Enzyme activation influencing factor

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). 

The biodisposition of sedative-hypnotics can be influenced by several factors, particularly alterations in hepatic function resulting from disease or drug-induced increases or decreases in microsomal enzyme activities (see Chapter 4). 

Several factors must be considered when the experimental assay condi tions are developed. The reaction rate depends on the concentrations ol substrate, enzyme, and necessary cofactors. In addition, the reaction rate is under the influence of environmental factors such as pH, temperature, anti ionic strength. Enzyme activity increases with increasing temperature until the enzyme becomes denatured. The enzyme activity then decreases until all enzyme molecules are inactivated by denaturation. During kinetic mea surement, it is essential that the temperature of all reaction mixtures lx maintained constant. 

There are many factors that influence the outcome of enzymatic reactions in carbon dioxide. These include enzyme activity, enzyme stability, temperature, pH, pressure, diffusional limitations of a two-phase heterogeneous mixture, solubility of enzyme and/or substrates, water content of the reaction system, and flow rate of carbon dioxide (continuous and semibatch reactions). It is important to understand the aspects that control and limit biocatalysis in carbon dioxide if one wants to improve upon the process. This chapter serves as a brief introduction to enzyme chemistry in carbon dioxide. The advantages and disadvantages of running reactions in this medium, as well as the factors that influence reactions, are all presented. Many of the reactions studied in this area are summarized in a manner that is easy to read and referenced in Table 6.1. 

Relatively innocuous factors can also sometimes influence liver enzyme activity. For example, the metabolic elimination of the bronchodilator theophylline has been reported to be prolonged in patients with influenza A or adenovirus infections. In 1990, an influenza epidemic in Seattle resulted in the admission of 11 children with high serum levels of theophylline and confirmed drug toxicity. These effects appear to be confined to cytochrome P450-based drug biotransformation. They may be related to the generation of interferons as a result of these infections, which, presumably, are causally related to the inhibitory effect on hydroxylases and demethylases. 

As mentioned before, people tend intuitively to turn to the one-variable-at-a-time technique for its conceptual simplicity, and ignore the possible interaction between independent variables. A good example of the interaction between factors is that between enzyme concentration (E) and reaction temperature (T). Assuming E and T are the chosen factors for optimization, one possible interaction will be that T tends to influence the way E affects the conversion yield and vice versa. Since reaction temperature increased, enzyme activity was suppressed than at low temperature and the rate of enzyme-catalysis is affected by temperature this will inevitably affect conversion yield of the product. Should the interaction be minor or negligible, a one-factor-at-a-time search will give a satisfactory result. 

In general, null alleles are associated with the classic early-onset phenotype, whereas missense mutations which lead to defective proteins that exhibit residual enzyme activity lead to attenuated phenotypes (Froissart et al., 2002). However, studies of genotype-phenotype correlation have revealed a lack of perfect concordance, which suggests other factors may be involved that influence disease outcome (Froissart et al., 2002). At present, the putative factors that modify LSD-phenotypes among patients with identical genotypes remain obscure. 

---