Inhibition noncompetitive

Noncompetitive inhibition occurs when the inhibitor binds to the enzyme at sites other than the active sites, as shown in Eqnation 4.34. This does not prevent snbstrate binding bnt resnlts in [ 57] being inactive and so slows down the reaction rate. The extent of inhibition in this case depends on the inhibitor, not on the snbstrate concentration. This decreases the value of but does not interfere with Kg. Equation 4.35 shows the reaction rate expression. 

Noncompetitive inhibition is a special case of linear mixed inhibition where 5 = 1 and a = p. Thus, the expression for the velocity of an enzymatic reaction in the presence of a noncompetitive inhibitor becomes 

TABLE 4.1 Summary of the Effects of Reversible Inhibitors on Apparent Enzyme Catalytic Parameters and K  

for noncompetitive inhibition, an apparent decrease in V ax is observed while Ks remains unaffected. A summary of the effects of reversible inhibitors on the catalytic parameters Ks and Vi ax is presented in Table 4.1. 

In noncompetitive inhibition, the inhibitor is presumed not to bind to an active site on the enzyme, but rather to bind at some other site. This complex formation may involve some change in the conformation of the enzyme, which makes it impossible for the substrate to bind at the active site. The inhibition of urease by Ag+, Pb2+, or Hg2+ is believed to be the result of these metal ions binding to the sulfhydryl (—SH) groups on the enzyme. For this type of action, we can write the equilibria 

In this equation, K represents the combined effects of both Kf and Kf. This equation indicates that a plot of 1 /i versus 1 / [S] should be linear with a slope that represents (Km/Rmax)(l + [I]/Ki) and an intercept of (l/i -max)(l + [I]/Ki)- A different line will be obtained for each initial 

FIGURE 6.9 A Lineweaver-Burk plot for the case of noncompetitive enzyme inhibition at three concentrations of the inhibitor. 

Pure noncompetitive inhibition (decrease in V ax with no change in K ) is seldom observed in enzyme kinetics studies, except in the case of very small inhibitors, such as protons, metal ions, and small anions. For noncompetitive 

Noncompetitive inhibitors interact with enzymes in many different ways. They can bind to the enzymes reversibly or irreversibly at the active site or at some other region. In any case the resultant complex is inactive. The mechanism of noncompetitive inhibition can be expressed as follows  

Since substrate and inhibitor do not compete for a same site for the formation of enzyme-substrate or enzyme-inhibitor complex, we can assume that the dissociation constant for the first equilibrium reaction is the same as that of the third equilibrium reaction, as 

As shown in the previous section, the rate equation can be derived by employing the Michaelis-Menten approach as followsrwhere 

Several variations of the mechanism for noncompetitive inhibition are possible. One case is when the enzyme-inhibitor-substrate complex can be decomposed to produce a product and the enzyme-inhibitor complex. This mechanism can be described by adding the following slow reaction to Eq. (2.49) 

This case is known as partially competitive inhibition. The derivation of the rate equation is left as an exercise problem. 

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. 

In the simplest monosubstrate case, a noncompetitive inhibitor would have no effect on substrate binding and vice versa. The inhibitor and the substrate bind reversibly, randomly, and independently at different sites. The substrate binds to E and El, and the inhibitor binds to E and EA however, the resulting EAI complex is inactive. The binding of one ligand has no effect on the dissociation constant of the other therefore, both binding reactions E + A EA and El + A EAI have the same dissociation constant. The kinetic model for this type of inhibition would be 

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). 

This Idnetic model is a usual description of a noncompetitive inhibition in biochemical textbooks, usually without specification that it is a very rare model. 

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. 

The general rate equation can be rearranged to show either A or B as the varied hgand. 

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Effect of the concentration of inhibitor on the Lineweaver-Burk plots for (a) competitive inhibition, (b) noncompetitive inhibition, and (c) uncompetitive inhibition. The inhibitor s concentration increases in the direction shown by the arrows. 

Fig. 1. Inhibition of porcine pancreatic a-amylase. Substrates, an inhibitor, and their binding orientations in the active site are shown schematically. The arrows denote the catalytic site in each case, (a) The small substrate, G2PNP [17400-77-0] (3) (b) the large substrate, G OH [13532-61 -1] (4) and (c) the inhibitor, 4-phenyl imidazole (5) and the substrate G2PNP (3) in the binding orientation for noncompetitive inhibition. The binding orientation of G2PNP...

Noncompetitive inhibitors interact with both E and ES (or with S and ES, but this is a rare and specialized case). Obviously, then, the inhibitor is not binding to the same site as S, and the inhibition cannot be overcome by raising [S]. There are two types of noncompetitive inhibition pure and mixed. 

Pure noncompetitive inhibition occurs if Ki = Ki. This situation is relatively uncommon the Lineweaver-Burk plot for such an instance is given in Eigure 14.15. Note that K is unchanged by I (the x-intercept remains the same, with or without I). Note also that Tmax decreases. A similar pattern is seen if the amount of enzyme in the experiment is decreased. Thus, it is as if I lowered [E],... 

FIGURE 14.16 Lineweaver-Burk plot of mixed noncompetitive inhibition. Note that both intercepts and the slope change in the presence of I. (a) When Ki is less than Ki (b) when Ki is greater than Ki. ... 

Presumably, these effects are transmitted via alterations in the protein s conformation. Table 14.6 includes the rate equations and apparent and 14iax values for both types of noncompetitive inhibition. 

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. 

Inhibition. Consider the case of competitive inhibition. Show a family of Lineweaver-Burk lines as [I] increases, including the line for [I] = 0. Construct the plots carefully so as to show how the x- and y-intercepts vary. Do the same for noncompetitive inhibition. 

Noncompetitive inhibition occurs when the inhibiting molecule is adsorbed after the substrate molecule has been absorbed. The assumed mechanism is... 

Denominator constant for noncompetitive inhibition 12.6 Rate constant for monomer X reacting with a Sec. 13.4.4... 

KINETIC ANALYSIS DISTINGUISHES COMPETITIVE FROM NONCOMPETITIVE INHIBITION... 

In noncompetitive inhibition, binding of the inhibitor does not affect binding of substrate. Formation of both EI and EIS complexes is therefore possible. However, while the enzyme-inhibitor complex can still bind substrate, its efficiency at transforming substrate to product, reflected by is decreased. Noncompetitive... 

For simple noncompetitive inhibition, E and EI possess identical affinity for substrate, and the EIS complex generates product at a negligible rate (Figure 8-10). More complex noncompetitive inhibition occurs when binding of the inhibitor does affect the apparent affinity of the enzyme for substrate, causing the tines to intercept in either the third or fourth quadrants of a double reciprocal plot (not shown). 

Figure 8-10. Lineweaver-Burk plot for simple noncompetitive inhibition.

Figure 3.2 Cartoon representations of the three major forms of reversible inhibitor interactions with enzymes (A) competitive inhibition (B) noncompetitive inhibition (C) uncompetitive inhibition. Source-. From Copeland (2000).

Because noncompetitive inhibitors bind to both the free enzyme and the ES complex, or subsequent species in the reaction pathway, we would expect these molecules to exert a kinetic effect on the E + S —> ES" process, thus effecting the apparent values of both VmdX/KM (influenced by both the K and al, terms) and Vmax (influenced by the aK term). This is reflected in the velocity equation for noncompetitive inhibition ... 

For noncompetitive inhibition, the value of k bs will vary with substrate concentration in different way, depending on the value of a (see Chapter 3). When a = 1, kobs is independent of substrate concentration ... 

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