COFACTOR REGENERATION This invention relates generally to cofactor regeneration. More specifically, although not exclusively, this invention relates to confocal recycling of plural cofactors. Many of the complex chemical pathways of living cells involve catalysis by enzyme cascades confined in zones such as the mitochondrion or Golgi body. ^{1, 2} Enzyme crowding and substrate channelling, resulting from this confinement, are two reasons proposed to account for high rates, efficiency and selectivity. ^3-7 Considering that total macromolecule levels are typically 400 gL ⁻¹ in mitochondria and 200 gL ⁻¹ in the cytosol of a eukaryotic cell, ^8-10 it seems certain that enzyme concentrations in vivo far exceed the high dilutions applying in conventional steady-state enzyme kinetic studies; yet the cellular concentration of any single enzyme is much less important than the systematic corralling of different interdependent enzymes. There is increasing interest in mimicking nature’s nanoconfinement in vitro for enhanced cascade biocatalysis. ^{3, 4, 6, 11} Strategies fall under two approaches: (i) surface-confined, in which enzymes are tethered to a surface ^12-15 and (ii) volume-confined, in which the enzymes are encapsulated in compartments such as microdroplets ^16-18 or protein cage-like structures, incorporated within the matrix of a metal organic framework (MOF) ^{21, 22} or immobilised within a hydrogel ²³ . The various efforts focus on the synthesis of often intricate enzyme nanoconfinement platforms and measurements of their performance. We previously reported how enzyme cascades can be confined, driven, controlled and monitored (in ‘real time’) within the nanopores formed naturally when indium tin oxide (ITO) nanoparticles are deposited on a conducting support. ^24-33 Indium tin oxide (typical composition 4% Sn) is electronically conductive. The basic concept is reported in our earlier patent application PCT/GB2017/050771 and is illustrated as a scheme in Figure 1A. If one of the entrapped enzymes is able to catalyse the direct electrochemical interconversion of nicotinamide cofactors, a new way emerges for exploiting enzyme cascades and investigating enzyme reactions. One such enzyme, ferredoxin NADP ⁺ reductase (FNR), exhibits rapid and reversible direct electron exchange between its active site flavin adenine dinucleotide (FAD) cofactor and the ITO material at which it binds tightly, enabling it to recycle NADP(H) locally to drive the reactions of a neighbouring dehydrogenase. ^{24, 26, 27} In green plants, FNR is responsible for channelling the energetic electrons generated by photosynthesis into organic chemistry: accordingly, the nanoporous electrode loaded with different enzymes which we call the Electrochemical Leaf (e-Leaf (RTM)) is able to mimic practical traits of catalysis in living cells. ^{24, 26, 27, 31} Nanoconfinement of enzyme cascades in such a way offers important advantages over experiments carried out on solutions: (a) in providing a high local concentration of adjacent (producer/receiver) enzyme pairs and by extension, teams of enzymes; (b) in promoting the local retention of intermediates and exchangeable cofactors (such as NAD(P)(H)) so that they are processed or recycled before they diffuse away; (c) in enabling the power of dynamic electrochemical methods to energize, control and observe the processes in a highly interactive way. The experiments are run from a dashboard control D, which includes the electrochemical workstation (direction, driving force, rate, progress) and the means to add or remove reagents to/from the immobilized catalysts. Accordingly, there is a desire to broaden the applicability of our previously-described systems to access different enzymes and/or to drive different chemistries. The advantage of our previously described system is that it does not require expensive chemical reagents; does not require subsequent removal of reagents before re-use; is selective; has large turnover numbers; and which may be deployed to effectively drive important industrially relevant chemical reactions. As such, broadening the applicability of the previously- described systems will bring about manifold advantages. Accordingly, a first aspect of the invention provides an electrochemical system comprising an electrode having a porous layer and plural enzymes and a cofactor located within the pores of the porous layer, a first of said plural enzymes being operable to recycle nicotinamide cofactors in response to an electrical stimulus, a second of said plural enzymes being operable to convert a second species to a first species in the presence of a nicotinamide cofactor and a third of said plural enzymes being operable to convert a reactant to a third species, wherein one or both of the second and third of said plural enzymes are operable to convert the respective second species and reactant in the presence of the cofactor. Advantageously, the nanoconfinement of the further enzymes in the electrochemical system provides the ability to link an electrochemically-mediated reaction to a cooperating chemically-mediated reaction. The term “cofactor” is intended to take its normal definition in biochemistry, i.e. a non-protein chemical compound that is required for an enzyme’s role as a catalyst. IUPAC defines a cofactor as follows: Organic molecules (cf. coenzymes) or ions (usually metal ions) that are required by an enzyme for its activity. They may be attached either loosely or tightly (prosthetic group) to the enzyme. A cofactor binds with its associated protein (apoenzymes), which is functionally inactive, to form the active enzyme (holoenzyme). The porous layer may be formed from nanoparticles and/or from particles having an average diameter in the range of 10 to 1000 nm, for example in the range of 20 to 200 nm, e.g. from 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 or 190 nm to 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 nm. The second of said plural enzymes may be operable to convert the second species to the first species in the presence of the nicotinamide cofactor and/or the second of said plural enzymes is operable to convert the second species to the first species in the presence of the cofactor. There may be further provided one or more further enzymes of said plural enzymes, wherein said one or more further enzymes is or are operable to convert the third species to the second species. For example, the third of said plural enzymes may be operable to convert the reactant to the third species in the presence of the cofactor. The third of said plural enzymes may be operable to convert the reactant and a fourth species to the third species. There may be further provided a fourth and fifth enzyme of said plurality of enzymes, wherein the fourth enzyme may be operable to convert a fifth species to the third species and the fifth enzyme may be operable to convert a sixth species to the fourth species. The plural enzymes may together provide a branched or linear enzyme cascade reaction. In some embodiments more than one of said plural enzymes may be operable to react in the presence of the cofactor. The may also be provided a source of chemical fuel, for example to at least partially drive the recycling of the cofactor. There may also be provided one or more further enzymes, for example to at least partially drive the recycling of the cofactor. In embodiments, the cofactor may be an organic molecule. In embodiments, the cofactor may comprise the nucleotide adenosine monophosphate (AMP). In an embodiment the cofactor may comprise or be ATP (adenosine triphosphate). In embodiments, ATP and/or AMP may be located in the pores of the porous layer of the electrode. In embodiments, the system may comprise chemical fuel to recycle a cofactor, for example a source of phosphoenolpyruvate to recycle ATP. The one or more further enzymes may include pyruvate kinase. In some embodiments the first of said plural enzymes is FNR. In some embodiments, the second of said plurality of enzymes is operable to convert the second species into the first species in the presence of both NADPH and ATP. In some embodiments, the second of said plurality of enzymes is configured to perform an oxidation or a reduction reaction on the second species, e.g. conversion of the second species to a first species in the presence of a nicotinamide cofactor comprise or is an oxidation or reduction reaction, wherein the second species is either oxidised or reduced to produce the first species. In embodiments, the second of said