Enzymes steric hindrance

An important consideration in the design of a BFC is the method for retaining enzymes in a device, in most cases in close proximity to the eleetrode surface. A BFC that requires constant addition of soluble enzyme would be prohibitively costly to operate, and the rate of electron transfer between the enzymes and electrode surface would likely be poor. Instead, enzymes are commonly immobilized in a BFC through adsorption [50], covalent attachment [51], confinement within a polymer matrix (such as Nafion [52], chitosan [53], or other synthetic polymers) [54], or other means [55]. The method of immobilization used can have a profound effect on enzyme activity through denaturation or inactivation of the enzyme, steric hindrance (blocking) of the active site, imposing mass transport limitations on substrate and/or cofactor diffusion, or significant modification of the local environment of the enzyme. Immobilization... 

FIGURE 7.1 Enzyme ortho- and allosterism as presented by Koshland [2], Steric hindrance whereby the competing molecules physically interfered with each other as they bound to the substrate site was differentiated from a direct interaction where only portions of the competing molecules interfered with each other. If no direct physical interaction between the molecules occurred, then the effects were solely due to effects transmitted through the protein structure (allosteric). 

The immobilization of enzymes for sensing purposes frequently provides several important advantages an increase of its stability, operational reusability and greater efficiency in consecutive multistep reactions. Sometimes immobilization is accompanied by a certain degree of denaturalization however, the enzyme-matrix interactions may assist in stabilization preventing conformational transitions that favor such process. In some cases excessive bond formation affects the conformation of the active site and the steric hindrances caused by the polymer matrix may render an inactive sensor. 

N-Dealkylation reactions are not restricted to tertiary amines. Secondary amines as well as primary amines can also be dealkylated although both types are less favored than tertiary amines. In the case of primary amines, the lone pair of electrons of the amino group can interact and complex with the Fe3+ of heme. Thus primary amines tend to be inhibitors of P450 activation and for that reason are generally poor substrates. Secondary amines have metabolic properties intermediary between those of tertiary amines and primary amines. They are less-effective inhibitors because of increased steric hindrance to complex formation but are also better substrates because they are less-effective inhibitors and thereby increase the effective concentration of enzyme. 

The most common of these systems is the enzyme-multiplied immunoassay technique or EMIT, which is particularly suited to the measurement of small molecules (haptens) such as drugs. EMIT is a trade mark of the Syva Corporation of Palo Alto, California. Although it does not involve the separation of bound fraction from free it is nevertheless a competitive assay system. The antigen is labelled with an enzyme in such a way that the enzyme retains its catalytic activity. When the antigen binds to the antibody the enzyme becomes inhibited, probably by an induced conformational change or by steric hindrance of the enzyme active site (Figure 7.15). 

If the biological activity of compound Y is less in enzyme A than that of related enzyme B, expect possible steric hindrance about substituent X. 

The means of achieving stereoselectivity due to steric hindrance toward the attack of a reagent or cosubstrate. The less hindered face will be the more susceptible side. Many enzymes utilize this method of product control. 

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