Enzyme molecules

Enzymes are basically specialty proteins (qv) and consist of amino acids, the exact sequence of which determines the enzyme stmcture and function. Although enzyme molecules are typically very large, most of the chemistry involving the enzyme takes place in a relatively small region known as the active site. In an enzyme-catalyzed reaction, binding occurs at the active site to one of the molecules involved. This molecule is called the substrate. Enzymes are... 

Effect of Temperature and pH. The temperature dependence of enzymes often follows the rule that a 10°C increase in temperature doubles the activity. However, this is only tme as long as the enzyme is not deactivated by the thermal denaturation characteristic for enzymes and other proteins. The three-dimensional stmcture of an enzyme molecule, which is vital for the activity of the molecule, is governed by many forces and interactions such as hydrogen bonding, hydrophobic interactions, and van der Waals forces. At low temperatures the molecule is constrained by these forces as the temperature increases, the thermal motion of the various regions of the enzyme increases until finally the molecule is no longer able to maintain its stmcture or its activity. Most enzymes have temperature optima between 40 and 60°C. However, thermostable enzymes exist with optima near 100°C. 

Although the mechanisms may be complicated and varied, some simple equations can often describe the reaction kinetics of common enzymatic reac tions qiiite well. Each enzyme molecule is considered to have an active site that must first encounter the substrate (reactant) to form a complex so that the enzyme can function. Accordingly, the following reaction scheme is written ... 

Kinetic behavior becomes complicated when there are two chemical species that can both complex with the enzyme molecules. One of the species might behave as an inhibitor of the enzyme reac tion with... 

When an enzyme molecule is attached to a carrier, its active site might be sterically blocked and thus its activity becomes unobservable (inactivated). 

The Michaehs-Menten equation and other similar nonhnear expressions characterize immobihzed enzyme kinetics. Therefore, for a spherical porous carrier particle with enzyme molecules immobilized on its external as well as internal surfaces, material balance of the substrate will result in the following ... 

The Michaelis-Menten scheme nicely explains why a maximum rate, V"max, is always observed when the substrate concentration is much higher than the enzyme concentration (Figure 11.1). Vmax is obtained when the enzyme is saturated with substrate. There are then no free enzyme molecules available to turn over additional substrate. Hence, the rate is constant, Vmax, and is independent of further increase in the substrate concentration. 

Inhibitors as well as substrates bind in this crevice between the domains. From the numerous studies of different inhibitors bound to serine pro-teinases we have chosen as an illustration the binding of a small peptide inhibitor, Ac-Pro-Ala-Pro-Tyr-COOH to a bacterial chymotrypsin (Figure 11.9). The enzyme-peptide complex was formed by adding a large excess of the substrate Ac-Pro-Ala-Pro-Tyr-CO-NHz to crystals of the enzyme. The enzyme molecules within the crystals catalyze cleavage of the terminal amide group to produce the products Ac-Pro-Ala-Pro-Tyr-COOH and NHs. The ammonium ions diffuse away, but the peptide product remains bound as an inhibitor to the active site of the enzyme. 

The turnover number of an enzyme, is a measure of its maximal catalytic activity, is defined as the number of substrate molecules converted into product per enzyme molecule per unit time when the enzyme is saturated with substrate. The turnover number is also referred to as the molecular activity of the enzyme. For the simple Michaelis-Menten reaction (14.9) under conditions of initial velocity measurements, Provided the concentration of... 

The catalytically active enzyme substrate complex is an interactive structure in which the enzyme causes the substrate to adopt a form that mimics the transition-state intermediate of the reaction. Thus, a poor substrate would be one that was less effective in directing the formation of an optimally active enzyme transition-state intermediate conformation. This active conformation of the enzyme molecule is thought to be relatively unstable in the absence of substrate, and free enzyme thus reverts to a conformationally different state. 

Allosteric enzymes have an oligomeric organization. They are composed of more than one polypeptide chain (subunit) and have more than one S-bind-ing site per enzyme molecule. 

Case 2 When [S] is small compared to K not all the enzyme molecules have S bound, and the kinetics are first order in S. 