plurality of enzymes is configured to convert a carboxylic acid to an aldehyde. In some embodiments the second of said plurality of enzymes is carboxylic acid reductase (CAR). One or more of said plurality of enzymes may be selected from carbonic anhydrase, phosphoglycerate kinase, rubisco, glyceraldehyde-3-phosphate dehydrogenase, glycerol kinase, glycerol phosphate dehydrogenase. The system may also have the nicotinamide cofactor located in the pores of the electrode and/or may further comprise a counter electrode. The system may also include a source of electrical power. In a specific aspect of the invention there may be provided an electrochemical system comprising an electrode having a porous surface of conductive porous material, wherein plural different enzymes, the enzymes preferably including FNR, nicotinamide cofactor and a second co-factor are located in the pores of the porous surface. Further enzymes may be provided to facilitate the re-cycling of the co-factor. The further enzymes may be located in the pores of the electrode. The nicotinamide cofactor preferably comprises NADP(H). The second co-factor may comprise or be ATP. The electrode may be immersed in a bulk solution. Chemical fuel and one or more reactants may be located in the bulk solution. It will be appreciated that enzymes are able to catalyse reactions reversibly. As such, the reactions in a cascade can be reversed by the simple expediency of reversing the polarity of the electrical source. As such, starting materials or reactants operated upon by the second enzyme can be sequentially converted into products by the n ^th enzyme. There is also provided a method of reacting species in an enzyme cascade, the method comprising: a. locating within the pores of a porous surface of an electrode a plurality of enzymes, a nicotinamide cofactor and a second cofactor, the first of said enzymes being operable to recycle the nicotinamide cofactor is response to an electrical stimulus, a second of said plural enzymes being operable to convert a first species to a second species in the presence of the nicotinamide cofactor and a third of said plural enzymes being operable to convert a reactant to a third species, one or both of the second and third of said plural enzymes being operable to convert the respective second species and reactant in the presence of the second cofactor, and b. providing a chemical fuel to at least partially help drive the recycling of the second cofactor. The importance of energized nanoconfinement for facilitating the study and execution of enzyme cascades that feature multiple exchangeable cofactors may be demonstrated by experiments with carboxylic acid reductase (CAR), an enzyme that requires both NADPH and 2 ATP during a single catalytic cycle. Conversion of cinnamic acid to cinnamaldehyde by a package of four enzymes loaded into and trapped in the random nanopores of an indium tin oxide electrode is driven and monitored through simultaneous delivery of electrical and chemical energy. The electrical energy is transduced by ferredoxin NADP+ reductase which exploits rapid, direct electron transfer to regenerate NADP(H). The chemical energy, provided by phosphoenolpyruvate, a fuel contained in the bulk solution, is co-transduced by adenylate kinase (AK) and pyruvate kinase (PK) which efficiently convert the AMP product back into ATP that is required for the next cycle. Use of the two- kinase system allows the recycling process to be dissected to evaluate the separate roles of AMP removal and ATP supply under pre-steady-state and steady-state catalysis. Embodiments of the invention will now be described by way of example only with reference to the accompanying drawings in which: Figure 1A is a schematic representation of an enzyme cascade energized, observed and controlled under nanoconfinement, according to the prior art; Figure 1B is a schematic representation of a general cascade in which a two-kinase recycling system is co-located; Figures 2A to 2E are schematics of general linear cascade arrangements; Figures 3A and 3B are schematics of general branched cascade arrangements; Figures 4 and 5 are cascade arrangements according to examples of the invention; Figures 6A to 6C are linear cascade arrangements with CAR, according to embodiments of the invention; Figures 7A and 7B are branched cascade arrangements with CAR, according to embodiments of the invention; and Figure 8 is a schematic representation showing confocal recycling of NADP(H) and ATP by a nanoconfined cascade in an Electrochemical Leaf; Figures 9A and 9B are cyclic voltammograms showing the reduction of cinnamic acid by CAR coupled to the interconversion of NADP+/NADPH by FNR with and without kinase (PK and AK); Figures 10A to 10D are cyclic voltammograms showing the development of electrocatalytic activity of the complete four-enzyme cascade of Figure 8 under four different kinase (PK:AK) ratios; Figure 10E is a graph showing the time dependences of the increase in electrocatalytic current density for each kinase ratio shown in Figures 10A to 10D; Figures 11A and 11B are graphs showing the production of cinnamaldehyde by CAR driven via FNR and NADP(H) and by the in situ generation of ATP by PEP catalyzed by AK and PK; Figure 12A is a schematic representation of CAR illustrating the capture of the tightly bound AMP product; Figure 12B shows outcomes resulting from the catalytic cycle of CAR; Figure 12C shows schematic representations of a nanoconfined system without the kinase cascade (left) and with the kinase cascade with a constant amount of PK but different levels of AK (middle and right). In our system, the rapid, simple and inexpensive electrophoretic deposition of ITO nanoparticles on to a conductive support such as graphite or titanium foil results in a robust layer 1-3 µm thick, depending on deposition time (2-10 min.) which is rich in pores less than 100 nm in diameter (preferably demonstrating a mesoporous (2-50nm pores) structure) into which enzymes permeate. ^{24, 27, 34} The procedure creates random nanospace to confine enzyme cascades that can now be energized and observed through FNR. Molecules of FNR (MW 32 kDa) bind with high affinity and are visible and quantifiable through the two- electron reversible cyclic voltammetry due to the FAD cofactor, the reduction potential being as expected for the free enzyme measured in solution. ^{24, 27} The electroactive coverage at pH 7.5 equates to many tens of monolayers, and estimations based on a 1 µm penetration depth show that its local concentration may approach 1 mM. As set out below, we have now demonstrated the use of the above system in enzyme cascade reactions where plural cofactors are recycled. The scheme shown in Figure 1A is the minimal cascade unit - an enzyme pair consisting of a first enzyme (E1), e.g. FNR and a second enzyme (E2), e.g. a dehydrogenase enzyme each of which are tightly bound in the nanopores of an electrode, along with a molecule of a nicotinamide cofactor N, (e.g. NADP(H)) that exchanges between the two. ²⁶ Examples of E2 that we have demonstrated so far include glutamate dehydrogenase, native and variant isocitrate dehydrogenases and alcohol dehydrogenases. ^{26-28, 30, 32, 33} By its tight coupling to E1 via localized and bidirectional nicotinamide (e.g. NADP(H)) recycling (E1↔hydride↔E2) E2 is itself rendered electroactive. Accordingly, our system (which we call the “e-Leaf” (RTM)) now extends Protein Film Electrochemistry (PFE), which has provided a unique insight into properties of enzymes that use long-range electron transfer, ^35-38 to the investigation of a class that includes 1/10th of all enzymes. The electrode is thus described by the notation (E1 + E2 + …)@ITO/support, where E1 acts as transducer, translating the rate of chemical flow into electrical current (and vice versa). An extended cascade has been described, in which E1 = FNR, E2 = L-malate NADP ⁺ -oxidoreductase, E3 = fumarase and E4 = L-aspartate ammonia lyase: this linear chain catalyzes the electrosynthesis of L- aspartate from pyruvate, CO ₂ and NH ₄ ⁺ , with carbonic anhydrase (E2A – comprising a branch linked to E2) allowing bicarbonate (HOCO ₂ ⁻ ) to be used in place of CO ₂ . ³¹ The two non-redox enzymes in the linear chain could be driven in either direction, synthesis or oxidation of aspartate, simply by varying the electrode potential, while the rate and progress of the overall reaction were monitored as current and accumulated charge. Such confinement of all enzymes in an inexpensive electrode material, along with