Enzyme hydrophobic forces

As we have just seen, the initial encounter complex between an enzyme and its substrate is characterized by a reversible equilibrium between the binary complex and the free forms of enzyme and substrate. Hence the binary complex is stabilized through a variety of noncovalent interactions between the substrate and enzyme molecules. Likewise the majority of pharmacologically relevant enzyme inhibitors, which we will encounter in subsequent chapters, bind to their enzyme targets through a combination of noncovalent interactions. Some of the more important of these noncovalent forces for interactions between proteins (e.g., enzymes) and ligands (e.g., substrates, cofactors, and reversible inhibitors) include electrostatic interactions, hydrogen bonds, hydrophobic forces, and van der Waals forces (Copeland, 2000). 

The predominant interaction of CYP3A4 is via hydrophobic forces and the overall lowering of lipophilicity can reduce metabolic lability to the enzyme. Figure 7.14 shows the relationship between unboimd intrinsic clearance in man and lipophilicity for a variety of CYP3A4 substrates. The substrates are cleared by a variety of metabolic routes including N-dealkylation, aromatization and aromatic and aliphatic hydroxylation. The trend for lower metabolic lability with lower lipophilicity is maintained regardless of structure or metabolic route. 

The preparation and characterization of short peptidic molecules that adopt a stable and predictible structure in solution is a prerequisite for the construction of de now-designed artificial enzymes and proteins. In natural polypeptides, the secondary structures are parts of a larger system and their conformational stability is due to several intra- and interchain non-covalent interactions such as van der Waals forces, electrostatic forces, hydrogen bonding, and hydrophobic forces [2], However, these interactions are less important in short... 

Adsorbing enzymes onto a variety of matrices is a fairly gende procedure for immobilizing them. The enzyme is mixed with an insoluble matrix whereupon the enzyme binds to the matrix with weak interactions (H bonds, van der Waals forces, hydrophobic forces, electrostatic forces). In general, the binding is based on a combination of such interactions. 

Tritylagarose (29) binds enzymes in a reversible fashion through hydrophobic forces, providing another kind of reversible immobilization. Several enzymes, such as alkaline phosphatase, fl-galactosidase, lactate dehydrogenase, and spleen phosphodiesterase, have been immobilized (29). 

Andrews [76] reported that plasteins were formed by an association of predominantly hydrophobic peptides via hydrophobic and possibly ionic bonding. Aso and coworkers [77,78] concluded that hydrophobic forces were a major factor in plastein chain assembly. They found that, compared with the substrate, the water-insoluble product contained smaller ratios of hydrophilic and larger ratios of hydrophobic amino acid residues. The results of Sukan and Andrews [58] showed that hydrophobic amino acids such as phenylalanine, leucine, isoleucine, tyrosine, valine, and proline were preferentially incorporated into plastein at the expense of hydrophilic amino acids. Also others have reported on a trend of preferential incorporation of hydrophobic amino acids into the protein product in enzyme-catalyzed reactions [46,60,79,80,81]. 

Chitosan is widely used as supports for enzyme and cell immobilization due to its appropriate characteristics. Immobilization is the process in which the enzyme, cells or organelles is confined in a definite position thus rendering an insoluble form that retains the catalytic activity and can be reused several times. The enzymes or cells are bound to the carrier material via reversible surface interactions. The forces involved are van der Waals forces and ionic and H-bonding interaction as well as hydrophobic forces. Chitosan support being a positively charged polymer binds negatively charged proteins bind easily [43, 70]. 

All enzymes are proteins, which are linear sequences of amino acids linked by peptide bonds. The folding of these sequences determined the secondary structure (such as a-helix, p-sheet or p-turn) and tertiary structure. Therefore, the properties of an en me are actually presumed from its sequence of amino acids. Some amino acids, dubbed hot spots , especially the ones in the active site where substrate binds, are sensitive to catalytic properties of an enzyme. Substitution of these important amino acids can significantly improve the activity or enantioselectivity toward a certain reaction. Protein stability is also maintained by the intramolecular and intermolecular interactions between residues of amino acids, including van der Waals forces, hydrophobic forces, electrostatic forces, hydrogen bonds and disulfide bonds. Detailed analysis of these amino acids, usually located in the protein surface, sheds light on the protein design for better thermostability. 

Cold denaturation is an interesting phenomenon that results from hydration of polar and nonpolar proteins and weakening of hydrophobic forces, all of which have a significant effect on protein folding and stability. Intracellular enzymes from psychrophiles are protected from cold denaturation by compatible solutes, such as potassium glutamate and trehalose (67). Psychrophiles also have intracellular cold shock proteins that act as chaperones, cryoprotectors, and antifreeze molecules. There is no explanation yet for protection from cold denaturation for extracellular psychrophilic enzymes, but exopolymeric substances may be involved (67). 

As this chapter indicates water is not simply a cheap and environmentally benign solvent, important though that is. Instead, the hydrophobic effect seen in water permits chemistry otherwise not accessible. It permits the construction of enzyme mimics that use hydrophobic forces to bind the substrates to the catalysts. It permits those reactions, and others, to achieve... 

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