Binding to transition state

For techniques not using mutation, the minimum affinity needed in the initial library is that which would be an adequate result from the search. There are no estimates for the repertoire sizes that cover target sequence space with higher minimal affinities however, arguments based on catalytic task space suggest that 10s molecules are sufficient for saturation [4], If enzymes are considered to bind to transitional states between reactants and products, then 10s ligands may be adequate for techniques without mutation. However, there is no estimate for the minimal affinity towards a transitional state needed to catalyze a reaction, so it is unclear what exactly is achieved by a library of this size. 

Enzymes have been successfully enriched from libraries by selecting variants that bind to transition state analogs, or by covalent trapping with surface bound suicide inhibitors,20 but this approach does not usually select the most active enzymatic variants. 

All ribozymes described so far were isolated by the method of direct selection, whereby the partly or completely randomized pool of nucleic acids is subjected to a competitive situation in which only those molecules survive that can catalyze a particular reaction. A different strategy by which catalysis can be achieved is the indirect selection for binding to transition state analogs (TSAs), a tech-... 

Consider the thermodynamic cycle that relates substrate binding to transition-state binding ... 

FIGURE 14.25 Catalytic antibodies are designed to specifically bind the transition-state intermediate in a chemical reaction, (a) The intramolecnlar hydrolysis of a hydroxy ester to yield as products a S-lactone and the alcohol phenol. Note the cyclic transition state, (b)... 

There are important consequences for this statement. The enzyme must stabilize the transition-state complex, EX, more than it stabilizes the substrate complex, ES. Put another way, enzymes are designed by nature to bind the transition-state structure more tightly than the substrate (or the product). The dissociation constant for the enzyme-substrate complex is... 

Destabilization of the ES complex can involve structural strain, desolvation, or electrostatic effects. Destabilization by strain or distortion is usually just a consequence of the fact (noted previously) that the enzyme is designed to bind the transition state more strongly than the substrate. When the substrate binds, the imperfect nature of the fit results in distortion or strain in the substrate, the enzyme, or both. This means that the amino acid residues that make up the active site are oriented to coordinate the transition-state structure precisely, but will interact with the substrate or product less effectively. 

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. 

Figure 1. Enzymes bind the transition state (ES) more tightly than the ground state (ES) by a factor approximately equal to the rate of acceleration...

The present review deals with a particular aspect of the chemistry of cyclodextrins the effects that they can have on organic reactions by virtue of their abilities to bind to many organic and inorganic species (Bender and Komiyama, 1978 Saenger, 1980 Szejtli, 1982). It is a considerable expansion of an earlier work (Tee, 1989) which first showed how the Kurz approach to transition state stabilization can be employed profitably in discussing reactions mediated by cyclodextrins. Most of the large amount of data that are analysed is collected in tables in the Appendix so as to avoid breaking up the discussion in the main text too frequently. 

The cleavage of /7-nitrophenyl alkanoates (222 n = 1-8) at high pH is modestly catalysed by micelles formed from cetyltrimethylammonium bromide (CTAB) in aqueous solution. Rate constants exhibit saturation behaviour with respect to [CTAB], consistent with substrate binding in the micelles. The strength of substrate binding and transition state binding to the micelles increases monotonically with the acyl chain length, and with exactly the same sensitivity. As a result, the extent of acceleration... 

The pathway from enzyme-bound substrate to the transition state involves changes in the electronic configuration and geometry of the substrate. The enzyme itself is also not static. The ability to tightly bind the transition state requires flexibUity in the active site. Such flexibility has been experimentally demonstrated in many cases. A corollary to this is that the effectivity of enzyme catalysis can easily be influenced and regulated by conformational changes in the enzyme. An extensive consideration of the mechanisms of enzymes can be found in the works by J. Kraut (1988) and A. Fersht (1998). 

In the last section we showed that enzymes have evolved to bind the transition states of substrates more strongly than the substrates themselves. It will now be seen that it is catalytically advantageous to bind substrates weakly. 

Figure 12.5 Two cases of enzyme evolution. In both cases the enzymes bind the transition states equally well, but in (a) the substrate is bound strongly, and in (b) the enzyme has evolved to bind the substrate weakly ([S] is the same in both graphs). The activation energy in (a) is for ES —> ES, i.e., AG + AG, whereas in (b) it is for E + S — ES, i.e., AG. (The changes in Gibbs free energies are for the concentration of substrate used in the experiment, and not for standard states of 1 M.)...

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