The active site of an enzyme

The active site of an enzyme (Fig. 4.5) is a 3D shape. It has to be on or near the surface of the enzyme if substrates are to reach it. However, the site could be a groove, hollow, or gully allowing the substrate to sink into the enzyme. 

Because of the overall folding of the enzyme, the amino acid residues which are close together in the active site may be extremely far apart in the primary structure. For example, the important amino acids at the active site of lactate dehydrogenase are shown in Fig. 4.6. The numbers refer to their positions in the primary structure of the enzyme. 

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Molecular volumes are usually computed by a nonquantum mechanical method, which integrates the area inside a van der Waals or Connolly surface of some sort. Alternatively, molecular volume can be determined by choosing an isosurface of the electron density and determining the volume inside of that surface. Thus, one could find the isosurface that contains a certain percentage of the electron density. These properties are important due to their relationship to certain applications, such as determining whether a molecule will fit in the active site of an enzyme, predicting liquid densities, and determining the cavity size for solvation calculations. 

This class of inhibitors usually acts irreversibly by permanently blocking the active site of an enzyme upon covalent bond formation with an amino acid residue. Very tight-binding, noncovalent inhibitors often also act in an irreversible fashion with half-Hves of the enzyme-inhibitor complex on the order of days or weeks. At these limits, distinction between covalent and noncovalent becomes functionally irrelevant. The mode of inactivation of this class of inhibitors can be divided into two phases the inhibitors first bind to the enzyme in a noncovalent fashion, and then undergo subsequent covalent bond formation. 

Uncovering of the three dimentional structure of catalytic groups at the active site of an enzyme allows to theorize the catalytic mechanism, and the theory accelerates the designing of model systems. Examples of such enzymes are zinc ion containing carboxypeptidase A 1-5) and carbonic anhydrase6-11. There are many other zinc enzymes with a variety of catalytic functions. For example, alcohol dehydrogenase is also a zinc enzyme and the subject of intensive model studies. However, the topics of this review will be confined to the model studies of the former hydrolytic metallo-enzymes. 

The equilibrium constant for the second-order attachment of a substrate to the active site of an enzyme was found to be 326 at 310 K. At the same temperature, the rate constant for the second-order attachment is 7.4 X 107 L-mol-s. What is the rare constant for the loss of unreacted substrate from the active site (the reverse of the attachment reaction) ... 

Figure 7-1. Planar representation of the "three-point attachment" of a substrate to the active site of an enzyme. Although atoms 1 and 4 are identical, once atoms 2 and 3 are bound to their complementary sites on the enzyme, only atom 1 can bind. Once bound to an enzyme, apparently identical atoms thus may be distinguishable, permitting a stereospecific chemical change.

The probing of the active site of an enzyme by using multiple crystal structures containing different small molecules was originally described using different organic solvents as the probe molecules [21]. This technique showed how the information derived from the small molecules binding in... 

The active site of an enzyme is small relative to the total volume of the enzyme. 

The active site of an enzyme is generally a pocket or cleft that is specialized to recognize specific substrates and catalyze chemical transformations. It is formed in the three-dimensional structure by a collection of different amino acids (active-site residues) that may or may not be adjacent in the primary sequence. The interactions between the active site and the substrate occur via the same forces that stabilize protein structure hydrophobic interactions, electrostatic interactions (charge-charge), hydrogen bonding, and van der Waals interactions. Enzyme active sites do not simply bind substrates they also provide catalytic groups to facilitate the chemistry and provide specific interactions that stabilize the formation of the transition state for the chemical reaction. 

Organizing a reaction at the active site of an enzyme makes it go faster. 

Metal ion-catalyzed hydrolytic processes have been studied for a long time, and many interesting systems have been explored which give valuable information about catalysis. However, with very few exceptions the catalysis afforded by these systems in water is disappointing when compared with enzymatic systems where a metal ion cofactor activates a substrate and a nucleophilic or basic group in an acyl or phos-phoryl transfer process. It has been noted that bulk water may not be a good medium to approximate the medium inside the active site of an enzyme where it is now known that the effective dielectric constants resemble those of organic solvents rather than water. 

The Probing by Fischer of the Active Site of an Enzyme (Modern Formula)... 

