Enzymes active sites and

FIGURE 6.11. Comparison of the environment around the transition state of lysozyme in the enzyme-active site and in the reference solvent cage. 

As discussed in the early sections it seems that there are very few effective ways to stabilize the transition state and electrostatic energy appears to be the most effective one. In fact, it is quite likely that any enzymatic reaction which is characterized by a significant rate acceleration (a large AAgf +p) will involve a complimentarity between the electrostatic potential of the enzyme-active site and the change in charges during the reaction (Ref. 10). This point may be examined by the reader in any system he likes to study. 

The structures of enzyme active sites, and other ligand binding pockets on enzymes, are ideally suited for high-affinity interactions with drug-like inhibitors. 

All enzymatic reactions are initiated by formation of a binary encounter complex between the enzyme and its substrate molecule (or one of its substrate molecules in the case of multiple substrate reactions see Section 2.6 below). Formation of this encounter complex is almost always driven by noncovalent interactions between the enzyme active site and the substrate. Hence the reaction represents a reversible equilibrium that can be described by a pseudo-first-order association rate constant (kon) and a first-order dissociation rate constant (kM) (see Appendix 1 for a refresher on biochemical reaction kinetics) ... 

Some nonreactive molecules are recognized by the target enzyme as pseudosubstrates. These bind to the enzyme active site and are chemically transformed into reactive species that then covalently inactivate the enzyme. 

In this case, [I] represents the inhibitor concentration at the enzyme-active site and K is the inhibition constant for a single enzyme ... 

The basis of many biochemical processes within a cell lies in the shape relationships that exist between the reacting molecules, e.g. an enzyme active site and its substrate. The affinity and specificity that such molecules show for each other form the basis of methods such as immunoassays, and they can also be exploited in affinity chromatography. 

Interpretation of KIEs on enzymatic processes (see Chapter 11) has been frequently based on the assumption that the intrinsic value of the kinetic isotope effect is known. Chemical reactions have long been used as models for catalytic events occurring in enzyme active sites and in some cases this analogy has worked quite well. One example is the decarboxylation of 4-pyridylacetic acid presented in Fig. 10.9. Depending on the solvent, either the zwitterionic or the neutral form dominates in the solution. Since the reaction rates in D20/H20 solvent mixtures are the same (see Section 11.4 for a discussion of aqueous D/H solvent isotope effects), as are the carbon KIEs for the carboxylic carbon, it is safe to assume that this is a single step reaction. The isotope effects on pKa are expected to be close to the value of 1.0014 determined for benzoic acid. This in mind, changes in the isotope effects have been attributed to changes in solvation. 

In MET, a low-molecular-weight, redox-active species, referred to as a mediator, is introduced to shuttle electrons between the enzyme active site and the electrode.In this case, the enzyme catalyzes the oxidation or reduction of the redox mediator. The reverse transformation (regeneration) of the mediator occurs on the electrode surface. The major characteristics of mediator-assisted electron transfer are that (i) the mediator acts as a cosubstrate for the enzymatic reaction and (ii) the electrochemical transformation of the mediator on the electrode has to be reversible. In these systems, the catalytic process involves enzymatic transformations of both the first substrate (fuel or oxidant) and the second substrate (mediator). The mediator is regenerated at the electrode surface, preferably at low overvoltage. The enzymatic reaction and the electrode reaction can be considered as separate yet coupled. 

Enzyme active sites and receptors rarely interact with hgands without some attendant change in conformation, and the ability to detect and quantify a conformational change hes at the heart of contemporary biochemical kinetics. See Induced Fit Model Fluorescence Spectroscopy Linked Functions Flemoglobin Cooperativity... 

Fig. 34. Glu-72- Zn interactions in native carboxypeptidase A and in carboxypep-tidase A-inhibitor complexes (inhibitors have been reviewed by Christianson and Lipscomb, 1989). When substrates or inhibitors bind to the enzyme active site and interact with the zinc ion, the interaction of the metal with Glu-72 tends from bidentate toward uniden-tate coordination. The flexibility of protein-zinc coordination may be an important aspect of catalysis in this system, and the Glu-72->Zn - coordination stereochemistry observed here is consistent with the stereochemical analysis of carboxylate-zinc interactions from the Cambridge Structural Database (Carrell et al., 1988 see Fig. 4).

The (3-lactam antibiotics structurally resemble the terminal D-alanyl-D-alanine (o-Ala-o-Ala) in the pen-tapeptides on peptidoglycan (murein) (Fig. 45.1). Bacterial transpeptidases covalently bind the (3-lactam antibiotics at the enzyme active site, and the resultant acyl enzyme molecule is stable and inactive. The intact (3-lactam ring is required for antibiotic action. The (3-lactam ring modifies the active serine site on transpeptidases and blocks further enzyme function. 

They showed that despite the fact that lower activities were generally observed, significant improvements of enantioselectivity in the oxidation of thioanisole by PAMO and EtaA could be induced by the addition of short-chain alcohols such as methanol and ethanol. Remarkably, methanol was able to cause a reversal of PAMO enantiopreference in the case of several substrates. Reversal of enantio-preference was also observed with EtaA when using t-BuOMe. The authors hypothesize that in these enzymes solvents exert their influence on enantioselectivity by binding in or near the enzyme active site and, depending on their structure, interfere with the association of the substrate. 

The Michaelis constant, KM, for an enzyme-substrate interaction has two meanings (1) Ku is the substrate concentration that leads to an initial reaction velocity of V" /2 or, in other words, the substrate concentration that results in the filling of one-half of the enzyme active sites, and (2) KM = (k2 + ki)/kv The second definition of Ku has special significance in certain... 

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