Enzyme flexibility

Considering these results and the contributions by Broos [125], Watanabe [126], and Ueji [55, 77], it may be concluded that the relation between enzyme flexibility and enanhoselective performance in organic solvents is now firmly established. Molecular dynamic simulations on the flexibility of subtilisin and the mobility of bound water molecules in carbon tetrachloride corroborate the idea that organic solvents reduce molecular flexibility via interactions at specific binding sites [127]. Whether predictive tools can be developed on the basis of this knowledge remains to be seen. 

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]. 

Regarding the enzyme polymer ratio, high polymer amounts may provide higher immobilisation yields but may restrict the enzyme flexibility and functionality, and high enzyme amounts may provide higher responses but may limit the sensitivity of the biosensor. The dilemma has appeared and the choice will depend on the own particular interests. The 1 2 ratio provided higher absorbance values (0.259 coefficient of variation (CV) — 17%) than those obtained with the 2 1 (0.041 CY — 9%) and the 1 3 (0.189 CV = 15%) ratios. In the same way, the 1 2 ratio also provided the highest immobilisation yields after 30-min incubation in buffer (62% as compared to 16% and 41% for the 2 1 and the 1 3 ratios, respectively). In our case, the results clearly demonstrate that the 1 2 enzyme polymer ratio is the optimum one for the biosensor construction. 

The activity of lyophilized HL-ADH in several solvents increased by orders of magnitude upon addition of small amounts of water to solvents of dielectric constant e from 1.9 to 36 (Guinn, 1991). Enzyme flexibility, as measured by electron spin resonance (ESR) spectroscopy with a spin label at the active site, did not depend on water content but on of the pure solvent HL-ADH turns less flexible with decreasing . 

A. van Hoek, and D. N. Reinhoudt, Flexibility of enzymes suspended in organic solvents probed by time-resolved fluorescence anisotropy. Evidence that enzyme activity and enantioselectidty are directly related to enzyme flexibility,... 

R. V. Rariy and A. M. Klibanov, On the relationship between enzymatic enantioselectivity in organic solvents and enzyme flexibility, Biocat. Biotransf. 2000, 18, 401-407. 

Broos J, Bisser AJW, Engbersen JI, Verboom W, van Hoek A, and Reinhoudt DN. Flexibility of Enzymes Suspended in Organic Solvents Probed by Time-Resolved Fluorescence Anisotropy. Evidence that Enzyme Activity and Enan-tioselectivity are Directly Related to Enzyme Flexibility. / Am Chem Soc 1995 117 12657-12663. 

VI. Pyridoxal Phosphate- and Pymvy 1-Dependent Enzymes Flexible Response to... 

It is important to note that conformational flexibility can occur in the substrate as well as the enzyme. Flexibility can allow a substrate to be tucked into an active site rotation around single bonds can position the reactive part of the substrate in proximity to catalytic groups, while allowing other parts of the molecule to avoid steric clashes. However, substrate flexibility could have been detrimental for early enzymes, as well. If a flexible substrate binds in an active site in a manner that doesn t orient the molecule properly with respect to the catalytic groups, a potential substrate becomes an inhibitor. 

Engh R A, Brandstetter H, Sucher G, et al. (1996). Enzyme flexibility, solvent and weak interactions characterize thrombin-ligand interactions Implications for drug design. Structure. 4 1353-1362. 

Enzyme Flexibility as a Molecular Basis for Metabolic Control... 

In a similar manner, Latif et al. [104] utilized MD simulations in order to gain insight into the structural properties and dynamics of a-chymotrypsin in imidazo-lium-based ILs with different types of anions ([bmim]PF, [bmim]BF, [bmim]Cl, [bmim]TfO, and [bmim]NTf2). At a low water content, the conformation of enzyme was closer to its native structure in the presence of ILs, presenting also a bell-shaped dependency on water content. However, no major conformational changes at the active site of the enzyme were observed. The solvation of the enzyme in water-immiscible ILs led to a higher enzyme flexibility at increased water content. 

The properties of stationary structures of enzymatic processes can be different from those obtained in gas phase calculations because, obviously, the interaction with the environment are not considered in the latter. Then, a more realistic picture of enzyme catalyzed reactions can be obtained including a small part of the active centre into the calculations. The problem is that in this cluster or supermolecule models, the optimised stmetures do not necessary fit into the enzyme active site and computing artefacts can be obtained. A common strategy is to anchor some key atoms of the enzyme to their crystallographic positions and then optimize the rest of the coordinates of the model [39]. Nevertheless, this is an approximate solution that presents several deficiencies. First, the result will be dependent on the initial X-ray stmeture that, in many cases, is far from the real stmeture of the protein-substrate complex at the TS. Second, long-range effects on the nuclear and electronic polarisation of the chemical system are not included in the calculations. Third, and probably the most dramatic deficiency, the enzyme flexibility is not properly taken into account. And finally, the computational cost of these calculations rapidly increases, as more atoms of the environment are explicidy included. 

Enzyme flexibility may influence the reaction in different ways (see Conformational Search Proteins and Molecular... 

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