Enzyme cosubstrate

In DET, the enzymatic and electrode reactions are coupled by direct (mediatorless) electron transfer. In this case, the electron is transferred directly from the electrode to the substrate molecule (or vice versa) via the active site of the enzyme. In such a system, the coupled overall process is the redox transformation of the substrate(s), which can be considered as an enzyme-catalyzed electrode process. According to this mechanism, the electrode surface acts as the enzyme cosubstrate, and the enzymatic and electrode reactions cannot be considered as separate, but as formal stages of the bioelectrocatalytic reaction mechanism. The catalytic effect of the enzyme is the... 

Calculate the overall Kiq and AG at pH 7 and 25 C for the conversion of fumaric acid to citric acid in the presence of the appropriate enzymes, cosubstrates, and cofactors. 

Regeneration enzyme Cosubstrate/ coproduct Specific Activity [U mg 1] Stability Coenzyme E 0 [V] vs. NHEa i1 ... 

Several enzymes can be immobilized within the same reaction layer, in order to increase the range of possible biosensor analytes, provide efficient regeneration of enzyme cosubstrates, or to improve the biosensor selectivity by decreasing the local concentration of electrochemical interfering substances. 

Fig. n Cyclic voltammetry of the avidin-biotin monolayer enzyme-cosubstrate integrated system (Sch. 5) in the absence (a) and presence of 0.5 M substrate in a phosphate buffer (pH = 8) at 25 and a scan rate of 0.04 V sec . (c) Variation of the inverse of the plateau current with the inverse of substrate concentration. 

The E. coli enzyme accepts substitution on either cosubstrate propanal, acetone or 1-fluoro-2-propanone can replace the donor and a variety of aldehydes can replace the acceptor moiety 3. Shortcomings are the relatively low conversion rates obtained for any substrate analog and the as yet unidentified level of relative stereocontrol induced upon substitution at the nucleophilic carbon. 

All NOS isoforms utilize L-arginine as the substrate, and molecular oxygen and reduced nicotinamide adenine dinucleotide phosphate (NADPH) as cosubstrates. Flavin adenine dinucleotide (FMN), flavin mononucleotide (FAD), and (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) are cofactors of the enzyme. All NOS isoforms contain heme and bind calmodulin. In nNOS and eNOS,... 

Interestingly, the E. coli enzyme s relaxed acceptor specificity allows for substitution of both cosubstrates, albeit at strongly reduced (<1% of v, catalytic rates. Propanal, acetone, or fluoroacetone can replace ethanal as the donor in the synthesis of variously substituted 3-hydroxyketones such as (112) or (113) (Figure 10.41)... 

A mutant E. coli strain LS5218 (fadR atoC) was employed for the synthesis of P(3HB-co-3HV) copolymer since this mutant strain constitutively expresses the enzymes involved in the transport and utilization of short chain fatty acids [58, 59]. P(3HB-co-3HV) could be synthesized by a recombinant E. coli strain LS5218 harboring the R. eutropha PHA biosynthesis genes when propionic acid or valeric acid was added as a cosubstrate [58,60]. The P(3HB-co-3HV) copolymer consisting of up to 40 mol% of 3HV could be produced. An alternative method that allowed synthesis of P(3HB-co-3HV) using propionic acid or valeric... 

The class II secreted fungal heme peroxidases include the LMPs LiP, MnP and VP [70]. All of these enzymes are extracellular and contain protoporphyrin IX (heme) as prosthetic group. They use H2O2 or organic hydroperoxides as electron accepting cosubstrates during the oxidation of diverse compounds. They are secreted as glycosilated, 35-38 kDa size proteins. 

For luciferin, a firefly luciferase cosubstrate, another method of retention has been evaluated which consisted of incorporating the substrate in acrylic microspheres during their formation, these last being then confined in a polymeric matrix31. Using the suitable co-immobilized enzymes (adenylate kinase and creatine kinase), the three adenylic nucleotides (ATP, ADP and AMP) could be assayed continuously and reproducibly with a selfcontainment working time of 3 h. 

S, substrate R, product P, reduced form of the cosubstrate (mediator) Q, oxidized form of the cosubstrate (mediator) Ei, reduced form of the enzyme E2, oxidized form of the e... 

