b and suggests that, even at 2 nm from the scaffold surface, the pH is one unit lower than that of

FC

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Figure 4 | Synthesis and the kinetics of GOxHRP conjugate. (a) Synthesis of GOxHRP conjugate. (b) SDS-PAGE analysis of the conjugate. The ve lanes to the right of the standard marker correspond to native HRP, modied HRP (HRP-SMCC), native GOx, GOxSH and the conjugate. Each lane is loaded with 1 mM protein. (c) Kinetics of HRP in the conjugate; the measurement was carried out with 1 nM conjugated HRP and 2 mM ABTS in PBS buffer.

(d) Activities of free GOx, GOxSH and the conjugated GOx. (e) Activities of free HRP, HRP-SMCC and the conjugated HRP. (f) The time courses of ABTS production and the model prediction based on the known activity of conjugated GOx and the Vmax and Km of conjugated HRP. (g) The differential curve of panel (f) presenting the H2O2 consumption rate. Error bars in panels (d) and (e) represent the s.d. calculated from three replicates.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13982 ARTICLE

a b 100

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Figure 5 | GOxHRP cascade with catalase competition. (a) Schematic of the GOxHRP cascade coupled with a competing reaction catalysed by catalase. (b) The suppression of ABTS production from the GOxHRP conjugate by addition of catalase; here the reaction velocity of GOx is 11.8 nM s 1 and

Vmax of HRP is 25.3 nM s 1. For the free GOxHRP experiment, see Supplementary Fig. 15.

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Figure 6 | Inuence of DNA on the enzymatic activity. (a) Schematic of the distribution of positive ions near the surface of DNA origami. (b) pH-distance prole near the DNA origami. (c) pH-activity prole of GOx in 1 mM glucose and (d) pH-activity prole of HRP in 200 mM H2O2 and 2 mM ABTS. Error bars represent the s.d. of three replicates.

the bulk solution. Steric effects and penetration of counterions into the DNA layer will have large effects at these high charge densities. As a result, the pH prole in Fig. 6b is only a very rough approximation. Its only message is that any enzyme bound to the charged surface spends a non-negligible amount of time in a lower pH regime; this may in turn affect its catalytic activity.

To test whether GOx and HRP activity may be affected, we determined the pH dependence of HRP and GOx activity. Both HRP and GOx show an increased maximal turnover rate in more acidic conditions (Fig. 6c,d and Supplementary Fig. 17). The activity of 1 nM of HRP dramatically increases from 32 to 250 nM s 1 when the pH changes from 7.4 to 4.6, which is very close to the observation by Zhao et al.21 for HRP activity enhancement in a DNA nanocage. Also, according to the studies by Goldstein et al.3638 if the GOx and HRP are anchored on a polyanionic carrier that can decrease the local pH by 2 units (from 7.5 to 5.5), then their activity will increase by1.2- and 4-fold, respectively, leading to an enhanced overall

activity. In the previous publications10,11,17, if the HRP is the limiting enzyme, the lower local pH will signicantly enhance the cascade activity; if the GOx is the limiting enzyme, the combination of the increased activity of GOx and the shortened transient time by the much more active HRP will also result in enhanced cascade activity.

The precise localization of enzyme cascades on DNA scaffolds and the resulting enhancement of the cascade throughput has been one of the most exciting advances in nanobiotechnology in recent years. However, the origin of the observed throughput enhancement, and the contribution of proximity to it, has been debated. Using the canonical GOxHRP cascade, we demonstrated that the nal activity of the cascade reaction is determined by the slower enzyme, which means that throughput enhancement beyond the initial phase requires an increase in the activity of the rate-limiting enzyme. By conjugating GOx and HRP, measuring the kinetics of the individual enzymes and the conjugate and measuring the kinetics of the conjugate in the

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13982

presence of an enzyme competing for the intermediate substrate, we were able to experimentally demonstrate that proximity has no effect on the nal cascade activity, thereby conrming theoretical predictions. We further show that the activity of GOx and HRP increases with decreasing pH and suggest that the origin of the throughput enhancement of the GOxHRP cascade on a DNA scaffold lies in the highly negatively charged DNA lowering the local pH experienced by the enzymes. Based on these insights, three questions should be carefully evaluated in order to support the claim that proximity causes activity enhancement in cascades: is there a steady state and has it been reached? which enzyme is the rate-limiting one? did the individual activity of this enzyme change? Our ndings provide a new insight into DNA scaffold multienzyme systems and point out a novel strategy to optimize the pH in the microenvironment of enzymes.

Methods

Materials. GOx from Aspergillus niger (type VII), HRP, D-glucose, H2O2(30 wt.% in water) and ABTS were purchased from Sigma Aldrich Co. LLC,St Louis, MO, USA; SPDP and sulfo-SMCC were purchased from ThermoFisher Scientic, Inc., USA. For all the experiments, the purchased HRP was rst puried by size exclusion chromatography to remove the impurities (Supplementary Figs 7 and 8). The RZ value (A403/A280) of puried HRP is 2.50.1.

Enzymatic assays. All of the enzyme quantications were carried out on a ultravioletvisible spectrophotometer (Evolution 201, Thermo Scientic, USA). The extinction coefcients for GOx at 280 and 450 nm are 2.67 105 and

2.61 104 M 1 cm 1, respectively. The extinction coefcient for HRP at

403 nm is 1.0 105 M 1 cm 1. Enzymatic activities were measured from

the changes in absorbance at 415 nm (or 600 nm) by the spectrophotometer (for the nal product ABTS radical cation, e415nm 3.6 104 M 1 cm 1 and

e600 nm

1.1 104 M 1 cm 1).

