Enzyme-inhibitor encounter complex

In a two-step enzyme isomerization mechanism, as in scheme C, the affinity of the inhibitor encounter complex and the affinity of the final E I complex are reflected in the diminutions of v, and of vs, respectively, that result from increasing concen-... 

As we have just seen, the initial encounter complex between an enzyme and its substrate is characterized by a reversible equilibrium between the binary complex and the free forms of enzyme and substrate. Hence the binary complex is stabilized through a variety of noncovalent interactions between the substrate and enzyme molecules. Likewise the majority of pharmacologically relevant enzyme inhibitors, which we will encounter in subsequent chapters, bind to their enzyme targets through a combination of noncovalent interactions. Some of the more important of these noncovalent forces for interactions between proteins (e.g., enzymes) and ligands (e.g., substrates, cofactors, and reversible inhibitors) include electrostatic interactions, hydrogen bonds, hydrophobic forces, and van der Waals forces (Copeland, 2000). 

We can now relate the kinetic constants kCM, Ku, and kcJKM to specific portions of the enzyme reaction mechanism. From our discussions above we have seen that the term kCM relates to the reaction step of ES conversion to ES. Hence experimental perturbations (e.g., changes in solution conditions, changes in substrate identity, mutations of the enzyme, and the presence of a specific inhibitor) that exclusively affect kCM are exerting their effect on catalysis at the ES to ES transition step. The term KM relates mainly to the dissociation reaction of the encounter complex ES returning to E + S. Conversely, the reciprocal of Ku (1IKU) relates to the association step of E and S to form ES. Inhibitors and other perturbations that affect the... 

For the enzyme isomerization mechanism illustrated in scheme C of Figure 6.3, there are two steps involved in formation of the final enzyme-inhibitor complex an initial encounter complex that forms under rapid equilibrium conditions and the slower subsequent isomerization of the enzyme leading to the high-affinity complex. The value of kohs for this mechanism is a saturable function of [/], conforming to the following equation ... 

Any substance that can diminish the velocity of an enzyme-catalyzed reaction is called an inhibitor. Reversible inhibitors bind to enzymes through noncovalent bonds. Dilution of the enzyme-inhibitor complex results in dissociation of the reversibly bound inhibitor, and recovery of enzyme activity. Irreversible inhibition occurs when an inhibited enzyme does not regain activity on dilution of the enzyme-inhibitor complex. The two most commonly encountered types of inhibition are competitive and noncompetitive. 

The Brownian Dynamics (BD) simulation technique can be used to simulate the diffusion and the association of molecules in solution. BD simulations have been widely used to simulate protein-small molecule and protein-protein association (62). This method may be exploited to simulate the hrst step of molecular recognition when two molecules diffuse from a distance. From such simulations, it is possible to compute the structure and the diffusional encounter complex ensemble and to calculate the bimolecular association rate constant for two diffusing proteins or enzymes and their substrates or inhibitors. In these calculations, the effects of mutations and variations in ionic strength, pH, and viscosity can be investigated (63). 

The less frequently encountered uncompetitive inhibition occurs when a chemical binds to the enzyme-substrate complex. The catalytic function is affected without interfering with substrate binding. The inhibitor causes a structural distortion of the active site and inactivates it (Voet and Voet 2004). This has the effect of reducing the available enzyme for the reaction, and hence reduces Vmax, and also drives the reaction (E + S ES) to the right, hence decreasing Km. 

In textbooks dealing with enzyme kinetics, it is customary to distinguish four types of reversible inhibitions (i) competitive (ii) noncompetitive (iii) uncompetitive and, (iv) mixed inhibition. Competitive inhibition, e.g., given by the product which retains an affinity for the active site, is very common. Non-competitive inhibition, however, is very rarely encountered, if at all. Uncompetitive inhibition, i.e. where the inhibitor binds to the enzyme-substrate complex but not to the free enzyme, occurs also quite often, as does the mixed inhibition, which is a combination of competitive and uncompetitive inhibitions. The simple Michaelis-Menten equation can still be used, but with a modified Ema, or i.e. ... 

Several models have been proposed to account for the non-random distribution of oligosaccharides formed when polysaccharides are hydrolysed by a-amylase. The preferred-attack model assumes that the probability of bond-cleavage depends on the position of the bond in the chain the repetitive (or multiple-attack) model assumes that a-amylase can form a cage-like complex with the substrate and attack it several times during a single encounter the multiple-enzyme (or dual-site) model assumes that the substrate is hydrolysed by the combined actions of exo- and en /o-enzymes. The effects of pH, inhibitors, and the chain length of the substrates have been studied in an attempt to decide which of the three models best fits the action of a-amylase. The effects of these variables on either the distribution of products or the action pattern of the enzyme were incorporated into the models, which were then used to interpret experimental data obtained with porcine pancreatic a-amylase. 

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