Phillips, D., 1966. The three-dimensional. structure of an enzyme molecule. Scientific American 215 75—80. 

Despite the large size of an enzyme molecule, there is reason to believe that there are only one or a few spots on its surface at which reaction can occur. These are usually referred to as active centers. The evidence for this view of enzyme reactions comes from many kinds of observations. One of these is that we can often stop or slow down enzyme reactions by adding only a small amount of a false substrate. A false substrate is a molecule that is so similar to the real substrate that it can attach itself to the active center, but sufficiently different that no reaction and consequently no release occurs. Thus, the active center is blocked by the false substrate. 

The stability of the enzyme-polymer complex and its dissociation upon the variation of pH depends on the structural and other physico-chemical properties of CP and enzyme molecule. Thus, a Biocarb-T heteroreticular biosorbent (Fig. 26) is characterized by a stability of its complex with ot-amylase (under the condition of its stabilization) in acid solutions and a complete dissociation of the complex during isolation of the active enzyme at pH 7-8. 

FIGURE 1119 The lysozyme molecule is a typical enzyme molecule. Lysozyme is present in a number of places in the body, including tears and the mucus in the nose. One of its functions is to attack the cell walls of bacteria and destroy them. This "ribbon" representation shows only the general arrangement of the atoms to emphasize the overall shape of the molecule the ribbon actually consists of amino acids linked together (Section 19.13). 

Once in the active site, the substrate undergoes reaction. The product is then released for use in the next stage, which is controlled by another enzyme, and the original enzyme molecule is free to receive the next substrate molecule. One example... 

FIGURE 13.41 A plot of the rate of an enzyme-catalyzed reaction (relative to its maximum value, k2[E]0, when S is in very high concentration) as a function of concentration of substrate for various values of (CM. At low substrate concentrations, the rate of reaction is directly proportional to the substrate concentration (as indicated by the black line for KM = 10). At high substrate concentrations, the rate becomes constant at k2[E]0 once the enzyme molecules are "saturated" with substrate. The units of S are the same as those of KM. 

FIGURE 13.42 (a) An enzyme poison (represented by the blue sphere) can act by attaching so strongly to the active site that it blocks the site, thereby taking the enzyme out of action, (b) Alternatively, the poison molecule may attach elsewhere, so distorting the enzyme molecule and its active site that the substrate no longer fits. 

Adsorption on solid matrices, which improves (at optimal protein/support ratios) enzyme dispersion, reduces diffusion limitations and favors substrate access to individual enzyme molecules. Immobilized lipases with excellent activity and stability were obtained by entrapping the enzymes in hydrophobic sol-gel materials [20]. Finally, in order to minimize substrate diffusion limitations and maximize enzyme dispersion, various approaches have been attempted to solubilize the biocatalysts in organic solvents. The most widespread method is the one based on the covalent linking of the amphiphilic polymer polyethylene glycol (PEG) to enzyme molecules [21]. 

FIGURE 3 Scanning electron micrograph (1200x magnification) of the surface of a porous alumina particle coated with poly(diphenoxy-phosphazene). Surface nitration, reduction, and glutaric dialdehyde coupling immobilized enzyme molecules to the surface. (From Ref. 23.)... 

Enhanced thermal stability enlarges the areas of application of protein films. In particular it might be possible to improve the yield of reactors in biotechnological processes based on enzymatic catalysis, by increasing the temperature of the reaction and using enzymes deposited by the LB technique. Nevertheless, a major technical difficulty is that enzyme films must be deposited on suitable supports, such as small spheres, in order to increase the number of enzyme molecules involved in the process, thus providing a better performance of the reactor. An increased surface-to-volume ratio in the case of spheres will increase the number of enzyme molecules in a fixed reactor volume. Moreover, since the major part of known enzymatic reactions is carried out in liquid phase, protein molecules must be attached chemically to the sphere surface in order to prevent their detachment during operation. 

The catalytic capacity of the rate-fimiting reaction in a metabolic pathway is the product of the concentration of enzyme molecules and their intrinsic catalytic efficiency. It therefore follows that catalytic capacity can be... 

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