For the GOx kinetic assay, 20 ml of 50 nM GOx was added to 980 ml of substrate solution (in PBS buffer, pH 7.4) to initiate the reaction. The nal assay solution contains 2 mM ABTS, 1 mM glucose, 1 nM GOx and 20 nM HRP. The increase in absorbance at 415 nm was recorded. The activity was computed from the slope of the absorbance versus time curve during the rst 1 min.

The consumption of oxygen by GOx was measured by a dissolved oxygen meter (Mettler Toledo, FiveGo) in PBS buffer containing 1 mM D-glucose in a sealed vessel. We placed the probe of the dissolved oxygen meter in a glass vessel fully lled with 3 ml substrate buffer. As soon as 10 ml of 300 nM GOx was added to the solution (nal concentration is 1 nM), the vessel is immediately sealed with a piece of plastic parafn lm. The oxygen concentration was recorded for 1 h under magnetic stirring.

For the HRP activity assay, 20 ml of 50 nM HRP, 20 ml of 10 mM H2O2 and

20 ml of 2 mM ABTS solution was added to 940 ml of PBS buffer (10 mM, pH 7.4). The absorbance at 415 nm (or 600 nm) was recorded for the rst 2 min.

For the GOxHRP cascade reaction, the nal assay solution contains1 nM GOx, 1 nM HRP, 2 mM ABTS and 1 mM D-glucose. The absorbance at 600 nm was recorded for 1 h.

All the assays were replicated at least twice. We also carried out the experiments with different batches of samples. For the time-course curve, as the zero point drift and the time offset for mixing slightly vary from parallel experiments, we do not average them but analyse them individually.

All the assays were carried out at 231 C.

Synthesis of HRP and GOx conjugate. The HRP and GOx was conjugated with a bi-functional linker, sulfo-SMCC. As the two free cysteine residues on GOx are hard to access, free sulfhydryl groups were rst grafted from the lysine residues on the surface of GOx. In a typical reaction, 40 ml of 80 mM SPDP solution (in dimethyl sulfoxide) was added into 1 ml of 40 mM GOx (in 50 mM phosphate buffer, pH 7.5) with a ratio of 80:1. After stirring for 1 h at room temperature,

20 ml of 1 M dithiothreitol was added to the mixture for 30 min. The excess linker and dithiothreitol were removed by passing the solution twice through a Hitrap desalting column (GE Healthcare Bio-Sciences, PA, USA). The concentration of GOxSH was determined from its absorbance at 280 nm.

HRP was functionalized via the maleimide group with sulfo-SMCC. Typically, 80 ml of 40 mM sulfo-SMCC solution (in 50 C water) was added dropwiseinto 500 ml of 40 mM HRP solution (50 mM phosphate buffer, pH 7.5). The mixture was stirred for 1 h at room temperature. The HRPSMCC was puried by passing it through a Hitrap desalting column twice. The concentration of HRPSPDP was determined by its absorbance at 403 nm.

For the conjugation of GOx and HRP, GOxSH solution was added into the puried HRPSMCC solution at a molar ratio of 1: 1. The mixture was incubated

at room temperature for 1 h and stored at 4 C before separation. The conjugated enzymes were separated by size exclusion chromatography with a HiLoad 16/600 Superdex 200 prep grade column on anKTA system.

Numerical simulation of GOxHRP coupled reaction. As the H2O2 (diffusion coefcient: 1.71 10 9 m2 s 1) can diffuse the average distance

between enzyme pairs (about 1 mm for the system containing 1 nM GOx and1 nM HRP) in 0.6 ms, the H2O2 solution can be considered well mixed on the timescale of the experiment, and the cascade reaction is determined by the kinetics of GOx and HRP. The production rate of H2O2 depends on the GOx activity (equation (3)), the consumption of H2O2 is described by MichaelisMenten kinetics of HRP (equation (4)), the concentration of H2O2 in the solution is described by equation (5) and the production of ABTS follows equation (6).

The initial H2O2 concentration is zero (equation (7)).

r1 kcat;1E1 3

r2

Vmax;2I t

I t Km;2

4

I t r1t Z

t0 r2dt 5

P 2 Z

t0 r2dt 6 I t 0 7

where [I]t is the concentration of the intermediate (H2O2) at time t; r1 is the generation rate of H2O2 by GOx, which is approximately constant in our experiment; kcat,1 is the turnover number of GOx; and Vmax,2 and Km,2 are the maximal velocity and Michaelis constant of HRP, respectively.

Data availability. Data supporting the ndings of this study are available within the article and its Supplementary Information Files and from the corresponding author upon reasonable request.

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Acknowledgements

We thank Miss Kristen Garcia for the purication of the conjugate. This work was supported by the Defense Threat Reduction Agency under award number HDTRA 1-14-1-0051.

Author contributions

Y.Z. and H.H. conceived and designed the research. Y.Z. performed all the experiments. S.T. performed the statistical analysis and analysed the pH prole at the surface of DNA origami. All the authors discussed the results and commented on the manuscript.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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How to cite this article: Zhang, Y. et al. Proximity does not contribute to activity enhancement in the glucose oxidasehorseradish peroxidase cascade. Nat. Commun. 7:13982 doi: 10.1038/ncomms13982 (2016).

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