NADP(H) which is required only at low levels, lends itself to scalable electrochemical reactors. ³⁰ The other major exchangeable cofactor for biocatalysis is ATP. Accordingly, we have investigated whether NADP(H) and ATP recycling might be coupled and engaged in a confocal manner, whereby the recycling of both cofactors is required to occur in the same or different regions in an enzyme cascade. Clearly, this would open further chemical avenues and provide means to exploit the chemical selectivity of many more enzymes in both linear and branched cascades. Referring now to Figures 1B, 2 and 3, there are shown general cascades in which a kinase recycling system is co-entrapped in an electrode system as described above, Figure 1B depicts a linear cascade arrangement comprising a 2-kinase recycling system in which enzymes E1 and E2, a first co-factor N and a second cofactor, e.g. ATP, are co- located in the pores of an electrode. A chemical fuel phosphoenolpyruvate (PEP) is provided to facilitate the recycling of the second cofactor, e.g. ATP, and to allow the second enzyme E2 to convert reactants R into products P. It will be appreciated that the second enzyme requires both a first and second cofactor to facilitate the conversion of reactants R to products P. Typically, the chemical fuel will be provided in a bulk solution with reactants. Figures 2A to 2E show schemes of various enzyme cascades in which the second cofactor, e.g. ATP, can help drive an enzymatic cascade reaction. For example, in Figure 2A the second cofactor cycle applies to the third enzyme E3 in the cascade. In Figure 2B, the second cofactor cycle applies to the third enzyme E3 in the cascade but the third enzyme E3 operates on the product of a fourth enzyme E4 in the cascade. Similarly, in Figure 2C the second cofactor cycle applies to the fourth enzyme E4 in the cascade the output of which is supplied to the third enzyme E3. In Figure 2D both a second cofactor cycle (ATP and ATP’) applies to both the third enzyme E3 and the fifth enzyme E5 in the cascade. As will be appreciated, the second cofactor applied to the third enzyme E3 may be the same or different to the second cofactor applied to the fifth enzyme E5. It will be further appreciated that these examples are non-limiting and the second cofactor, or further cofactors, may be applied at any point within an enzyme cascade by utilisation of the appropriate enzymes. In each of Figures 2A to 2D the first enzyme is FNR and the first co-factor is NADP(H). In Figure 2E, the first enzyme is FNR and the first co- factor is NAD(H) demonstrating that the system can be used for both the NADP(H) and NAD(H) nicotinamide cofactors. Clearly, because of the specificity of enzymes and because of their co-location within pores of an electrode it is possible to design enzyme cascades of any length. The co-location in nanopores ensures that all of the enzymes, and especially the successive enzymes within a cascade are in close proximity. At such distances diffusion is practically instantaneous meaning that transport issues do not limit the overall rate of reaction. Figures 3A and 3B show branched cascades which might be employed applying cofactors at different enzymes. Again in a branched cascade the second cofactor, or further cofactors may be applied at any point depending on the enzyme system required. For example, in Figure 3A, the products of enzymes E4 and E5 are reacted by enzyme E3, to which the second co-factor is applied. In contrast, in the cascade reaction of Figure 3Bthe products of Enzymes E3 and E5 are reacted by enzyme E2, the second (or further) cofactors ATP, ATP’ operate on the enzymes E3 and E5. Examples 1 and 2 The enzymes used in Examples 1 and 2 are enzymes from the Calvin cycle. Referring now to Figure 4A, there is shown an example according to the invention, wherein the linear cascade exploited is that of Figure 2A, where enzyme E1 is FNR, enzyme E2 is glyceraldehyde-3-phosphate dehydrogenase, enzyme E3 is phosphoglycerate kinase. In this system, the enzymes E2 and E3 and cofactors NADP+ and ATP are located on the electrode. In a cascade reaction phosphoenolpyruvate (PEP) is provided in the bulk solution together with 3-phosphoglycerate which is sequentially converted to 1,3- bisphosphoglycerate and glyceraldehyde-3-. This can happen with significant turnover of the cofactors due to the presence of electrons at the electrode and PEP in the bulk solution allowing for the regeneration of the cofactors NADP+ and ATP respectively. Referring now to Figure 4B, there is shown an example according to the invention, wherein the branched cascade exploited is that of Figure 3A, where enzyme E1 is FNR, enzyme E2 is glyceraldehyde-3-phosphate dehydrogenase, enzyme E3 is phosphoglycerate kinase, enzyme E4 is rubisco and enzyme E5 is carbonic anhydrase. In this cascade reaction, ribulose 1.5 biphosphate is converted to 3-phosphoglycerate in the presence of enzymes E4 and E5. In the sequential steps, as described above for Figure 4A,, 3-phosphoglycerate is converted to 1,3-bisphosphoglycerate in the presence of phosphoglycerate kinase, and ATP; and 1,3 bisphosphoglyercate is converted to glyceraldehyde-3-phosphate in the presence of glyceraldehyde-3-phosphate dehydrogenase, enzyme E2 and NADP ⁺ . As above, all of the enzymes and the cofactors are located on the electrode, with PEP and ribulose 1,5 biphosphate being provided in the bulk solution in which the electrode is immersed. Examples 3 and 4 The enzymes used in these examples are enzymes from gluconeogenesis. Referring to Figure 5A, there is shown an example according to the invention, wherein the linear cascade which is exploited is as shown in Figure 2E. In this system, phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase are located on the electrode, along with ATP and NADH as cofactors. The enzyme cascade sequentially converts 3-phosphoglycerate to 1, 3-biphosphoglycerate into glyceraldehyde-3-phosphate which may be subsequently processed at a further enzyme in the cascade. Referring now to Figure 5B, there is shown an example according to the invention, wherein the linear cascade which is exploited is that of Figure 2E. In this system, glycerol kinase and glycerol phosphate dehydrogenase are located on the electrode, along with ATP and NAD+. The enzyme cascade sequentially converts glycerol to glycerol phosphate to dihydroxyacetone phosphate. To gain mechanistic insight into how recycling of two cofactors (e.g. NADP(H) and ATP) can be achieved, an enzyme requiring both NADPH and ATP was sought. Carboxylic acid reductase (CAR) (EC 1.2.1.30) catalyzes the reduction of carboxylic acids to their respective aldehydes, a reaction that consumes both NADPH and ATP (which is converted to AMP). CAR obtained from Segniliparus rugosus (Sr) has been characterized by x-ray crystallography: it has two mobile domains, the N-terminal domain containing the adenylation site and the C-terminal domain housing the reductase. ^39-41 In the proposed mechanism, ATP binds with the carboxylic acid substrate and a bound phosphoester intermediate is formed with the release of pyrophosphate (PP _i ): in the subsequent step, a thioester intermediate is formed, and AMP is released. The arm of the enzyme then flips to the reductase domain where the thioester is reduced by NADPH to give the aldehyde product. ⁴¹ The crystal structure reveals a molecule of AMP, presumably bound tightly in the state of the enzyme that is purified. It has been reported how catalysis by CAR could be carried out in solution, using a glucose dehydrogenase to regenerate NADPH and polyphosphate kinases to regenerate ATP. ⁴² The enzyme offered an ideal subject with which to examine how electrochemically-driven NADP(H) recycling and chemically-driven ATP recycling can be combined simultaneously under the condition of nanoconfinement. The resulting system (for example as shown in Figure 1B) incorporated adenylate kinase (AK) K1 to convert AMP and low-level ATP to ADP, and pyruvate kinase (PK) K2 to convert ADP back to ATP using phosphoenolpyruvate PEP as a small chemical fuel molecule. The primary enzyme cascade, consisting of FNR and CAR, is thus linked to a service branch that recycles ATP. The recycling system operates very locally and offers insight into the importance of two separate tasks; (a) providing ATP to CAR, and (b) assisting in the removal of AMP from CAR. Referring now to Figures 6 and 7 there is shown linear cascade arrangements (Figures 6A to 6C) and branched cascade arrangements (Figures 7A to 7B) wherein enzyme