Substrates cosubstrates

Measurement and modeling of the influence of concentrations of substrates, cosubstrates, coenzymes and inhibitors on enzyme activity form the central subject of enzyme kinetics and are discussed in Sect. 7.4. 

Radiometric Assays Traditionally, sulfonated metabolites for many small chemicals have been detected with a radiometric assay that utilizes S -labeled PAPS (Anderson and Weinshilboum, 1980 Foldes and Meek, 1973). The reaction involves incubation of the substrate, cosubstrate, and enzyme in an appropriate buffer. The incubation is terminated by the addition of barium hydroxide, barium acetate, and zinc sulfate, which cause the unreacted PAPS to precipitate out. Thus, the unprecipitated radioactivity is associated with the sulfonated product and can be quantitated with liquid scintillation counting. A variation of this assay has been developed for larger molecules such as flavonoids, where the incubation is terminated by the addition of ethyl acetate under acidic pH conditions and in the presence of an ion-pairing agent, whereby the sulfonated product can then be detected in the organic phase upon liquid-liquid phase separation (Varin et al., 1987). 

Enzyme inhibition caused by the high concentrations of the auxiliary substrate -cosubstrate inhibition - is common. 

Substrates Cosubstrates Enzymes activity Prosthetic groups Inhibitors /Act i vato rs Signal generation and amplification in binding assays... 

The measure of the catalytic activity of an enzyme is the rate of the reaction catalyzed by the enzyme. The conditions of an enzyme activity assay are optimized with relation to type and ionic strength of the buffer, pH, and concentrations of substrate, cosubstrate and activators used. The closely controlled assay conditions, including the temperature, are critical because, in contrast to substrate analysis, the reliability of the results in this case often can not be verified by using a weighed standard sample. 

With the radical reversal of the maximum current trend at 45% GOx, it is enticing to ask whether the same processes control the sensor response after the maximum, as do before it. It is possible to argue that under limiting glucose conditions (Figure 8) and at low enzyme loadings (<40%), the system is kinetically limited by the enzyme-sUbstrate-cosubstrate reaction (reactions i and ii. Scheme I). There are several pieces of evidence that tend to support this assumption. Previously... 

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,... 

The complexity introduced by exposure of an established mixed culture growing with a single substrate to an alternative cosubstrate is illustrated by the following. A stable mixed culture of Pseudomonas putida mt-2, P. putida FI, P. putida GJ31, and Burkholderia cepacia G4 growing with limited concentrations of toluene was established. Exposure to TCE for a month resulted in the loss of viability of the last three organisms, and resulted in a culture dominated by P. putida mt-2 from which mutants had fortuitously arisen (Mars et al. 1998). 

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. 

The Ping-Pong Mechanism. Kinetic Control by Substrate and/or Cosubstrate... 

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... 

The two limiting situations are not symmetrical Whereas the cosubstrate concentration comes into play alone in the first case, the concentrations of both the substrate and cosubstrate are present in the second. 

It is also interesting to examine how the plateau current varies with the substrate concentration. For simplicity, we assume that the substrate and cosubstrate concentrations are small enough as compared to the Michaelis-Menten constants for saturation effects to be negligible for both reactions. Then, as illustrated in Figure 5.2, the variations of the plateau current are given by... 

It is also worth examining how the entire catalytic wave depends on kinetic control by the substrate and/or cosubstrate in terms of shape and location on the potential axis. From Figure 5.1 we see that there is a small positive shift of the wave as kinetic control passes from reaction (1) to reaction (2). The shape of the wave also changes, going from... 

Another case of interest is the transition between no catalysis and the pure kinetic conditions leading to plateau-shaped responses. In the kinetic zone diagram of Figure 2.17, it corresponds to the extreme right-hand side of the diagram, where the cyclic voltammogram passes from the Nernstian reversible wave of the cosubstrate to the plateau-shaped wave, under conditions where the consumption of the substrate is negligible. The peak... 

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... 

The most striking feature of the peak or plateau current variation with substrate concentration is that the initial, and expected, increase is followed be a strong decrease that eventually annihilates catalysis. This decrease results from inhibition by the substrate, H202, which converts the E2 complex in the complex E3. E3 is the iron(II) dioxygen complex classically considered in dioxygen transport. It may regenerate E spontaneously by expulsion of the superoxide ion. This process is, however, very slow and the most important regeneration pathway involves the cosubstrate as pictured in Scheme 5.3. 

As in the homogeneous case, expression of the plateau current in equation (5.20) gives a simple representation of the competition between substrate and cosubstrate in the kinetic control of the enzymatic reaction. Equation (5.19) suggests the construction of primary and secondary plots allowing the derivation of the kinetic constants, as will be shown in the next section. 

The competition between substrate and cosubstrate also shows up in the variations of the half-wave potential. The wave is centered on the standard potential pQ when reaction (2) is the RDS (i.e., when k2Cp(l/k2>2+ l/ i, 2 + 1/kiCg) —>0). The wave then shifts toward positive values as kinetic control passes from reaction (2) to reaction (1), with, for example, decreasing substrate concentration. When these conditions are fulfilled [i.e., when fc2Cp(l/k2>2 + l/h, 2 + 1/kiCg) — oo], the wave equation becomes... 

FIGURE 5.25. Avidin-biotin construction of a monolayer glucose oxidase electrode with an attached ferrocenium cosubstrate and cyclic voltammetric response in a phosphate buffer (pH 8) at 25°C and a scan rate of 0.04 V/s. a attached ferrocene alone, h In the presence of 0.5 M glucose, c Variation of the inverse of the plateau current with the inverse of substrate concentration. Adapted from Figure 1 in reference 24, with permission from the American Chemical Society. 

All these experiments were carried out at such low scan rates that the outside diffusion layer of the cosubstrate (on the order of 105 A) is much larger than the film thickness. An experimental test for knowing whether this condition is fulfilled is that the plateau of S-shaped catalytic current then observed is much larger than the reversible cosubstrate peak observed in the absence of substrate i icat. Under these conditions, the concentration profiles within the film (bottom of Figure 5.30) do not depend on time. 

---