Enzyme solvent polarity

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). 

Solvents can cause enzyme inactivation (decrease the number of active enzyme molecules). The exact mechanisms are not so well known, but it is clear that solvent polarity plays an important role. Several solvent parameters have been used to try to rationalise the influence of solvents on enzymes. The parameter which has been used most for this purpose is the log P value, which is defined as the logarithm of the partition coefficient of a substance in the standard 1-octanol/water two-phase system (Table 9.4). Log P values can be determined experimentally by measuring the partitioning of the solvent between octanol and water. Alternatively, log P values... 

Solvent polarity is known to affect catalytic activity, yet consistent correlations between activity and solvent dielectric (e) have not been observed [12,102]. However, a striking correlation was found between the catalytic efficiency of salt-activated subtilisin Carlsberg and the mobility of water molecules (as determined using NMR relaxation techniques) associated with the enzyme in solvents of varying polarities (Figure 3.11) [103]. As the solvent polarity increased, the water mobility of the enzyme increased, yet the catalytic activity of the enzyme decreased. This is consistent with previous EPR and molecular dynamics (MD) studies, which indicated that enzyme flexibility increases with increasing solvent dielectric [104]. 

Enzyme flexibility is greater in solvents with high polarity because of weaker electrostatic interactions in these solvents [54, 104, 105]. The loss in enzyme activity seen in the NMR study described above may be attributed to the water stripping model as water is stripped from the enzyme, locations in and on the enzyme previously inaccessible to the solvent may become accessible, thus permitting increased solvent-enzyme interactions [103]. As a result, enzyme structure may be disrupted (e.g., partially denatured), and catalytic activity is decreased. The partially denatured enzyme appears to exhibit greater flexibility as solvent polarity increases [106, 107]. 

J. Kim, D. S. Clark, and J. S. Dordick, 2000, Intrinsic effects of solvent polarity on enzymic activation energies, Biotechnd. Bioeng. 67, 112-116. 

Surprisingly, it has been shown that many enzymatic reactions can advantageously be carried out in nonaqueous solutions, that is, in carefully selected organic solvents containing little or no added water, sometimes with dramatic changes in enzyme specificity as a function of solvent polarity [309-312]. 

Attempts have been made to correlate the influence of solvents on enzyme activity, stability, and selectivity with physicochemical solvent characteristics such as relative permittivity, dipole moment, water miscibility, and hydrophobicity, as well as empirieal parameters of solvent polarity. However, no rationale of general validity has been found, except the simple rule that nonpolar hydrophobic solvents are generally better than polar hydrophilic ones. The best correlations are often obtained with the logarithm of the 1-octanol/water partition coefficient, Ig Pq/wj a quantitative measure of the solvent s hydrophobicity cf. Section 7.2). 

Similarly, enzyme activity has been correlated to solvent polarity. Oxidation of cinnamyl alcohol by horse liver alcohol dehydrogenase (LADH) (pH 7.5) was observed in anhydrous hexane, methylene chloride and acetonitrile (Guinn et al., 1991). The oxidation rates were observed to increase as the dielectric constant decreased (Table 5). Electron paramagnetic spectroscopy (EPR) and two active site directed spin labels were used to examine the effect of solvent dielectric on structural stability. As the dielectric constant of the solvent decreased, the spectra broadened, indicative of an increase in rigidity or stability. 

Effect of pH. If ionic strength changes cannot render PAH/PSS capsules permeable to larger species (e.g., macromolecules, enzymes, nanoparticles), then manipulation of pH or solvent polarity can be used. The point about the (PAH/PSS) system is that PSS is a strong polyelectrolyte and remains fully ionized, whereas PAH is a weak polyelectrolyte and so its dissociation is dependent on pH. 

An alternative approach is to combine QM and MM methods such that the reacting system (or the active site in an enzyme) is treated explicitly by a quantum mechanical method, while the surrounding environmental solvent molecules (or amino acids), which constitute the most time-consuming part in the evaluation of the potential energy surface, are approximated by a standard MM force field. " Such a method takes advantage of the accuracy and generality of the QM treatment for chemical reactions - and of the computational efficiency of the MM calculation.Because the reactant electronic structure and solute-solvent interactions are determined quantum mechanically, the procedure is appropriate for studying chemical reactions, and there is no need to parameterize potential functions for every new reaction. Furthermore, the solvent polarization effects on the solute are naturally included in the... 

Although sugars are reasonably soluble in hydrophilic organic solvents such as those used above, most enzymes demonstrate a low activity in these systems which require long reaction times and induce rapid inactivation of the biocatalyst. The instability of enzymes in polar solvents can be partially overcome by immobilization on hydrophilic supports [30]. Overall, this approach is unattractive for large-scale manufacturing. 

If a catalyst is to work well in solution, it (and tire reactants) must be sufficiently soluble and stable. Most polar catalysts (e.g., acids and bases) are used in water and most organometallic catalysts (compounds of metals witli organic ligands bonded to tliem) are used in organic solvents. Some enzymes function in aqueous biological solutions, witli tlieir solubilities detennined by the polar functional groups (R groups) on tlieir outer surfaces. 

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