Enzyme cavity

Recently, several CYP crystal structures have been deposited in the P D B and could be used to understand the different metabolic properties. From the analysis of the different protein structures, it could be concluded that in general CYPs maintain the secondary elements across the different subfamilies, but they have a quite high flexibility, accommodating the protein structures to the ligand bound in the enzyme cavity. 

The same model indicates, of course, that only (S)-lactate [not (R)-lactate] is formed in the reduction. By the same token it explains why, in the reverse reaction, the enzyme is substrate stereoselective for (S)-lactate (R)-lactate, if locked into the enzyme cavity, would have CH3 rather than C—H juxtaposed with the NAD+ and could thus not be oxidized. 

Enzymes owe their superb adivity and seledivity to the spatial and chemical configuration of the adive site. The enzyme cavity fits around the substrate (or substrates), and the multipoint contad directs it precisely to the desired reaction center. The lock-and-key model, introduced in 1894 by the German chemist and 1902 Nobel laureate Emil Fischer [17], is an excellent analogy (Figure 5.3a). This model was... 

An important stmctural factor influencing the reactivation process could also be the rigidity of the linking chain. Owing to the rigidity of the connection chain, spatial orientation of the pyridinium rings in the enzyme cavity is limited. Compounds with a certain level of rigidity in the connection chain were synthesized with the aim of... 

In agreement with the GRID findings, site-directed mutagenesis experiments demonstrated that lipophilic interactions are extremely important for binding to take place in the enzyme cavity CYP2C9. In turn, flexibility of sidechains modifies the physicochemical enviroment of the cavity, as well as the protein pharmacopho-ric pattern. 

Another family of superimposed surfaces is used in the study of active sites of enzymes. By superimposing approximate molecular surfaces of several molecules showing similar activity with respect to the given enzyme, a part of their envelope surface, called their union surface, can be taken as an object that approximately fills out the cavity of the enzyme [167,311,338]. The shape of the union surface (in fact, the shape of its complement) is expected to provide more information on the shape of the enzyme cavity than a surface of a single active molecule. 

Consider a given molecular contour surface G(a). If the size s of the cubes is chosen small enough, then any finite polycube P can fit within G(a). As in the two-dimensional case, we do not consider orientation constraints and we assume that the contour surface G(a) and polycube P may be translated and rotated with respect to one another the relative orientation of G(a) and the cubic grid is not fixed. In this model, the identity of a polycube is independent of its orientation. Two polycubes P and F are regarded identical if and only if they can be superimposed on one another by translation and rotation in 3D space. Note, however, that the polycube method of shape analysis and determination of resolution based similarity measures can be augmented with orientation constraints, suitable for the study of molecular recognition and shape problems in external fields or within enzyme cavities [240,243]. 

Pi-complexes, also called donor-acceptor complexes, are often a weak association of an electron-rich molecule with an electron-poor species. The donor is commonly the electron cloud of a pi bond or aromatic ring the acceptor can be a metal ion, a halogen, or another organic compound. In the absence of solvent, as can occur in an enzyme cavity, the cation-pi interaction can be stronger than hydrogen bonding. The cation snuggles into the face of the aromatic pi cloud (see aromaticity. Sections 1.9.3 and 12.3). 

Figure 3 A schematic presentation of materials for affinity chromatography and enzymes immobilized in acrylamide gel beads and how the two fields led to the idea of molecular imprinting. In affinity chromatography, specific affinity ligands on polymeric beads rebind antigens with high selectivity and strength. It was believed that such specific sites could also be obtained from beads with embedded enzymes. Upon extraction and removal of the embedded enzymes cavities that are complementary to the original enzyme would be left behind and these should theoretically rebind the enzyme with high selectivity and strength [17].

