Enzymatic catalysis stabilization

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. 

We have just discussed several common strategies that enzymes can use to stabilize the transition state of chemical reactions. These strategies are most often used in concert with one another to lead to optimal stabilization of the binary enzyme-transition state complex. What is most critical to our discussion is the fact that the structures of enzyme active sites have evolved to best stabilize the reaction transition state over other structural forms of the reactant and product molecules. That is, the active-site structure (in terms of shape and electronics) is most complementary to the structure of the substrate in its transition state, as opposed to its ground state structure. One would thus expect that enzyme active sites would bind substrate transition state species with much greater affinity than the ground state substrate molecule. This expectation is consistent with transition state theory as applied to enzymatic catalysis. 

In this chapter we have seen that enzymatic catalysis is initiated by the reversible interactions of a substrate molecule with the active site of the enzyme to form a non-covalent binary complex. The chemical transformation of the substrate to the product molecule occurs within the context of the enzyme active site subsequent to initial complex formation. We saw that the enormous rate enhancements for enzyme-catalyzed reactions are the result of specific mechanisms that enzymes use to achieve large reductions in the energy of activation associated with attainment of the reaction transition state structure. Stabilization of the reaction transition state in the context of the enzymatic reaction is the key contributor to both enzymatic rate enhancement and substrate specificity. We described several chemical strategies by which enzymes achieve this transition state stabilization. We also saw in this chapter that enzyme reactions are most commonly studied by following the kinetics of these reactions under steady state conditions. We defined three kinetic constants—kai KM, and kcJKM—that can be used to define the efficiency of enzymatic catalysis, and each reports on different portions of the enzymatic reaction pathway. Perturbations... 

Fig. 10. Participation of a bi-nuclear metal site in enzymatic catalysis (a) the substrate coordinates to one metal ion while the other activates the nucleophile (b) the substrate is stabilized by amino acid side chains, and the metal ions provide the bridging hydroxide as the nucleophile (c) the bridging hydroxide is displaced by the substrate, and the nucleophile is another water, which may be activated by an amino acid side chain in the active site...

Several review articles have been published on the catalytic functions of micelles and related systems (Fendler and Fendler, 1970, 1975 Menger, 1977 Berezin et al., 1973 Cordes and Dunlap, 1969 Cordes and Gitler, 1973 Kunitake, 1977 Kunitake and Okahata, 1976 Bunton, 1979). The conventional catalytic functions of micelles are, in most cases, related to (i) the concentration of reactants and catalytic acid-base species in the micellar phase due to electrostatic and/or hydrophobic forces and (it) the stabilization of transition states and/or destabilization of initial states by the micellar environments. The situation is more complex when one of the reagents is hydrophilic (Bunton et al., 1979). However, the last few years have witnessed several novel advances in this field especially in relation to enzymatic catalysis. 

An ionic liquid can be used as a pure solvent or as a co-solvent. An enzyme-ionic liquid system can be operated in a single phase or in multiple phases. Although most research has focused on enzymatic catalysis in ionic liquids, application to whole cell systems has also been reported (272). Besides searches for an alternative non-volatile and polar media with reduced water and orgamc solvents for biocatalysis, significant attention has been paid to the dispersion of enzymes and microorganisms in ionic liquids so that repeated use of the expensive biocatalysts can be realized. Another incentive for biocatalysis in ionic liquid media is to take advantage of the tunability of the solvent properties of the ionic liquids to achieve improved catalytic performance. Because biocatalysts are applied predominantly at lower temperatures (occasionally exceeding 100°C), thermal stability limitations of ionic liquids are typically not a concern. Instead, the solvent properties are most critical to the performance of biocatalysts. 

At the outset it is difficult to comprehend this variability, if, as we have postulated, the effect of the charge on the metal ion in distorting the electrons of the substrate is of paramount importance to the function of the metal. It would then be expected that the stronger the bonds between the metal and the substrate, the greater will be the pull on the electrons and therefore the greater the catalytic effect. The order of activating ability of the metals should therefore be the same as the order of complex stability. Such an order is indeed frequently followed in nonenzymatic catalysis (21, 52). Why, then, is enzymatic catalysis so relatively unpredictable in comparison with nonenzymatic catalysis ... 

It is apparent that the use of enzymatic catalysis continues to grow Greater availability of enzymes, development of new methodologies for their utilization, investigation of enzymatic behavior in nonconventional environments, and the design and synthesis of new biocatalysts with altered selectivity and increased stability are essential for the successful development of this field. As more is learned about selectivity of enzymes toward unnatural substrates, the choice of an enzyme for a particular transformation will become easier to predict. It should simplify a search for an appropriate catalyst and help to establish biocatalytic procedures as a useful supplement to classical organic synthesis. 

Alanine racemase, as another PLP-dependent enzyme, is a bacterial enzyme used to create D-alanine from L-alanine for incorporation into the bacterial cell wall. Its role is to act as an electron sink to stabilize carbanionic intermediates generated in enzymatic catalysis. 

FIGURE 5.4 A schematic showing the qualitative difference between enzymatic and nonenzymatic catalysis. In enzymatic catalysis, the transition state of a specific pathway is stabilized. Typical nonenzymatic catalysis, such as protic or Lewis acid, results in increasing the energy of the starting material, making several pathways energetically accessible. This difference is the source of the selectivity found in enzymatic catalysis as well as the presence of by-products in nonenzymatic catalysis. 

The amazing rate enhancement observed in enzymatic catalysis results from stabilization of the transition states and/or destabilization of the substrates. These effects are achieved by interactions of the reactants with the protein residues. Depending on the particular reaction some interactions may play a dominant role, or many different types, such as electrostatics, hydrophobic interactions, geometric distortion, or hydrogen bonds, may concurrently contribute to catalysis. Understanding these interactions is the key factor in exploiting enzymatic reactions for the purpose... 

In these two examples, the principle of transition-state stabilization as a source of enzymatic catalysis is successfully exploited for the selection of active mutants. Selection with a TSA appears to be able to enrich libraries in variants with high catalytic activity. 

The available data support a mechanism involving catalysis by distortion in which the enzyme binds and stabilizes a transition state that is characterized by partial rotation about the C-N amide bond. The energy that is required to distort this bond out of planarity with the C=0 bond, thereby destroying the resonance stabilization of the amide linkage, is supplied by favorable transition state binding interactions between enzyme and substrate. As Lumry states (1986), mechanical distortion as a source of small-molecule reactivity is attractive as a basis for enzymatic catalysis. It is quite realistic to assume that a distorted substrate will have enhanced reactivity, either because its ground state or the activated complex for its chemical reaction or both are altered by strain and stress in the protein conformation. However, as mentioned previously, this distortion need not be the result of mechanical deformation but could also be the result of desolvation or electrostatic destabilization. In fact, the current data support contributions from all three mechanisms for distortion. 

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