These values are then plotted against 1/C . The slope of this linear secondary plot provides k, while the intercept gives the value of I /k 2 + 1/ 2 2- It follows that the two rate constants k 2 and k22 may not be derived separately from this type of experiment. The same is, of course, true for the two Michaelis constants. One has to know the value of one of them independently, or at least know that one is much larger than the other. Dealing with redox enzymes, the variations of the intercept in a series of cosubstrates of increasing reducing power may be used to solve the problem. Indeed, if for the most reducing cosubstrates, the intercept becomes independent of the cosubstrate, one is entitled to conclude that it represents the value of l/k t2. The procedure is illustrated with an experimental example in the next section. 

Molecular Recognition of an Enzyme by Artificial One-Electron Cosubstrates... 

Specific recognition of enzymes by their natural substrates and cosubstrates is a common rule but what about recognition of an enzyme by artificial cosubstrate The cyclic voltammetric investigation of glucose oxidase provides an answer to the question.11 Because the flavin prosthetic group... 

FIGURE 5.7. Effect of changing the cosubstrate and the pH on the kinetics of an homogeneous redox enzyme reaction as exemplified by the electrochemical oxidation of glucose by glucose oxidase mediated by one-electron redox cosubstrates, ferricinium methanol ( ), + ferricinium carboxylate ( ), and (dimethylammonio)ferricinium ( ). Variation of the rate constant, k3, with pH. Ionic strength, 0.1 M temperature 25°C. Adapted from Figure 3 in reference 11, with permission from the American Chemical Society. 

This behavior, as well as complementary observations, can be explained on the basis of the reaction mechanism depicted in Scheme 5.3. The main catalytic cycle involves three successive forms of the enzyme in which the iron porphyrin prosthetic group undergoes changes in the iron oxidation state and the coordination sphere. E is a simple iron(III) complex. Upon reaction with hydrogen peroxide, it is converted into a cation radical oxo complex in which iron has a formal oxidation number of 5. This is then reduced by the reduced form of the cosubstrate, here an osmium(II) complex, to give an oxo complex in which iron has a formal oxidation number of 4. 

The Ping-Pong Mechanism with an Immobilized Enzyme and the Cosubstrate in Solution... 

The reaction scheme shown in Scheme 5.4 is the same as in the homogeneous case except that all forms of the enzymes are now immobilized onto the electrode surface. The cosubstrate is still in solution. The current is composed of two terms, one pertaining to the diffusion of the cosubstrate and the other to the catalytic reaction ... 

Immobilization of Both the Enzyme and the Cosubstrate. Electron Transfer and Electron Transport in Integrated Systems... 

Two examples of attachment of the cosubstrate to the electrode surface together with a monolayer of enzyme are shown in Figures 5.25 and 5.26, based on the avidin-biotin24 and antigen-antibody253 technologies, respectively. 

The concentrations of the two forms, P and Q, of the cosubstrate may be regarded as approximately uniform within the film containing the enzyme molecules, the attaching chains, and the ferrocene and ferricenium moieties. Under such conditions, the current may be expressed by... 

FIGURE 5.27. Equilibrium profile of the cosubstrate heads (reduced + oxidized forms) in the integrated system depicted in Figure 5.26, also showing the location of the enzyme sites. Adapted from Figure 5 in reference 25b, with permission from the American Chemical Society. 

In the monomolecular layer systems described so far, diffusion of the cosubstrate through the film is not a rate-limiting factor. This is true in the case of a free-moving cosubstrate, but also, at least at low scan rates, with cosubstrates attached to the structure. When several layers are coated on the electrode, diffusion of the cosubstrate may become rate limiting even if it is not attached to the structure. The diffusion rate of the two cosubstrate forms increases with its concentration. One may thus expect that the enzymatic reaction, rather than diffusion, tends to be the rate-determining step upon raising the cosubstrate concentration and that this situation is reached all the more easily that the number of layers is small. Under such conditions, the separation of the cyclic voltammetric current in two independent contributions [equation (5.29)] is still valid. icat is thus proportional to the total amount of enzyme contained in the film per unit surface area and therefore to the number, N, of monomolecular layers deposited on the electrode ... 

Although this class of enzymes is involved in most electrochemical approaches, other enzymes may be investigated electrochemically indirectly. For example, the system can be arranged such that the product of the targeted nonredox enzyme serves as substrate for an appropriately selected redox enzyme. Detection then involves the redox cosubstrate of the redox enzyme. 

The diffusion-reaction problem in the more general case occurs in a system containing n — 1 inactivated enzyme layers adjacent to the electrode surface on top of which N — n active layers have been deposited. Table 6.9 lists the equations that govern the fluxes of the two forms of the cosubstrate in such systems. 

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