More specifically we analyze the similarities and differences between bio- and chemo-catalytic systems in the sections on oxidation and reduction catalysis. We highlight the important mechanistic concepts and energetic requirements that were described in the previous chapters such as pre-transition-state orientation and solvation (the dielectric constant of an enzyme is as low as that of a zeolite) and the match of the shape and size of the transition state with the enzyme cavity. In this context, it will be important to compare enzyme action concepts such as lock and key and induced Ht with some of the related ideas discussed in Chapter 2. We refer back to concepts in transition-metal surface catalysis in order to establish ideas on possible mechanisms. 

Upon the adsorption of the glucose molecule into the enzyme cavity, the cavity closes as observed in Fig. 7. lb. The cavity continues to change as the reaction proceeds, which helps to drive the reaction over the potential energy surface to the product state. Desorption of the product molecules requires reopening of the cavity in order to release them. This process can be aided by the coadsorption of an additional reactant molecule at a second peptide binding site (allosteric effect). This reduces the interaction between product molecules and cavity, assists desorption and decreases the tendency of the enzyme to become deactivated by product poisoning. 

A unique feature of the enzyme is multi-point bonding of reaction intermediates to the enzyme cavity and the participation of several protein substituents often belonging to different amino acids in the activation of a substrate (Fig. 7.1c). 

The optimum induced fit between the enzyme cavity and the reacting substrate enables the enzyme for a particular reaction to discriminate readily between reactants that differ in size. This is illustrated in Fig. 7.2 for the conversion of glucose and closely related molecules by the hexakinase enzyme. 

When a reactant molecule adsorbs on a particular site, entropy is lost compared with the reactant state in solvent or gas phase. This was described earlier in the chapter on zeolites. Within the rigid lock and key model, this entropy loss would be maximum, thus reducing the free energy gained upon adsorption. This is an additional reason why an optimum fit between reactant and enzyme cavity is not preferred. When the fit between the reactant and the cavity is not optimum, the reactant will maintain some mobility in the adsorbed state, hence the entropy loss is less. The basic mechanistic principles for enzyme catalysis discussed so far include the induced fit of the enzyme cavity as a response to substrate shape and size, pretransition-state stabilization of activated molecules and the principle of optimum motion. A reaction that proceeds through intermediates via transient covalent bonds is preferred. 

Tight(T), loose(L) and open(O) phases of three catalytic centers are proptosed. ATP will only be released from Fi once ATP is formed at a different site by adsorption of ADP and phosphate. The conformational changes that occur in the enzyme cavities when the reaction proceeds are communicated through the subunits to the other sites. This allotropic communication implies synchronization of the different phases of the reaction at the different reaction centers. 

The reactions can also be of the acid base type induced by the electrostatics of the enzyme cavity. 

Fischer l suggested at the end of the 19th century that unique activity of enzymes is related to the need for reactant molecules to fit optimally in the enzyme cavity. This is the lock and key molecular recognition model. Later Koshlandl l postulated the concept of induced fit the enzymes assume shapes that are complementary to that of the substrate after the substrate is bound. 

Paulingl l suggested in 1948 a strategy for developing enzyme cavities that stabilize the transition state of the rate-limiting step. This can be recognized as the need for a substrate to have an optimum interaction with the enzyme. We have seen that the stabihzation of pretransition-state structures is usually the essential step that stabilizes transition states. 

The microenvironment within the enzyme cavity has a great influence on the reaction catalyzed by enzymes. Most of the effective parameters on enzyme operation—such as substrate preorganization, restricted substrate motion, protein dynamics, covalent binding of the transition state, and desolvation of the substrate—are induced by this microenvironment [5]. Therefore, designing a binding cavity in the structure of artificial enzymes is of great importance [2,6]. 

Scheme 12.9. A retroaldol-like cleavage for the removal of a one-carbon nnit as an eqniva-lent of formaldehyde CH2O from serine (Ser, S) to generate the amino acid glycine (Gly, G). The enzyme cavity contains the cofactor pyridoxal as well as the tetrahydrofolate. The latter is converted to 5,10-methylenetetrahydrofolate in the process.

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