E1 is FNR and enzyme E2 is CAR. In each cascade, enzyme E3 provides CAR with a substrate that will lead to a desired product, e.g. cinnamic acid or for the conversion of benzoic acid to benzaldehyde. These cascades are not limited to one substrate. Additional ATP-requiring enzymes can also be used simultaneously or successively and may be applied at any location within a cascade. Referring now to Figure 8, there is shown a schematic of a flow chart showing confocal recycling of NADP(H) and ATP by a nanoconfined cascade in an Electrochemical Leaf (e- Leaf (RTM)). Electrical energy supplied to the porous ITO electrode is transduced by entrapped FNR to regenerate NADP(H) (arrows A1) locally; chemical energy supplied as a fuel in the form of PEP (arrows A2), is transduced by the kinase pair, adenylate kinase (AK, E.C.2.7.4.3) and pyruvate kinase (PK, E.C.2.7.1.40) also trapped in the pores. The central reaction, the reduction of a carboxylic acid to an aldehyde catalysed by CAR, is simultaneously energised by both branches. Table 1 lists sizes and kinetic characteristics of the enzymes used in examples of the invention. Of the four enzymes, only FNR can be quantified on the electrode, through the size of the prominent two-electron voltammetric peaks due to FAD; quantities of each of the other enzymes present on the electrode are varied by adjusting the loading ratio. Notably, CAR is an inherently slow enzyme, and the one most likely to limit the rate (current), whereas the kinases are very active. For FNR the turnover frequency for NADP ⁺ /NADPH interconversion correlates with the rate of direct electron tunnelling between the ITO surface and the FAD active site, which depends strongly on the electrode potential that is applied. Two types of electrochemical experiment were used to monitor electrocatalysis, cyclic voltammetry to study potential and waveshape, and chronoamperometry to conduct cinnamaldehyde electrosynthesis and reveal the effect of periodic refuelling with PEP. As with PFE applied to electron-transport enzymes, it was expected that the shapes of the electrocatalytic voltammograms obtained with the e-Leaf (RTM) under different times and conditions would provide important basic insight into the operation of the cascade and factors limiting catalysis. Three scenarios were anticipated: (1) Control by FNR (electron transfer and NADP ⁺ reduction). The current would have a strong and persistent potential dependence showing that interfacial electron transfer is rate limiting. Since FNR displays fast electron exchange with the ITO surface and rapid, quasi- reversible NADP(H) recycling when studied in isolation, this condition would apply only if turnover by E2 is sufficiently fast to produce a high demand on the rate of NADPH recycling. (2) Steady state catalysis without an intermediate limiting the rate. A sigmoidal wave would result showing a current plateau: this result would be observed if the reaction rate is limited by reactions occurring at E2 or other enzymes. (3) An intermediate is depleted. A peak-like current response would indicate that an essential reactant is being depleted (consumed) faster that it can be replaced. Table 1. Parameters for the enzymes used in this investigation, with references giving further information. * Commercially prepared from rabbit muscle (Merck (Sigma)) Experiments were carried out to investigate how effectively the catalytic activity of CAR could be coupled to NADP(H) recycling by FNR without PK and AK also being loaded and present in the electrode nanopores, ATP instead being present in the external cell solution and thus being required to diffuse into the pores. Figure 9A shows a series of cyclic voltammograms recorded at a scan rate of 1 mVs ⁻¹ (each cycle taking 10 min.) as the concentration of ATP was increased. (The solution was mixed briefly at each addition of ATP). All other reactants were present in solution from the start (20 µM NADP ⁺ in standard reaction buffer). Electrode surface area: 4 cm ² (1 double-sided Ti foil) stationary electrode; 25 °C. The electroactive coverage of FNR determined before introducing NADP ⁺ indicated a value of 22 pmolcm ⁻² : this value equates to a concentration in the region of 0.2 mM throughout a 1 μm depth, but this ignores the space taken up by ITO itself and the local concentration may be significantly higher. Scan 1A corresponds to the background activity of the NADP ⁺ /NADPH interconversion by FNR. At the lower ATP concentrations 1B (0.1 mM- 3.9 mM) no coupling was observed even after several cycles, although interestingly, both the reduction and oxidation peaks corresponding to the bidirectional catalysis of NADP ⁺ /NADPH interconversion by FNR, were enhanced. This effect was found to be reproducible. Although the origin of the ATP enhancement of FNR activity was not pursued further in this study (since it soon became clear that the NADPH regeneration stage, [electrons→FNR→NADPH], is not a bottleneck in the overall process) the observation confirmed that ATP has easy access into the electrode pores to reach the trapped enzymes. A sigmoidal reduction wave 1C due to the catalytic recycling of NADPH back to FNR by CAR, eventually became evident after further additions of ATP resulted in a total concentration of 9.0 mM. The current continued to increase in subsequent successive scans without further ATP additions 1D graduating to black 1E, stabilizing after 2.5 h (> 15 cycles) whereupon an additional injection of ATP to give a total concentration of 18.8 mM did not result in any further increase. A very different result was obtained if PK and AK were included in the loading solution (at a 10:1 PK/AK ratio) with the aim of achieving in situ production and recycling of ATP (Figure 9B) instead of relying on using ATP as a solution-based single-use reactant. The cell solution contained 1 mM AMP along with a small quantity of ATP (10 μM) to act as primer, as well as 20 µM NADP ⁺ , (in standard reaction buffer). Electrode surface area: 6 cm ² (1 double-sided Ti foil); 1 mVs ⁻¹ scan rate; stationary electrode; 25 °C. The electroactive coverage of FNR determined before the addition of NADP ⁺ was comparable to that observed in Figure 9A, at approximately 27 pmolcm ⁻² , despite the additional loading of the kinases in the porous electrode. The catalytic reaction was initiated by introducing PEP to give a final concentration of 2.5 mM. Scan 1A’ corresponds to the background catalysis of NADP ⁺ /NADPH interconversion by FNR, and after initiation by PEP 1C’ the reductive current increased over 1.7 h 1D’ graduating to 1E’. A scan rate of 1 mVs ⁻¹ was used. Notable was the early appearance of a peak-type current response before a sigmoidal waveform was eventually established, indicating that ATP is initially consumed more rapidly than it can be replaced. The final current density obtained after 8 cycles (80 min.) was similar in magnitude to that in Figure 9A indicating that in situ generation of ATP by the kinase enzymes results in a comparable rate to that achieved using 8.8 mM ATP in the bulk solution. Having established the efficiency of in situ ATP recycling, experiments were carried out to investigate the effect of varying the amounts and ratios of the two kinases responsible, only FNR can be quantified inside the electrode, so variations were performed by keeping the quantities of FNR and CAR constant, while changing the concentrations of PK and AK in the loading solution. Importantly, instead of including a low level of ATP as a primer, only the trace level of ATP contaminant present in commercial preparations of AMP was exploited. ⁵³ To detect and quantify this trace of ATP in the AMP preparation ³¹ P NMR spectroscopy was carried out on a solution containing 20 mM AMP and for comparison, a separate solution containing 20 mM AMP and 20 mM ATP. There were no detectable peaks corresponding to ATP in the spectrum for the 20 mM AMP sample, therefore the trace level of ATP was concluded to lie below the detection limit, equating in this case to < 1% and thus < 10 μM in 1 mM AMP. In all subsequent experiments, AMP was present in solution at 1 mM with the exception of Figure 11A (5 mM). Figure 10 shows four sets of cyclic voltammograms in which the performance of the cascade was measured as a function of the amounts of AK and PK present in the loading solution. A scan rate of 1 mV s ⁻¹ was used and each cycle took approximately 10 minutes. All other substrates and cofactors were present from the start (20 µM NADP ⁺ and 1 mM AMP in standard reaction buffer). Kinase