Enzymatic reactions experimental conditions

Enzymatic reactions are influenced by a variety of solution conditions that must be well controlled in HTS assays. Buffer components, pH, ionic strength, solvent polarity, viscosity, and temperature can all influence the initial velocity and the interactions of enzymes with substrate and inhibitor molecules. Space does not permit a comprehensive discussion of these factors, but a more detailed presentation can be found in the text by Copeland (2000). Here we simply make the recommendation that all of these solution conditions be optimized in the course of assay development. It is worth noting that there can be differences in optimal conditions for enzyme stability and enzyme activity. For example, the initial velocity may be greatest at 37°C and pH 5.0, but one may find that the enzyme denatures during the course of the assay time under these conditions. In situations like this one must experimentally determine the best compromise between reaction rate and protein stability. Again, a more detailed discussion of this issue, and methods for diagnosing enzyme denaturation during reaction can be found in Copeland (2000). 

Moreover, the organized systems can be used to smooth the required experimental conditions of CL reactions, above all pH conditions. In this way, it is possible to carry out CL reactions more easily, as well as to develop simultaneously these reactions with other kinds of reactions, such as enzymatic ones, that are of great biochemical interest. 

In vitro Metabolism. Numerous variables simultaneously modulate the in vivo metabolism of xenobiotics therefore their relative importance cannot be studied easily. This problem is alleviated to some extent by in vitro studies of the underlying enzymatic mechanisms responsible for qualitative and quantitative species differences. Quantitative differences may be related directly to the absolute amount of active enzyme present and the affinity and specificity of the enzyme toward the substrate in question. Because many other factors alter enzymatic rates in vitro, caution must be exercised in interpreting data in terms of species variation. In particular, enzymes are often sensitive to the experimental conditions used in their preparation. Because this sensitivity varies from one enzyme to another, their relative effectiveness for a particular reaction can be sometimes miscalculated. 

Initial conditions for the compartmental model and the enzymatic reaction were set to n0 = [10 5], and s0 = 10, eo = 5, and c0 = 0, respectively. These values are very low regarding the experimental reality, but they were deliberately chosen as such to facilitate the computation of the exact solution. 

Considering the complex kinetic behavior of most enzymatically controlled reactions (41), the formal treatment of simple catalytic analogues should not pose additional problems. However, one consequence of the less perfect, but for most practical and mechanistical purposes sufficient performance of synthetic catalysts in comparison to enzymes is that in many kinetic studies, a large excess of substrate over the catalyst cannot be used, because then the uncatalyzed reaction will be too fast. Consequently, kinetic studies under catalyst saturation, or the steady-state methods that are most often used in the investigation of enzymes (18, 19, 41), are not suitable here. The formal treatment of the resulting, often quite complex, kinetics is greatly facilitated by computer-aided numerical simulations, which also help to design proper experimental conditions. 

Reaction rates in enzyme systems are usually extremely sensitive to variation of experimental conditions, i.e., to pH, temperature, ionic strength, specific ions, solvent, etc. We now consider in some detail the influence of pH and temperature on enzymatic reactions. 

The use of microemulsions or reverse micelles as media for chemical and enzymatic reactions has been reviewed in recent years [20,37,38]. Microemulsions, including those based on organogels, are also useful media for enzyme-catalyzed synthetic reactions [37,39-43] and for preparation of nanoparticles [44]. In a very different direction, Vanag and Hanazaki [45] showed that the ferroin-catalyzed Belousov -Zhabitinskii oscillatory reaction exhibits frequency-multiplying bifurcations in reverse AOT microemulsions in octane, A clear understanding of reactivity in microemulsions and insight into how to optimize the experimental conditions requires kinetic models with predictive power. We focus attention primarily on this problem. 

Vedejs and Chen [39] described an efficient non-enzymatic system able to approach the efficiency of some of the lipase methods in enantioselectivity. The reaction was carried out in a 2 1 ratio racemic secondary alcohol acylating agent, in contrast to Evans procedure. The pyridinium salt 8 was prepared by reaction of the chiral 4-dimethylaminopyridine (DMAP) 6 with the commercially available chloroformate 7. This pyridinium salt proved to be unreactive to secondary alcohols. The reactivity was found only upon strict experimental conditions addition of a Lewis acid, then the racemic alcohol, followed by addition of a tertiary amine gave the carbonate 9. Under these conditions (using MgBr2 and triethylamine), (2-naphthyl)- -ethanol was converted (room temperature, 20 h and 54% conversion) into the (S)-carbonate (82% ee). The recovered alcohol showed 83% ee, revealing a stereoselectivity s=39 for the process. A number of 1-arylalkanols have been resolved by this procedure in 20-44% yield (based on the racemic material) and 80-94% ee. For the use of this system in enantiodivergent reactions, see Schemes 6.1 and 6.32. 

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