The Active Site and Transition States

It is a major challenge to elucidate the mechanisms responsible for the efficiencies of enzymes. Jencks (1) offered the following classification of the mechanisms by which enzymes achieve transition state stabilization and the resulting acceleration of the reactions proximity and orientation effects of reactants, covalent catalysis, general acid-base catalysis, conformational distortion of the reactants, and preorganization of the active sites for transition state complementarity. 

Fig. 31.40 Active site and transition-state analogues (1) 7,7,7-triphenylheptanoic acid (2) w,(i),-triphenylalkyl-UDP (3) 3-5 -0-[[(2-decanoyl-3-phenylpropyloxycarbonyl)methyl]sulfonyl]uridine (DMSU). In insert, structure of a putative transition state analogue for the phenol glucuronidation reaction catalysed by UGT.

There are several factors through which anions can influence the pathway and O2 reduction kinetics. The main factors are competition with O2 for surface sites changes in the activity coefficients of the reactants, intermediates, and transition states and the acidity and dielectric properties of the electrolyte side of the interface [Adzic, 1998]. For example, perfluoro acids have higher O2 solubility and lower adsorbability than... 

The active site of an enzyme is generally a pocket or cleft that is specialized to recognize specific substrates and catalyze chemical transformations. It is formed in the three-dimensional structure by a collection of different amino acids (active-site residues) that may or may not be adjacent in the primary sequence. The interactions between the active site and the substrate occur via the same forces that stabilize protein structure hydrophobic interactions, electrostatic interactions (charge-charge), hydrogen bonding, and van der Waals interactions. Enzyme active sites do not simply bind substrates they also provide catalytic groups to facilitate the chemistry and provide specific interactions that stabilize the formation of the transition state for the chemical reaction. 

The concept of catalytic antibodies was suggested succinctly by Jencks. If complementarity between the active site and the transition state contributes significantly to enzymatic catalysis, it should be possible to synthesize an enzyme by constructing such an active site. One way to do this is to prepare an antibody to a haptenic group which resembles the transition state of a given reaction. The combining sites of such antibodies should be complementary to the transition state and should cause an acceleration by forcing bound substrates to resemble the transition state. ... 

For the tight binding of the transition state the binding surface of the enzyme must be complementary to the structure of the transition state, so that optimal interactions between the enzyme and the transition state are possible. This demand imphes that enzymes display a high affinity to molecules which are chemically similar to the transition state of the reaction. Complexes of such transition state analogues with enzymes are well suited for X-ray structure analysis to elucidate the structural principles of the active site and the catalytic mechanism. 

Early enzymatic theory emphasized the importance of high complementarity between an enzyme s active site and the substrate. A closer match was thought to be better. This idea was formally described in Fischer s lock and key model. The role of an enzyme (E), however, is not simply to bind the substrate (S) and form an enzyme-substrate complex (ES) but instead to catalyze the conversion of a substrate to a product (P) (Scheme 4.2). Haldane, and later Pauling, stated that an enzyme binds the transition state (TS ) of the reaction. Koshland expanded this theory in his induced fit hypothesis.5 Koshland focused on the conformational flexibility of enzymes. As the substrate interacts with the active site, the conformation of the enzyme changes (E — E ). In turn, the enzyme pushes the substrate toward its reactive transition state (E TS ). As the product forms, it quickly diffuses out of the active site, and the enzyme assumes its original conformation. 

Despite the differences, the two mechanisms show significant similarities both classes of enzymes employ a pair of carboxylic acids at the active site and both mechanisms operate via transition states with substantial oxocarbenium ion character. However, strong evidence indicates that a covalent intermediate is formed in retainers [Sinnot 1990 McCarter and Withers 1994],... 

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... 

Now, if we assume that the active sites of these enzymes have a hydrophobic pocket at Sj as well as discrete subsites for substrate amino acids, we can explain these results by assigning different levels of importance to these different modes of interaction for the two enzymes. To account for the Pi specificity of FKBP, we not only assume a more prominent role for Pi-Si interactions but also that these interactions are characterized by dehydration of the Michaelis complex, E S, as it proceeds to the transition state, [E S]t. What we are suggesting here is that in E S, the Pi residue is not yet buried in Si and that the active site and the substrate are still at least partially solvated. As E S proceeds to [E S], the Pi residue becomes buried in the Si pocket and the residual water of solvation is expelled from the active site. This scenario can reasonably account for the large values of A/ft and ASt that we observe for reactions of FKBP, since the formation of hydrophobic contacts between apolar groups in aqueous solution is known to be accompanied by positive enthalpy and entropy changes (Nemethy, 1967). Likewise, to account for the lack of Pi specificity for CyP, we assume that subsite interactions play a more prominent role than do Pi—Si interactions. Thus, the Pi-Si hydrophobic interactions that dominate the thermodynamic parameters for FKBP have a smaller role for this enzyme. 

Figure 4 Chemical tools for the study of y-secretase. Transition-state anaiog inhibitors inciude hydroxyi-containing moieties that interact with the catalytic aspartates of aspartyl proteases. Helical peptides mimic the APR transmembrane domain and interact with the substrate docking site on the protease. These potent inhibitors were converted into affinity labeling reagents that contain a chemicaiiy reactive bromoacetyi or photoreactive benzophenone for covalent attachment to the protein target and a biotin moiety to allow isolation and detection of the labeled protein. Both types of chemical probes interacted with the two presenilin subunits but at distinct locations, which suggests that both the active site and the docking site of y-secretase lie at the interface between these subunits.

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