Enzymes, inhibition, substrate reversibility

The three reversible mechanisms for enzyme inhibition are distinguished by observing how changing the inhibitor s concentration affects the relationship between the rate of reaction and the concentration of substrate. As shown in figure 13.13, when kinetic data are displayed as a Lineweaver-Burk plot, it is possible to determine which mechanism is in effect. 

The mechanisms which underlie enzyme inhibition are described more fully in Chapter 3. Suffice to say here that reversible inhibitors which block the active site are called competitive whilst those which prevent release of the product of the reaction are non-competitive. By preventing the true substrate accessing the active site, competitive inhibitors increase Km (designated by or K PParent). A non-competitive inhibitor decreases V mprime symbol ( ) here to imply physiological as it does for energy change. 

Binding of a reversible inhibitor to an enzyme is rapidly reversible and thus bound and unbound enzymes are in equilibrium. Binding of the inhibitor can be to the active site, or to a cofactor, or to some other site on the protein leading to allosteric inhibition of enzyme activity. The degree of inhibition caused by a reversible inhibitor is not time-dependent the final level of inhibition is reached almost instantaneously, on addition of inhibitor to an enzyme or enzyme-substrate mixture. 

Di- and trifluoromethyl ketones inhibit a great number of esterases and proteases with often very high inhibition constants (cf. Chapter 7). Although the fluorinated ketone is covalently bonded to the nucleophilic residue of the enzyme, the inhibition is reversible, as the inhibitor could be displaced by another nucleophile. The covalent nature of the interactions as well as the tetrahedral structure of the adducts have been demonstrated by kinetic studies, by NMR experiments, and by the X-ray diffraction of the enzyme-substrate complexes. ... 

A number of conformationally restricted fluorinated inhibitors have been synthesized and evaluated. These smdies show that (1) subtle conformational differences of the substrates affect the inhibition (potency, reversible or irreversible character) (Figure 7.50), (2) a third inhibition process involving an aromatization mechanism could take place (Figure 7.51). When the Michael addition and enamine pathways lead to a covalently modified active site residue, the aromatization pathway produces a modified coenzyme able to produce a tight binding complex with the enzyme, responsible for the inhibition (Figure 7.51). ... 

Carbary , a widely used methyl carbamate, is a pseudosubstrate of acetylcholinesterase that reacts 105 to 106 times more slowly than do normal substrates. The carbamoylated enzyme formed is not as stable as the phosphorylated enzymes and the inhibition is reversible. 

The rate of inactivation is inhibited by reversible inhibitors or substrates of the enzyme. 

FIGURE 2-50. The consequence of a substrate competing successfully for reversal of enzyme inhibition is that the substrate essentially displaces the inhibitor and shoves it off. Because the substrate has this capability, the inhibition is said to be reversible. 

TIs also inhibit the reverse transcriptase enzyme s ability to perform one of the initial steps in HIV replication. The NNRTIs, however, directly inhibit the active (catalytic) site on this enzyme, whereas zidovudine and other NRTIs serve as false substrates that take the place of the substance (thymidine) normally acted on by this enzyme (see Reverse Transcriptase Inhibitors Mechanism of Action ). Hence, NNRTIs provide another way to impair one of the key steps in HIV replication, and these drugs can be used along with other agents (NRTIs, protease inhibitors) to provide optimal benefits in preventing HIV replication and proliferation (see the next section). 

Xenobiotics compete with acetylcholine for binding to the substrate-binding site of the enzyme resulting in reversible or irreversible inhibition of the enzyme. 

Enzyme inhibitors are divided into two classes, irreversible and reversible. Irreversible inhibition implies destruction or permanent modification of chemical groups in the enzyme. In contrast, reversible inhibitors form a complex with the enzyme that can dissociate and release the active enzyme. An enzyme E can bind either to substrate S, to form an ES complex (which can go on to products) or to inhibitor I, to form the complex EL... 

