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How Enzymes Work The enzymatic catalysis of reactions is essential to living systems.

Under biologically relevant conditions, uncatalyzed reactions tend to be slowmost biological molecules are quite stable in the neutral-pH, mildtemperature, aqueous environment inside cells. Furthermore, many common reactions in biochemistry entail chemical events that are unfavorable or unlikely in the cellular environment, such as the transient formation of unstable charged intermediates or the collision of two or more molecules in the precise orientation required for reaction. Reactions required to digest food, send nerve signals, or contract a muscle simply do not occur at a useful rate without catalysis. An enzyme circumvents these problems by providing a specific environment within which a given reaction can occur more rapidly. The distinguishing feature of an enzyme-catalyzed reaction is that it takes place within the confines of a pocket on the enzyme called the active site (Fig. 61). The molecule that is bound in the active site and acted upon by the enzyme is called the substrate. The surface of the active site is lined with amino acid residues with substituent groups that bind the substrate and catalyze its chemical transformation. Often, the active site encloses a substrate, sequestering it completely from solution. The enzyme substrate complex, whose existence was first proposed by Charles-Adolphe Wurtz in 1880, is central to the action of enzymes. It is also the starting point for mathematical treatments that define the kinetic behavior of enzyme-catalyzed reactions and for theoretical descriptions of enzyme mechanisms.(blue paper)

FIGURE ddd Binding of a substrate to an enzyme at the active site. The enzyme chymotrypsin, with bound substrate in red (PDB ID 7GCH). Some key active-site amino acid residues appear as a red splotch on the enzyme surface.

Immobilized Enzymes Immobilized enzyme technology is the key scientific advance in which an enzyme immobilized and used in a packed column or a fluidized bed reactor so as to enable reuse and meet other purposes. Initial attempts to immobilize enzymes on naturally derived supports such as charcoal were conducted early in the twentieth century and eventually led to the development of more robust biocatalysts immobilized on synthetic resins by the mid-1950s. Immobilization often confers a number of advantages relative to the free biocatalyst including ease of removal from the process stream, potential for reuse, improvements in stability, favorable alterations in kinetic parameters, suitability for continuous production and in some cases the ability to operate in organic solvents. Immobilization needs to improve the performance of an enzyme enough to offset the costs associated with the procedure. Such gain can be measured in terms of improvement in the total amount of product produced per unit of enzyme, improvement in the ease of removing the biocatalyst, or through the enabling of new applications for a given enzyme. It is often the case, however, that the cheapest immobilization methods suffer from a number of drawbacks including lack of both enzymatic and mechanical stability, leaching of the enzyme, fouling of the support, and limited enzymatic activity. The many methods for producing immobilized enzymes can be divided into these subcategories: i. Adsorption to a matrix such as carbon, chitin, diatomaceous earth, and ion exchange resins. ii. Crosslinking enzyme crystals and whole cells with gluteraldehyde and other agents. iii. Gel entrapment in silica sol-gels, alginate and protein matrices. iv. Covalent attachment to resins and other carriers.

v. Encapsulation within a membrane or liposome.

Many factors influence the catalytic efficiency and kinetics of an immobilized enzyme. The immobilization process itself can lead to loss of enzymatic activity through incomplete capture of the enzyme or through denaturation of the enzyme protein. Immobilized enzymes often demonstrate dramatic improvements in stability over free enzymes, especially in organic solvents or at elevated temperatures. Kinetics of Immobilized Enzymes Another major factor in the performance of immobilized enzymes is the effect of the matrix on mass transport of substrates and products. Hindered access to the active site of an immobilized enzyme can affect the kinetic parameters in several ways. The effective concentration of substrates and products is also affected by the chemistry of the matrix especially with regard to the respective partition coefficients between the bulk solution and the matrix.