Transition state, enzyme binding

The active site of subtilisin is outside the carboxy ends of the central p strands analogous to the position of the binding sites in other a/p proteins as discussed in Chapter 4. Details of this active site are surprisingly similar to those of chymotrypsin, in spite of the completely different folds of the two enzymes (Figures 11.14 and 11.9). A catalytic triad is present that comprises residues Asp 32, His 64 and the reactive Ser 221. The negatively charged oxygen atom of the tetrahedral transition state binds in an oxyanion hole,... 

It is unlikely that such tight binding in an enzyme transition state will ever be measured experimentally, however, because the transition state itself is a... 

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. 

The AG for binding the substrate and the transition state is shown as a difference between the energies of the ES complex and E + S. The AG for binding the transition state is shown as a difference between the energies of the E TS complex and E + TS. If the transition state binds tighter (bigger AG) than the substrate, the enzyme-catalyzed reaction must have a lower activation energy. 

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 mentioned in the Introduction, various authors have been influenced (directly or indirectly) by the Kurz approach in their discussions of enzyme behaviour (e.g. Wolfenden, 1972 Lienhard, 1973 Jencks, 1975 Schowen, 1978 Fersht, 1985 Kraut, 1988 Wolfenden and Kati, 1991). Also, as noted earlier, the concepts of transition state binding and stabilization were crucial to the development of transition state analogues as enzyme inhibitors and hence as chemotherapeutic agents (Jencks, 1969 Wolfenden, 1972 Wolfenden and Frick, 1987 Wolfenden and Kati, 1991). 

The discussion of Krs values above is an attempt to show how they may be used to gain insights into transition state binding at or near the active sites of enzymes. For other examples of the explicit or implicit application of Kurz s ideas to enzymes, the reader is directed to the references cited at the start of this subsection and in the Introduction, particularly the reviews by Kraut (1988) and by Wolfenden and Kati (1991). 

B Experimental evidence for the utilization of binding energy in catalysis and enzyme transition state complementarity... 

The effects of enzyme-transition state complementarity on the binding of transition state analogues may be masked by extraneous binding artifacts. Chapter... 

The side chains of amino acid residues in a protein may be changed at will by protein engineering (Chapter 14) and the consequent effects on binding and catalysis studied directly. This is the subject of Chapter 15, where it will be seen how the equations derived so far actually hold in practice and are used to analyze the data. There is direct evidence for enzyme-transition state complementarity. 

Strain and stress in enzymes arise from several different causes. We have seen in this chapter, and we shall see further in Chapters 15 and 16, that stress and strain may be divided into two processes, substrate destabilization and transition state stabilization. Substrate destabilization may consist of steric strain, where there are unfavorable interactions between the enzyme and the substrate (e.g., with proline racemase, lysozyme) desolvation of the enzyme (e.g., by displacement of two bound water molecules from the carboxylate of Asp-52 of lysozyme) and desolvation of the substrate (e.g., by displacement of any bound water molecules from a peptide28). Transition state stabilization may consist of the presence of transition state binding modes that are not available for the... 

Specificity between competing substrates depends on the relative binding of their transition states to the enzyme. Enzyme-transition state complementarity maximizes specificity because it ensures the optimal binding of the desired transition state. This is also the criterion for the optimal value of kcatIKM, which is not surprising, since specificity is determined by kcatIKM. Maximization of rate... 

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... 

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. 

Sim6n, L. Goodman, J. M. Enzyme catalysis by hydrogen bonds The balance between transition state binding and substrate binding in oxyanion holes, J. Org. Chem. 2009, 75,1831-1840. 

We can not absolutely discount the possibility that the observed loss in transition state binding is indirectly due to minor structural aberrations in the enzyme as a result of the substitutions. However, the crystal structure of 6-galactosidase, which became available near the completion of this study, shows that His-357 and His-391 are near the known active site residues and probably line the active site cavity (24). Therefore, they have the potential to form direct interactions with the substrate in the transition state form. 

The idea of transition state binding has proved very fruitful in understanding enzymic catalysis in general, as researchers have looked for specific transition state interactions between enzyme and substrate. To the author, though, the frequently encountered statement that enzymes work by binding transition states , without elaboration, is a tautology - as we have seen, the concept of transition state binding follows immediately from elementary transition state theory. 

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