Enzyme inhibition can occur by the reaction of an enzyme-substrate complex with an inhibitor and the reaction of a free enzyme with an inhibitor, which is called noncompetitive inhibition. These inhibitions are not reversible by increasing the concentration of the substrate in the enzyme solution. The reaction of the enzyme-substrate complex with an inhibitor can be expressed as ... 

For competitive, reversible enzyme inhibition, the lowest measurable IC50 value is half of the enzyme concentration used in the assay (Cheng and Prusoff, 1973). From a practical view, kcJKM values of 104 M 1 s 1 and above are desirable for inhibitor profiling assays. With an enzyme-substrate pair characterized by a kcJKM value of 104 M 1 s, an assay can usually be run with a protease concentration in the single-digit nanomolar range in an automated setting. 

Most therapeutic drugs are reversible competitive inhibitors, which bind at the catalytic (active site) of the enzyme. Competitive inhibitors are especially attractive as clinical modulators of enzyme activity because they offer two routes for the reversal of enzyme inhibition, by decreasing the concentration of inhibitor or by raising the concentration of substrate. 

Competitive The catalytic site (usually), competing with substrate for binding in a dynamic equilibrium-like process. Inhibition is reversible by increasing substrate concentration. Vmax is unchanged Km is increased (the presence of inhibitor effectively decreases the affinity of the enzyme for its substrate). 

D-Lactate cytochrome c reductase is inhibited by p-mercuriphenyl sulfonate salts, metal chelators, and dicarboxylic acids such as oxalate and oxaloacetate (Table XVI) (312, 314, 315). According to Nygaard (314), salts (cations) inhibit at the acceptor site, and dicarboxylic acids at the substrate site. Cremona and Singer (315) have studied the inhibitions by metal chelators and by oxalate. They recognized two types of inhibition. One type of inhibition is that which is caused by EDTA or oxalate. This kind of inhibition is reversed immediately upon dilution of the enzyme-inhibitor mixture. The second is that which results from addition of o-phenanthroline. Enzyme preparations treated with o-phenanthroline bind 2 moles of the chelator per mole of Zn . This complex is stable and inactive, and does not result in the release of Zri . The inactive... 

The effect of NMMA is attributable to its prevention of NO formation by NOS and its reversal by excess substrate (l-arginine) is a classic example of competitive enzyme inhibition (Figure 2). 

The various kinds of reversible inhibition that have been identified all depend on non-covalent binding, but inhibitors differ in how they act, with consequent differences in their kinetic effects. Figure 8-6 depicts a general scheme for enzyme inhibition of a simple single substrate-single product reaction. 

Irreversible enzyme inhibition, also cahed enzyme inactivation (or active-site directed ineversible inhibition, because it is generally competitive with substrate), occurs when a compound blocks the enzyme activity for an extended period of time, generally via covalent bond formation. Therefore, even though some slow tight-binding inhibitors functionahy block the enzyme activity irreversibly, they are stih considered reversible... 

Measurement of the in vitro efficacy of compounds as substrates is usually deduced by comparison of their k JK ratios where is the first-order rate constant for product formation and is the Michaelis equilibrium constant [38]. For those compounds which are classical, reversible inhibitors, K, the dissociation (or inhibition) equilibrium constant, and (kassoc) the rate constant for enzyme inhibition, are the most commonly reported kinetic values. These values may be measured while using either a high-molecular-weight natural substrate or a low-molecular-weight synthetic substrate. For alternate-substrate inhibitors, that is, compounds which form a stable complex (an acyl-enzyme ) that dissociates to enzyme and intact inhibitor or to enzyme and an altered form of the inhibitor, the usually reported value is K, the apparent K. For compounds which irreversibly inactivate the enzyme, the kinetics are usually measured under conditions such that the initial enzyme concentration [E] is much lower than the inhibitor concentration [I] which in turn is much lower than the Ky Under these conditions the commonly reported value is obs/[I]> the apparent... 

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