Figure 6. Enzymes act as recycling catalysts in biochemical reactions. A substrate molecule binds (reversible) to the active site of an enzyme, forming an enzyme substrate complex. Upon binding, a series of conformational changes is induced that strengthens the binding (corresponding to the induced fit model of Koshland [148]) and leads to the formation of an enzyme product complex. To complete the cycle, the product is released, allowing the enzyme to bind further substrate molecules. (Adapted from Ref. 1). See color insert.

Figure 11. Allosteric regulation A conformational change of the active site of an enzyme induced by reversible binding of an effector molecule (A). The model of Monod, Wyman, and Changeux (B) Cooperativity in the MWC is induced by a shift of the equilibrium between the T and R state upon binding of the receptor. Note that the sequential dissociation constants Kr and KR do not change. The T and R states of the enzyme differ in their catalytic properties for substrates. Both plots are adapted from Ref. 140. See color insert.

The microenvironment of the micellar core inferred from fluorescent probes is said to be similar to some organic media (Turner and Brand, 1968 Cordes and Gitler, 1973). Similar conclusions have been obtained by other spectroscopic means (see previous sections). The active site of an enzyme is usually quite hydrophobic and the number of water molecules at the active site is limited. Therefore, it is very useful to study the behavior of the catalytic groups in organic media in relation to micellar and enzymatic catalysis. 

The binding sites of most enzymes and receptors are highly stereoselective in recognition and reaction with optical isomers (J, 2 ), which applies to natural substrates and synthetic drugs as well. The principle of enantiomer selectivity of enzymes and binding sites in general exists by virtue of the difference of free enthalpy in the interaction of two optical antipodes with the active site of an enzyme. As a consequence the active site by itself must be chiral because only formation of a diasteromeric association complex between substrate and active site can result in such an enthalpy difference. The building blocks of enzymes and receptors, the L-amino acid residues, therefore ultimately represent the basis of nature s enantiomer selectivity. 

Various mechanisms of reversible inhibition have been proposed. Competitive inhibition is conceptually the easiest to understand. Recall that the active site of an enzyme is complementary in shape to the shape of the substrate (crudely, the lock and key hypothesis). Suppose a compound which is not the true substrate, but structurally similar to it blocks the active site by binding to it. The true substrate cannot bind and so no reaction will occur. Hence, there is competition between the true substrate and the inhibitor for binding at the active site. 

If the overall reaction rate is controlled by step three (k3) (i.e. if that is the rate limiting step), then the observed isotope effect is close to the intrinsic value. On the other hand, if the rate of chemical conversion (step three) is about the same or faster than processes described by ks and k2, partitioning factors will be large, and the observed isotope effects will be smaller or much smaller than the intrinsic isotope effect. The usual goal of isotope studies on enzymatic reactions is to unravel the kinetic scheme and deduce the intrinsic kinetic isotope effect in order to elucidate the nature of the transition state corresponding to the chemical conversion at the active site of an enzyme. Methods of achieving this goal will be discussed later in this chapter. 

FIGURE 1.5. Schematic drawing showing the use of a temperature sensitive polymer to control access to the active sites of an enzyme. Access is denied when the polymer (dotted line) is in the crystalhne state (left), and allowed when the polymer is in the flexible state (right). 

Two models currently exist to explain how an enzyme and its substrate interact. One model, called the lock and key model, suggests that an enzyme is like a lock, and its substrate is like a key. The shape of the active site on the enzyme exactly fits the shape of the substrate. A second model, called the induced fit model, suggests that the active site of an enzyme changes its shape to fit its substrate. Figure 6.21 shows both models. 

Protein functions and interactions are infinitely varied in biological species— one of the major problems associated with complete classification of any proteome. Proteins may transport substances myoglobin and hemoglobin (discussed in Chapter 7) transport oxygen, and carbon dioxide, in mammalian blood. Proteins called enzymes catalyze necessary biochemical reactions. The active site of an enzyme contains those amino acids that come in direct contact... 

Figure 2. Alternative electron-transfer mechanisms, (a) Direct electron transfer (tunneling mechanism) from electrode surface to the active site of an enzyme, (b) Electron transfer via redox mediator.

Figure 3.1 Amino add side-chain groups involved in binding NAD at the active site of an enzyme. The enzyme is glyceraldehyde dehydrogenase. More than 20 amino acids, the position of which in the primary structure is indicated by the number, counting from the N-terminal amino acid, are involved in the binding. This emphasises the complexity of the binding that is responsible for the specificity of the enzyme for NAD (depicted in bold). The molecular structure of nicotinamide adenine dinucleotide (NAD ) provided in Appendix 3.3.

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