ratios were calculated as the number of mols based on tetrameric PK and monomeric AK where 1 represents 0.04 nmols of enzyme in the loading solution and 10 represents 0.4 nmols. Electrode surface area for each experiment: 5.8 cm ² (1 double-sided Ti foil). Other conditions: electrode stationary, 25 °C. For each experiment, the electroactive coverage of FNR was determined before the addition of NADP ⁺ and for PK:AK ratios of 1:1, 10:1, 1:10 and 10:10, the values (in pmolcm ⁻² ) were 24.427.5, 10.7, and 20.5 respectively. There is no trend in FNR coverage with increasing amount of kinase since the values for each extreme (1:1, Figure 10A and 10:10, Figure 10D) are comparable: the result for the 1:10 experiment (Figure 10C) is an outlier and shows that even with this lower amount of FNR present, the system is not FNR- limited since the current density is similar to the others and a sigmoidal shape is retained. Initial cyclic voltammograms in each panel 2A, 2A’, 2A’’, 2A’’’ correspond to the interconversion of NADP ⁺ /NADPH catalysed by FNR. Activation of the FNR/CAR/PK/AK cascade was initiated by adding PEP to a final concentration of 2.5 mM, all other reactants being present from the start (standard reaction buffer). In each experiment, the catalytic current increased with successive continuous cycles represented by 2B, 2B’, 2B’’ and 2B’’’ until a maximum current was reached 2C, 2C’, 2C’’, 2C’’’. The maximum current densities achieved in each case varied by only a factor of two, but the time taken to reach the maximum level decreased in the order: 1PK: 1AK (Figure 10A, 3.7 hours); 10PK: 1AK (Figure 10B, 2 hours); 1PK: 10AK (Figure 10C, 40 minutes) and 10PK: 10AK (Figure 10D, 20 minutes). Figure 10E shows the time dependences of the increase in electrocatalytic current density for each kinase ratio shown in Figures 10A to 10D. Current density was measured at -0.45 Volts vs SHE during the reductive sweep of the cyclic voltammograms and plotted against the corresponding time since initiation of the coupled reaction. The results show that although in situ ATP recycling requires both PK and AK, it is AK that is more important for shortening the lag phase and time required to reach an optimal steady state. Further, by comparing Figure 9B with Figure 10B (both 10PK: 1AK) it was established that the same result can be obtained without adding a known quantity of ATP as primer, relying only on the amount present as a contaminant in AMP. Similar final current densities were reached at 100 minutes and 120 minutes respectively. Figure 11 shows the results of two larger-scale chronoamperometry experiments for the synthesis of cinnamaldehyde by CAR driven via FNR and NADP(H) and by the in situ generation of ATP by PEP catalyzed by AK and PK, in which the enzyme cascade was driven at a fixed reducing potential of -0.42 Volts vs SHE to drive the reduction. Experiments were performed in an anaerobic glovebox to avoid any contribution to the current from the reduction of O2. A ‘book’ of five double-sided ITO@Ti foil electrodes (total surface area, 25 cm ² ; a booklet of 5 double-sided Ti foils) were used. After loading enzymes overnight, the electrodes underwent stringent rinsing in ultrapure water and were subsequently transferred to the glovebox in fresh buffer. In the experiment shown in Figure 11A, the electrode was loaded using the following mixture: 21.5 nmols of CAR, 9 nmols of FNR, 0.5 nmols of PK and 1.7 nmols of AK (resulting in concentrations of 2.6 µM, 1.1 µM, 0.2 µM and 0.06 µM respectively) in 100 mM TAPS pH 8. The reaction was initiated by the addition of NADP ⁺ to a final concentration of 20 µM (all other reagents were present from the start (5 mM AMP and 5 mM PEP in standard reaction buffer, 10 mM cinnamic acid, 10 mM MgCl ₂ , 10 mM KCl, 5 mM NaPi, in 100 mM HEPES, pH 7.5); the cell solution (5 mL) was stirred throughout with a magnetic flea. Injection of NADP ⁺ (shown by arrow A3) resulted in a rapid increase in current (over the duration of 1-2 minutes, see inset showing magnification of the live injection of NADP ⁺ ; electrode surface area: 25 cm ² ) which decreased gradually to a low level after 12 h. A sample for NMR analysis was taken at approximately 15 h., after which time the charge passed was equivalent to 1.29 x 10 ⁻⁵ moles – a concentration (in 5 mL) of 2.57 mM. Analysis of the NMR spectrum showed a concentration of 2.78 mM. The agreement between coulometric and NMR values is quite reasonable considering several factors associated with the small volume and current scales: (i) water evaporation over 15 h which would concentrate the NMR sample (the cell was not perfectly sealed and cinnamaldehyde is much less volatile than water); (ii) difficulty in allowing for background current (an estimation based on three background values, gives 1.29 x 10 ^-5 moles +/- 0.25 x 10 ⁻⁵ moles, equivalent to a concentration of 2.78 mM +/- 0.5 mM); (iii) migration of reactants and products into side arms. Since two moles of PEP should be consumed for each mole of cinnamaldehyde, the result demonstrates that the reaction can run until PEP is exhausted. Figure 11B shows the results of a parallel experiment in which the cascade reaction was initiated instead by introducing a substoichiometric amount of PEP (indicated by arrow A4) (to a final concentration of 1mM) and then ‘refuelled’ twice by further additions during the time course. The electrode was loaded as outlined for Figure 11A with one change – a lower amount of PK was used (0.2 nmoles). All other reagents were present from the start (20 µM NADP ⁺ and 1 mM AMP in standard reaction buffer, 10 mM cinnamic acid 10 mM MgCl ₂ , 10 mM KCl, 5 mM KP _i , in 100 mM HEPES, pH 7.5; electrode surface area: 28 cm2 (a booklet of 5 double-sided Ti foils); other conditions: 25 °C, solution stirred throughout.). In contrast to the experiment initiated by NADP ⁺ , a lag of approximately 25 min was observed before the current started to increase (Figure 11B, inset). The current then started to decrease, more rapidly than observed with the 5-fold higher PEP concentration. After a total of approximately 2.4 h it was estimated, from the charge passed, that the PEP level should have decreased from 1 mM to 0.38 mM: at this point, the cascade was refuelled (arrow A5) by adding a second equivalent addition of PEP (taking into account the PEP depleted during the first stage, this gave a total concentration in the bulk solution of ~ 1.38 mM). In contrast to the injection of PEP made initially, no delay was observed, the current increasing immediately. After approximately 4.3 h, a sample was removed for NMR quantification of cinnamaldehyde (arrow A6), the result showing a concentration of 0.65 mM, equivalent to 3.28 μmoles in the cell volume at that point, 5.05 mL. For comparison, the total charge passed up to this point was 0.59 C, equating to 3.1 μmoles cinnamaldehyde and a concentration of 0.60 mM. The cascade was refuelled again at 4.4 h (arrow A7) by the addition of PEP to a final concentration of ~ 5.15 mM (taking into account the amount of PEP estimated (by charge passed) to have already been consumed at this stage and the 20 volume change due to removal of the sample for NMR). Once again, the increase in current was immediate. The current was monitored for a further 7h. The total charge passed during the entire experiment was 1.28 C, equating to 6.6 µmoles of cinnamaldehyde. The final concentration of cinnamaldehyde from NMR analysis in ~4.55 mL of total remaining solution was 1.79 mM, thus equating to approximately 8.1 μmoles. Accordingly, there is good agreement between coulometric and NMR values. The strong catalytic current observed in CVs and controlled potential experiments is directly related to the rate at which cinnamic acid is converted to cinnamaldehyde. The results can be interpreted using basic electrochemical guidelines and they reveal considerable insight into how and why nanoconfinement of an enzyme cascade produces such efficiency. All voltammetric waves attributed to cinnamic acid reduction have onset potentials that coincide with the reduction of NADP ⁺ to NADPH. Further, the waveforms are either sigmoidal or peak-type, demonstrating that interfacial electron transfer between ITO and FNR is not rate-limiting: had this been the case, the current would continue to increase with potential rather than reach a plateau, as the rate of electrocatalytic regeneration of NADPH struggles to match demand by CAR. The clear implication is that the rate of cinnamic acid reduction is limited by subsequent chemical