Noncompetitive inhibition examples

If the inhibitor combines irreversibly with the enzyme—for example, by covalent attachment—the kinetic pattern seen is like that of noncompetitive inhibition, because the net effect is a loss of active enzyme. Usually, this type of inhibition can be distinguished from the noncompetitive, reversible inhibition case since the reaction of I with E (and/or ES) is not instantaneous. Instead, there is a time-dependent decrease in enzymatic activity as E + I El proceeds, and the rate of this inactivation can be followed. Also, unlike reversible inhibitions, dilution or dialysis of the enzyme inhibitor solution does not dissociate the El complex and restore enzyme activity. 

Nonetheless, note that such constraints hinge upon detailed knowledge of the functional form of the rate equation. For example, for noncompetitive inhibition, no restriction occurs Both saturation parameters may attain any value with in their assigned interval, independent of the saturation of the other reactant. We thus emphasize that choosing all saturation parameters... 

Full and partial noncompetitive inhibitory mechanisms, (a) Reaction scheme for full noncompetitive inhibition indicates binding of substrate and inhibitor to two mutually exclusive sites. The presence of inhibitor prevents release of product, (b) Lineweaver-Burk plot for full noncompetitive inhibition reveals a common intercept with the 1/[S] axis and an increase in slope to infinity at infinitely high inhibitor concentrations. In this example, K =3 IulM. (c) Replot of Lineweaver-Burk slopes from (b) is linear, confirming a full inhibitory mechanism, (d) Reaction scheme for partial noncompetitive inhibition indicates binding of substrate and inhibitor to two mutually exclusive sites. The presence of inhibitor alters (reduces) the rate of release of product by a factor p. (e) Lineweaver-Burk plot for partial noncompetitive inhibition reveals a common intercept with the 1/[5] axis and an increase in slope to a finite value at infinitely high inhibitor concentrations. In this example, /Cj= 3 iulM and P = 0.5. (f) Replot of Lineweaver-Burk slopes from (e) is hyperbolic, confirming a partial inhibitory mechanism... 

Figure 9-12 shows a plot of 1 /v against 1 / [S] at a series of fixed values of [I]. For the case that fC, = K2 (classical noncompetitive inhibition), a family of reciprocal plots that intersect on the horizontal axis at a value of -1 / Km is obtained. On the other hand, if K1 and K2 differ (the general case of noncompetitive inhibition), the family of curves intersect at some other point to the left of the vertical axis and, depending upon the relative values of fC, and K2, either above or below the horizontal axis. The example illustrated is for K2 = 0.5/C, that is, for the binding of M to ES being twice as strong as that to E. 

Product inhibition (Section A,12) can also provide information about mechanisms. For example, if 1 / v is plotted against 1 / [A] in the presence and absence of the product Q, the product will be found to compete with A and to give a typical family of lines for competitive inhibition. On the other hand, a plot of 1 / v vs 1 / [B] in the presence and absence of Q will indicate noncompetitive inhibition if the binding of substrates is ordered (Eq. 9-43). In other words, only the A-Q pair of substrates are competitive. Product inhibition is also observed with enzymes having ping-pong kinetics (Eq. 9-47) as a result of formation of nonproductive complexes. 

Fiqtire 3.5 (a) Competitive inhibition inhibitor and substrate compete for the same binding site. For example, indole, phenol, and benzene bind in the binding pocket of chymotrypsin and inhibit the hydrolysis of derivatives of tryptophan, tyrosine, and / phenylalanine, (b) Noncompetitive inhibition inhibitor and substrate bind simultaneously to the enzyme. An example is the inhibition of fructose 1,6-diphosphatase by AMP. This type of inhibition is very common with multisubstrate enzymes. A rare example of / uncompetitive inhibition of a single-substrate enzyme is the inhibition of alkaline phosphatase by L-phenylalanine. This enzyme is composed of two identical subunitjs, so presumably the phenylalanine binds at one site and the substrate at the other. [From N. K. Ghosh and W. H. Fishman, J. Biol. Chem. 241, 2516 (1966) see also M. Caswell and M. Caplow, Biochemistry 19, 2907 (1980). 

Enzyme inhibition Many types of molecule exist which are capable of interfering with the activity of an individual enzyme. Any molecule which acts directly on an enzyme to lower its catalytic rate is called an inhibitor. Some enzyme inhibitors are normal body metabolites that inhibit a particular enzyme as part of the normal metabolic control of a pathway. Other inhibitors may be foreign substances, such as drugs or toxins, where the effect of enzyme inhibition could be either therapeutic or, at the other extreme, lethal. Enzyme inhibition may be of two main types irreversible or reversible, with reversible inhibition itself being subdivided into competitive and noncompetitive inhibition. Reversible inhibition can be overcome by removing the inhibitor from the enzyme, for example by dialysis (see Topic B6), but this is not possible for irreversible inhibition, by definition. 

This article describes various approaches to inhibition of enzyme catalysis. Reversible inhibition includes competitive, uncompetitive, mixed inhibition, noncompetitive inhibition, transition state, and slow tight-binding inhibition. Irreversible inhibition approaches include affinity labeling and mechanism-based enzyme inhibition. The kinetics of the various inhibition approaches are summarized, and examples of each type of Inhibition are presented. 

