Transition structure binding

The rate acceleration achieved by enzymes is due to several factors. Particularly important is the ability of the enzyme to stabilize and thus lower the energy of the transition state(s). That is, it s not the ability of the enzyme to bind the substrate that matters but rather its ability to bind and thereby stabilize the transition state. Often, in fact, the enzyme binds the transition structure as much as 1012 times more tightly than it binds the substrate or products. As a result, the transition state is substantially lowered in energy. An energy diagram for an enzyme-catalyzed process might look like that in Figure 26.8. 

The capsules bind guest molecules with quite a high selectivity. Why should they not also be able to bind transition structures more strongly than reac-... 

Bartlett has derived a method181 for proving that a putative transition state analog exerts its inhibitory power from successfully mimicking the transition state. If a series of structurally-related inhibitors (all containing the identical core chemical structure meant to simulate the transition state) bind to the target enzyme with log (fQ) values that linearly correlate (slope = 1) with the log (KMlkcai) values of the same series of structurally-related substrates, then... 

As remarked already, kc/ku measures the maximal acceleration at levels of the CD sufficient to saturate complexation of the substrate. By looking carefully at the variations of this ratio with structure one may obtain insights into the mode of transition state binding (VanEtten et al., 1967a,b Bender and Komiyama, 1978). More useful is the ratio k2/ku (=kc/K ku) because it takes into account the effect of substrate binding and it scales the reactivity of S towards the CD to its intrinsic reactivity in the absence of CD. 

As pointed out above, values of KTS are obtainable from rate data without making any assumptions about the reaction mechanism. Therefore, one may use KTs and its variation with structure as a criterion of mechanism, in the same way that physical organic chemists use variations in other kinetic parameters (Brpnsted plots, Hammett plots, etc.). For present purposes, the value of Kts can be useful for differentiating between the modes of binding in the S CD complex and the TS-CD transition state, between different modes of transition state binding, and hence between different types of catalysis (Tee, 1989). 

In addition to structure stabilization and catalytic applications, transition metal-binding sites may be designed and exploited for regulatory purposes. For example, a regulatory metal-binding site has been engineered into the active site of trypsin, where metal binding inhibits proteo-... 

M ) is ideally positioned to lower the pATa of the hydroxyl group of the last nucleotide in the growing replicant and is believed to facilitate its deprotonation by a general base in the vicinity . The resulting hydroxide attacks the a-phosphate of the dNTP ". The other Mg + binds to the phosphate tail, holds it in a position favorable for a S v2-type reaction and stabilizes charge separation in the pentacoordinated transition structure (associative mechanism) ". Most likely this second Mg + also stabilizes the pyrophosphate (which is... 

How far a KM evolves relative to the substrate concentration depends on the change in structure when the substrate becomes the transition state. A limit must eventually be reached at this point, any increase in KM must be matched by a weakening of transition state binding. The problem will be most severe for large metabolites present at high concentrations. 

In one approach, the free energies of binding, out of water into the enzyme active site, of the reactant(s) and transition structure are computed, in order to see if rate acceleration can be explained by selective binding of the transition structure. However, there are several caveats associated with such an approach. First, it must be decided whether to use the same reactant and transition state structures in solution and in the enzyme. If the same structures are used, then the potential for catalysis specifically by selective transition state binding can be quantified. Of course, the actual enzyme-bound structures may be different than those in aqueous solution, and... 

In the ideal case, both of these approaches are applied to the same reaction. The reaction coordinate in the enzyme is computed directly, and then the binding energies of the reactant(s) and transition structure(s) obtained from these calculations are themselves computed. 

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