steps, either the turnover by CAR or the supply of ATP and reactants. Notably, there is a striking contrast between the two experiments shown in Figures 9A and 9B, i.e. ‘bulk’ ATP, and ‘in situ’ ATP. The first important point is that the final outcomes are similar in terms of waveshapes and current density; however, a very high solution concentration of ATP was required to achieve the final sigmoidal response for the simple FNR + CAR cascade, whereas only a trace (contaminating) amount of ATP (estimated to be no more than 10 μM) from the 1 mM AMP present in solution was required if PK and AK had been also been loaded into the electrode. A second point is more subtle. During the build-up of activity in the bulk-ATP experiment, the voltammogram is always sigmoidal, in other words a steady state pertains throughout. In contrast, during the build-up of activity in the in-situ experiment which uses PK and AK along with AMP and PEP in solution, peak-type voltammograms are observed early in the experiment, showing that an essential component is being depleted. The eventual transformation to a sigmoidal waveform shows that this component cannot be PEP (which is consumed having transferred from high (2.5 mM) concentration in bulk): instead, it is certainly ATP, initially present only at low concentration and prone to depletion until a sufficiently high level has been established. An initial priming quantity of ATP is essential as there is no mechanism whereby the PK/AK recycling system can produce it starting from AMP alone. The results shown in Figure 10 confirm that 10 μM ATP, now introduced intentionally, is able to prime the recycling process. The four CV experiments (Figures 10A to 10D), conducted with different amounts and ratios of PK and AK, reveal two facts. First, the waveforms increase in magnitude and transform from peak-type to sigmoidal, the rate of development increasing with total kinase loading and the transitory peak-type phase disappearing at the highest total loading. Consequently, early on, the available trace amount of ATP is quickly depleted, but eventually its production rate is sufficient to sustain a steady state. Second, the final sigmoidal current densities vary by less than a factor of two from very low (1:1, Figure 10A) to high (10:10, Figure 10D) and the final, marginal optimization is achieved by a tenfold increase in PK concentration. A high concentration of ATP is needed to achieve catalysis by CAR when it is supplied as a stoichiometric reactant. One key piece of evidence supporting this is the sensitivity of NADP(H) recycling by FNR, alone, to the presence of ATP, a result that could be reproduced in numerous separate experiments. Although the molecular interpretation of this enhancement of FNR activity is yet to be investigated, the result confirms the rapid arrival of ATP in the immediate pore locality. Attention is thus directed instead to AMP, which appears from solution kinetic studies to be a weak inhibitor of CAR (Ki= 8.2 mM). ⁵⁴ However, any such simple interpretation contradicts an important direct measurement, namely that the bound AMP that is identified in the crystal structure of CAR was not introduced separately but remained throughout purification. ⁴⁰ The logical explanation for this discrepancy is that in the absence of turnover conditions, CAR adopts a resting inactive state in which AMP is tightly bound. Referring now to Figure 12A, there is shown a proposal for how capture of the tightly bound AMP product, revealed in the crystal structure of CAR accounts for the efficiency of confocal ATP accumulation and recycling in situ compared to ATP supplied from the bulk solution. Referring to Figure 12B, there is shown possible outcomes throughout the catalytic cycle of CAR: NADPH binds to the enzyme species with the carboxylic acid intermediate and AMP both bound, the reduction step is then catalyzed to produce the aldehyde. At this point, E-AMP either dissociates, releasing AMP to regenerate active CAR, or switches to a ‘resting inactive state’ shown in orange, in which AMP is more tightly bound (which is the state revealed in the crystal structure, Figure 12A). Referring to Figure 12C, there is shown a nanoconfined system without the kinase cascade (left) and with the kinase cascade with a constant amount of PK but different levels of AK (middle and right). The special importance of adenylate kinase (AK) lies in its ability to sequester the AMP (arrows A8), allowing CAR to start the next cycle. Without the kinase cascade present, the probability that the E’AMP state persists in the pores is high since AMP sequestration is not possible, hence it is shown in bold. With the kinase cascade present, E’-AMP is less persistent, (shown by increasing transparency as the level of AK increases (middle to right); thus the system with more AK achieves an optimal steady state more rapidly than the system with less AK, as depicted in Figure 10B and 10D. The lag period is determined by how quickly active CAR is regenerated from the E’AMP inactive complex, aided by the removal of local AMP by AK. As illustrated in Figure 12, the tight binding of AMP in a resting state provides an explanation for why ATP introduced as a stoichiometric reagent is so ineffective compared to the localized recycling system. First, with regard to the lag period, in order to initiate the first catalytic cycle, AMP must first be released and sequestered. A negative effect of nanoconfinement would be to restrict its escape by diffusion into bulk solution, allowing it to re-bind. In the absence of AK to sequester AMP, the catalytic activity thus relies only on its escape through the pores. In contrast, when the entire AK/PK recycling system is in place, catalysis is initiated with just the trace amount of ATP that is present initially. The emphasis thus shifts to the fact that a crucial role of the recycling system is to remove AMP efficiently when nanoconfinement would retard its escape, an advantage that continues in subsequent catalytic cycles. The conclusion we reach is that local recycling not only supplies ATP but removes AMP, a function that is not performed when ATP is supplied as a stoichiometric reagent. The use of a two-enzyme ATP recycling branch as opposed to a single-enzyme system allows the roles of each stage to be dissected. The mechanism of CAR is thus more complicated than previously thought. The problem for precise modelling of the nanoconfined system lies in the uncertainty surrounding the actual quantities of CAR, AK and PK actually present in the ITO pores. The concept thus emerging also hinges on the advantage afforded by the fact that CAR is inherently the least active of the four enzymes. Were it to be highly active, then even once activity has started to increase, the ATP required to prime the PK/AK service cycle would be spent (by CAR) before it could be invested (by AK) to produce ADP and produce (by PK) more ATP. Locally generated ATP is clearly much more effective than relying on its diffusion from solution. The results of the controlled potential electrolysis (chronoamperometric) experiments shown in Figure 11 provide firm support for these cascade dynamics. Provided the PK/AK service cycle has already been primed after introducing PEP, reduction of cinnamic acid to cinnamaldehyde starts rapidly after injecting NADP+. In contrast, if NADP+ is already present and the reaction is initiated instead by injecting PEP, there is a delay (Figure 11B inset). Ultimately, the performance of the cascade depends on all enzymes and recyclable components being resident in the electrode. Given an optimal balance of all four enzymes, the catalytic rate is simply determined by the supply of PEP. The likelihood that CAR is the slowest enzyme allows us to place a lower limit on the local concentration of this enzyme that is active: based on a current density of 7 μA cm ⁻² and the published turnover frequency of 5 s ⁻¹ , the surface (2D) coverage must be at least 7 pmolescm ⁻² . Distributed across a depth of 1 μm (which ignores space taken up by ITO itself) the local concentration would need to be considerably higher than 0.07 mM. The e-Leaf (RTM) thus emerges as a confocal dual cofactor recycler to drive a complex cascade simultaneously by two sources of energy, electrical through FNR and NADP(H) and chemical via PEP, which serves as the fuel. In the light of recent work demonstrating that ITO electrodes can be scaled up to many hundreds of cm ² for small scale production, it is clear that the e-Leaf can be developed to exploit