Noncompetitive inhibitions result from combination of the inhibitor with an enzyme form other than the one the substrate combines with, and one that is present at both high and low levels of the substrate. An example is a dead-end inhibitor resembling the first substrate in an ordered mechanism. It is competitive versus A, but noncompetitive versus B, because B cannot prevent the binding of the inhibitor to free enzyme. In a random mechanism, an inhibitor binding at one site is noncompetitive versus a substrate binding at another site. 

Competitive and noncompetitive inhibitions are the most common types, especially for product inhibitors. The first product (P) released in an ordered mechanism, for example, gives noncompetitive inhibition versus either substrate A or B as the result of partially reversing the reaction. This result can occur at either low or high levels of substrate, and thus V/K as well as V is affected. A dead-end inhibitor combining with EQ in the same fashion, however, gives uncompetitive inhibition because it cannot reverse the reaction. 

Sometimes steady-state kinetics are insufficient to analyze the mechanism of inactivation for a given inhibitor. For example, irreversible enzyme inhibitors that bind so tightly to the enzyme that their dissociation rate ( ff) is effectively zero also exhibit noncompetitive inhibition patterns. They act by destroying a portion of the enzyme through irreversible binding, thereby lowering the overall enzyme concentration and decreasing Vmax- The apparent Km remains unaffected because irre-... 

Noncompetitive inhibitors interact reversibly with enzymes to form an inactive species, effectively removing active enzyme and thus interfering with the rate of conversion of substrate to product. The inhibitor may interact with free enzyme, or with the enzyme-substrate complex. The key feature of noncompetitive inhibition that distinguishes it from competitive inhibition is that inhibition does not affect the apparent affinity of the enzyme for its substrate (i.e., the apparent Km). For example, a noncompetitive inhibitor may bind in a region remote from the active site to cause a reversible change in enzyme tertiary structure that completely prevents substrate binding and product formation. In this type of inhibition, the quantity of active enzyme appears to decrease as inhibitor concentration increases, so that the apparent Fmax for the reaction decreases. 

Fig. 3. p-Glucosidase inhibition shown by Lineweaver-Burk plot (reproduced from [2]). Lineweaver-Burk plot of kinetic data from peak 2 cellobiase ((3-glucosidase) at several product inhibitor levels. This is an example of noncompetitive inhibition where the product is not only completing for binding in the active site but also binding to a secondary site on the enzyme that alters the enzyme catalytic ability... 

IRREVERSIBLE INHIBITION Inhibition may be reversible or irreversible. In reversible inhibition (i.e., competitive, uncompetitive, and noncompetitive inhibition), the inhibitor can dissociate from the enzyme because it binds through noncovalent bonds. Irreversible inhibitors usually bond covalently to the enzyme, often to a side chain group in the active site. For example, enzymes containing free sulfhydryl groups can react with alkylating agents such as iodoacetate ... 

Noncompetitive inhibition occurs when the inhibition depends only on the concentration of the inhibitor. This is usually caused by adsorption of the inhibitor at a site other than the active site but one which is necessary for activation. In other words, an inactive derivative of the enzyme is formed. Examples are the reaction of the heavy metals mercury, silver, and lead with sulfhydryl groups (—SH) on the enzyme. The sulfhydryl group is tied up by the heavy metal (ESH + Ag" " —> ESAg + H" ), and this reaction is irreversible. This is why heavy metals are poisons they inactivate enzymes in the body. 

However, this case is extremely rare in nature. An example is the noncompetitive inhibition of phenyllactate versus an amide substrate for carboxypeptidase. In this case, the initial collision complex of substrate and enzyme has an interaction with the terminal carboxyl and the arginine on the enzyme, as well as with the rest of the polypeptide chain, but the aromatic group of the terminal amino add is not in Ae specificity pocket. For it to seat itself requires twisting of the amide bond, which is the rate limiting and energy requiring step of the reaction. Thus, phenyllactate can slip into this pocket and prevent proper seating of the substrate. With an ester substrate, where rotation of the ester bond is not hindered, the collision complex has the specificity pocket filled, and phenyllactate is a competitive inhibitor (Auld Holmquist, 1974). 

As a mle, a noncompetitive inhibition occurs only if there are more than one substrate or product (Todhunter, 1979 Fromm, 1995). For example, a noncompetitive inhibition will take place in a random bisubstrate reaction, when an inhibitor competes with one substrate while the other substrate is varied. Thus, the equilibria shown below describe a Rapid Equilibrium Random bisubstrate system in which an inhibitor competes with A but allows B to bind. 

Hyperbohc noncompetitive inhibition. Case 6, represents a special example, hi this case, the inhibitor not only decreases the rate constant for product... 

In practice, the most common type of reversible inhibition relevant to httman P450s and drag metabolism is the competitive mechanism. Uncompetitive irrhibition is very rare one (non-P450) example is the inhibition of steroid 5a-reductase by the drag finasteride [194], An example of noncompetitive inhibition is that of cholesterol blocking the oxidation of nifedipine and quinidine by P450 3A4, even though cholesterol is also a substrate for the enzyme [157]. 

The derivation of the rate law is given in the Summary Notes on the Web and DVD-ROM. Equation (9-42) is in the form of the rate law that is given for an enzymatic reaction exhibiting noncompetitive inhibition. Heavy metal ions. such as Pb, Ag, and as well as inhibitors that react with the enzyme to form chemical derivatives, are typical examples of noncompetitive inhibitors. 

---