the immobilisation and nanoconfinement of entire enzyme-based synthetic pathways, avoiding steps and minimizing losses. The experiments described here were not carried out with the object of optimizing turnover numbers for NADPH and ATP, which are at best, just 90 for NADPH and > 180 for ATP (from Figure 11A); there is plenty of scope for improvement in these metrics, longer experiments or lower concentrations being obvious ways forward. In any case, as these cofactors are being recycled locally, within the ITO pores, the real turnover numbers (per entrapped molecule) must be orders of magnitude higher. With the introduction of nanoconfined ATP recycling, an obvious exploitation of the discovery would be in the area of cancer research where malfunctioning kinase enzymes are central. ⁵⁵ Finally, the ability to drive and study an enzyme that uses both reducing energy and ATP has further implications. In the case of CAR, simultaneous ATP consumption is required to increase the reducing power of NADPH, which is otherwise thermodynamically incapable of reducing a carboxylic acid to its aldehyde. Another enzyme of considerable current interest is nitrogenase, which uses electrons from a FeS protein and ATP, again in a coupled simultaneous process, to produce NH ₃ from N ₂ . ⁵⁶ The lessons learnt from this investigation and the practical ways in which electrochemical and chemical energy can be simultaneously supplied to complex immobilised enzyme systems may therefore be harnessed to advantage in several different ways. Methods All electrochemical experiments were performed under anerobic conditions in a glovebox (MBraun or Belle Technologies) containing a N ₂ atmosphere (O ₂ < 2 ppm), with a three- electrode configuration using a three- compartment glass cell for chronoamperometry or a two-compartment cell for cyclic voltammetry. Electrochemical measurements were made using an Autolab (PGSTAT128N) or EcoChemie potentiostat with Nova software. Potentials (E) are quoted with respect to the standard hydrogen electrode (SHE) using the correction ESHE = ESCE + 0.241 V at 25 °C or ESHE = EAg/AgCl + 0.21 V.52 Electrodes were prepared by electrophoretic deposition of ITO nanoparticles onto each side of pieces of Ti foil, as described in Chem. Sci. 2017, 8 (6), 4579-4586. ²⁴ Ferredoxin NADP ⁺ reductase (FNR) from Chlamydomonas reinhardtii was expressed in E. coli and purified as described in Angew. Chem. 2019, 58 (15), 4948-4952 and ChemElectroChem 2020, 7 (22), 4672- 4678 and as described below. Post-translational phosphopantetheinylation of CAR is required for maximum enzyme activity, therefore CAR from Segniliparus rugosus was co- expressed in E. coli with a phosphopantetheine transferase from Bacillus subtilis, as set out later. Rabbit muscle pyruvate kinase (Type VII) and adenylate kinase were obtained from Merck (Sigma). The enzymes, FNR, CAR and when included, AK and PK, were mixed and loaded as follows: a fresh ITO@Ti electrode was placed in a buffered solution (100 mM TAPS, pH 8) containing the specified number of moles of each enzyme and stirred overnight in a coldroom at 4 °C. The electrode was then rinsed thoroughly in a stream of ultrapure water (Millipore, 18 MΩ cm) to remove any unbound enzyme. This rinsing took place outside the glovebox, following which the electrode was immersed in fresh buffer (the same as that intended to be used in the experiment) and purged in the glovebox port for several minutes before being taken into the box. The amount of electroactive FNR loaded on the electrode was estimated, as described previously, by measuring the area of the background subtracted peaks observed in the absence of NADP ⁺ . ²⁴ The other enzymes are not electroactive, precluding such measurement. We thus adopted a strategy used previously, in which the ratio of enzymes in the loading solution was varied to optimize the electrocatalytic response. The concentration and retention of NADP ⁺ in the electrode pores allows for the use of concentrations typically in the range 5- 20 µM. ^{26, 27, 30, 31} In this work, we used 20 µM NADP ⁺ to optimise signal to noise. This concentration was chosen based on a double titration experiment (both ATP and NADP ⁺ ) for the coupling of CAR to FNR. For the experiments shown in Figures 9 and 10, the amount of FNR added to the loading solution was kept constant at 1.8 nmoles, resulting in a concentration of 0.6 μM for Figure 9A and 0.5 µM for Figures 9B and 10(A-D). With the exception of Figure 9A, the amount of CAR added to the loading solution was kept constant at 5.4 nmoles resulting in a loading concentration of 1.5 µM (4.3 nmoles for Figure 9A resulting in a concentration of 1.4 µM). Consequently, the FNR:CAR ratio was 1:3 for each experiment (apart from 1:2.4 for Figure 9A). The PK:AK ratios were calculated in terms of the number of moles based on tetrameric PK and monomeric AK where 1 (unity) represents 0.04 nmoles of enzyme in the loading solution and 10 represents 0.4 nmols. The reaction buffer for all experiments contained 10 mM cinnamic acid, 10 mM MgCl ₂ , 10 mM KCl, 5 mM phosphate (KP _i ) and 100 mM HEPES, pH 7.5, referred to throughout as ‘standard reaction buffer’. The NADP ⁺ concentration in the bulk solution was constant throughout at 20 µM. Further details of the methods are explained below: FNR Production A transformation of Escherichia coli cells (BL21(DE3)) was carried out by uptake of a vector (aLICator pLATE 51, Thermo Scientific) containing the gene which encodes FNR from Chlamydomonas reinhardtii with an N-terminal His-tag and the genes to confer ampicillin resistance. The cells were then plated on lysogeny broth (LB)-agar plates containing ampicillin at 0.3 mM and incubated overnight at 37 °C. Resistance to ampicillin was used to select positive transformants. A single colony was used to inoculate 100 mL of LB media (25 gL ^-1 ) containing ampicillin (100 µgmL ^-1 ) and grown overnight at 37 °C in a shaking incubator (Innova) at 200 rpm. The following day, 10 mL of this overnight culture was diluted into 500 mL of fresh LB containing ampicillin at 0.3 mM and grown at 37 °C, 200 rpm for approximately 3 hours at which point they were induced by the addition of IPTG to a final concentration of 1 mM and grown under the same conditions for a further 3-4 hours. The cells were harvested by centrifugation and the pellets resuspended in cold buffer (50 mM HEPES, 150 mM NaCl, 10% V/V glycerol, pH 7.4) and stored at -80 °C until purification. FNR purification: cells were lysed three times using a French press at 20 psi after which, cell debris was removed by centrifugation at 45000 rpm for 1 hour (Beckman Ultracentrifuge). The supernatant was loaded onto a Ni ^2□ HisTrap affinity column (GE Healthcare Life Sciences) using an Äkta purification system with dual wavelength absorbance detector. The column was washed with 50 column volumes of buffer (50 mM HEPES, 500 mM NaCl, 1 mM dithiothreitol (DTT), pH 7.4. A linear concentration gradient of imidazole reaching a maximum of 250 mM in ~30 min at a rate of 1.5 mLmin ^-1 , was used to elute 1 mL fractions. Those fractions containing FNR were based on the absorbance at 280 nm and 460 nm, the latter corresponding to the flavin cofactor. The fractions containing FNR can easily be detected by eye since they are bright yellow but attention to the peak at 280 is important for the identification of fractions at the beginning of the elution gradient which also may contain contaminant proteins. Fractions containing FNR were pooled and concentrated using a 10 KDa molecular weight cut-off centrifugal filter (Amicon®). Imidazole was removed by dialysis overnight at 4°C against 2 L of dialysis buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 1 mM DTT, pH 7.4). FNR was aliquoted into small portions, (5 – 10 µL), flash frozen in liquid nitrogen and stored at -80 °C. CAR Production Two plasmids encoding a His-tagged CAR from Segniliparus rugosus (Sr) and an untagged phosphopantetheine transferase (Sfp) from Bacillus subtilis were used to co-transform Escherichia coli cells (BL21(DE3)). The Sfp enzyme was required to carry out a post- translational modification of the co-expressed CAR (phosphopantetheinylation). The vector containing the gene to encode CAR also encoded the genes to confer resistance to kanamycin (kan) while the vector containing the gene to encode Sfp, also encoded resistance to streptomycin (strep); double resistance to both antibiotics was used to select positive transformants containing both plasmids. The cells were plated on a series of LB- agar plates containing three concentration sets for each antibiotic (plate A 0.03 mgmL ^-1 kan and 0.05 mgmL ^-1 strep; plate B 0.021 mgmL ^-1 kan and 0.035 mgmL ^-1 strep; plate C 0.015 mgmL ^-1 kan and 0.025 mgmL ^-1 strep). Positive transformants grew on all three plates and so the highest antibiotic concentration was chosen for subsequent expression. A single colony was used to inoculate 100 mL of LB containing 0.03 mgmL ^-1 kan and 0.05 mgmL ^-1 strep and the culture was grown overnight at 37 °C at 200 rpm; the overnight culture was then diluted (5 mL / 500 mL (X8)) into autoinduction media (~35 gL ^-1 ) (Formedium) containing 0.03 mgmL ^-1 kan and 0.05 mgmL ^-1 strep and grown for 72 hours at 20 °C (200 rpm). The cells were harvested by centrifugation, the pellets resuspended in cold buffer (50 mM HEPES, 150 mM NaCl, 10% V/V glycerol, pH 7.4) and stored at ^80 °C until purification. The CAR was purified as described for FNR but with a less steep imidazole elution gradient reaching a maximum of 250 mM in ~60 min at a rate of 1.5 mLmin ^-1 eluting in 1 mL fractions. The absorbance at 280 nm was used to identify the fractions containing CAR and this was confirmed by activity assay. UV-VIS spectroscopy was used to monitor the rate of NADPH depletion by CAR during its catalysis of the reduction of cinnamic acid to cinnamaldehyde. In brief, all reactants (200 µM NADPH, 10 mM cinnamic acid, 10 mM MgCl ₂ , 10 mM KCl, 10 mM ATP in 100 mM HEPES buffer pH 7.5) were added to a quartz cuvette and the absorbance at 340 nm measured for approximately 1 min before the addition of ~1-5 µL from each elution. A decrease in absorbance at 340 nm (due to the depletion of NADPH as it is oxidised to NADP ⁺ confirmed the presence of active CAR. Active fractions were pooled and concentrated using a 50 KDa molecular weight cut-off centrifugal filter (Amicon®). Imidazole was removed by dialysis overnight at 4°C against 2 L of dialysis buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 1 mM DTT, pH 7.4). CAR was aliquoted into 100 µL portions, flash frozen in liquid nitrogen and stored at -80 °C. Accordingly a further aspect of the invention provides a method of producing CAR and a composition comprising CAR, preferably with a 50 kDa maximum mass, which CAR has been modified post-translation by a Sfp enzyme, the CAR preferably being derived from cells with resistance to one or more of kanamycin and streptomycin. ITO Electrode Preparation Indium tin oxide (ITO) electrodes were made by electrophoretic deposition of ITO particles (Sigma) onto titanium foil (Sigma), which acted as a conductive support. A suspension of ITO (1 mgmL ^-1 ) and iodine (0.5 mgmL ^-1 ) was prepared in acetone and sonicated for at least 30-45 min. The Ti foil electrode was aligned to face an auxiliary electrode of similar size (typically ITO glass or another piece of Ti foil) with approximately 1-2 cm separation. Both were held in this orientation in the ITO suspension and a voltage of 10 V was applied for approximately 7 minutes per electrode side. The ITO electrode was removed and allowed to dry in air before use. For the large-scale chronoamperometry experiments shown in Figure 3, five ITO@Ti foil electrodes were made into a booklet by punching a hole through the top of each foil and threading Ti wire through several times to ensure electrical connection between each. This edge of the booklet was compressed using a vice to give stability and also to ensure all electrodes were connected to the wire. Before use, all electrodes were rinsed thoroughly in ultrapure water (Milli-Q™, 18 MΩcm). Buffer production For all experiments, the standard reaction buffer solution contained: 10 mM cinnamic acid, 10 mM MgCl ₂ , 10 mM KCl , 5 mM phosphate (K ⁺ ) and 100 mM HEPES (pH 7.5). Pyruvate kinase requires K ⁺ ions and is highly selective for K ⁺ over Na ⁺ therefore KCl was included in the reaction buffer and the solution pH was adjusted using KOH rather than NaOH. Inorganic phosphate was included based on its enhancement of PK activity. CAR and both kinase enzymes require Mg ²⁺ ions, therefore MgCl ₂ was included at 10 mM. A pH of 7.5 was chosen based on the activity of CAR previously measured in solution. For all additions of reactants to initiate or titrate, concentrated stocks were prepared in standard reaction buffer (for the PEP concentrated stock solution, cinnamic acid was omitted because a precipitate formed in this case) and the pH adjusted again to 7.5 so that the additions (typically 10 – 50 µL) to the cell solution did not dilute the other reactants or change the pH. Fresh concentrated stock solutions of NADP ⁺ , PEP and AMP were prepared for each experiment. For the ATP titration experiment, a 0.5 M stock ATP solution (dissolved in standard reaction buffer) was prepared and the pH adjusted to 7.5 so that the additions throughout the experiment did not alter the pH (the stock was flash frozen as single-use aliquots and stored at -80 °C). A concentrated stock solution of phosphate buffer (prepared by titrating the monobasic salt with dibasic salt to pH 7.5) was used as a stock solution such that a small volume addition would achieve the 5 mM final concentration in the cell solution. The concentration of NADP ⁺ used throughout was constant at 20 µM; AMP was 1 mM with the exception of the experiment shown in Figure 3A which contained 5 mM AMP; PEP was 2.5 mM with the exceptions set out above. Enzyme Loading The desired number of moles of each enzyme were added to a buffer solution (100 mM TAPS pH 8) in a glass vial (a sufficient volume was used to cover the submerged electrode). The vial was placed on a magnetic stirrer in a 4 °C cold-room where it was stirred overnight using a magnetic flea. All electrodes were rinsed thoroughly in a stream of ultrapure water for at least 1-2 minutes before use. The electrode was then submerged into fresh buffer containing no enzyme, for transport into the glovebox. Electrochemical Measurements All experiments were performed under anerobic conditions using a glovebox (MBraun for all cyclic voltammetry or GBT Technologies for chronoamperometry) containing an N ₂ atmosphere (O ₂ < 2 ppm). Cyclic voltammetry measurements were made using an Autolab potentiostat (PGSTAT128N) with Nova software; the potential was swept linearly between the upper and lower limits at a scan rate of 1 mVs ^-1 beginning at the more oxidising potential. Chronoamperometry experiments were controlled using an EcoChemie autolab with Nova software. Measurements were made using either a two-compartment or three-compartment glass cell for cyclic voltammetry experiments and large-scale synthesis chronoamperometry experiments respectively. The two-compartment cell consisted of a working electrode compartment (water-jacketed and plumbed to a circulating water bath to maintain a constant temperature of 25 °C) connected, via a Luggin capillary, to a nonisothermal side arm housing a standard calomel (SCE) reference electrode. The working electrode compartment also contained a platinum wire counter electrode which completed the three- electrode configuration. The three-compartment cell was configured for an Ag/AgCl reference electrode and its additional side arm housed the counter platinum mesh electrode and was separated from the main working compartment by a glass frit. The side arms in both configurations contained 0.1 M NaCl. Potentials (E) are quoted with respect to the standard hydrogen electrode (SHE) using the correction ESHE = ESCE + 0.241 V at 25 °C or ESHE = EAg/AgCl + 0.21 V. It will be appreciated by those skilled in the art that several variations to the aforementioned embodiments are envisaged without departing from the scope of the invention. It will also be appreciated by those skilled in the art that any number of combinations of the aforementioned features and/or those shown in the appended drawings provide clear advantages over the prior art and are therefore within the scope of the invention described herein. REFERENCES 1. Pareek, V.; Tian, H.; Winograd, N.; Benkovic, S. J., Metabolomics and mass spectrometry imaging reveal channelled de novo purine synthesis in cells. Science 2020, 368 (6488), 283-290. 2. Zhao, X.; Palacci, H.; Yadav, V.; Spiering, M. M.; Gilson, M. K.; Butler, P. J.; Hess, H.; Benkovic, S. J.; Sen, A., Substrate-driven chemotactic assembly in an enzyme cascade. Nature Chemistry 2018, 10 (3), 311-317. 3. Schoffelen, S.; van Hest, J. C. M., Multi-enzyme systems: bringing enzymes together in vitro. 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