Lineweaver-Burk plot competitive inhibition

Effect on Lineweaver-Burke plot Competitive inhibition shows a characteristic Lineweaver-Burke plot in which the plots of the inhibited and uninhibited reactions intersect on the y axis at 1/Vmax (Vmax is unchanged). The inhibited and uninhibited reactions show different x axis intercepts, indicating that the apparent Km is increased in the presence of the competitive inhibitor (see Figure 5.12). 

Figure II-10 Lineweaver-Burk plot competitive inhibition.

Effect on Lineweaver-Burke plot Noncompetitive inhibition is readily differentiated from competitive inhibition by plotting 1/v0 versus 1/[S] and noting that Vmax decreases in the presence of a noncompetitive inhibitor, whereas Km is unchanged (see Figure 5.14). 

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. 

Competitive and non-eompetitive inhibitions are easily distinguishable from the Lineweaver-Burk plot. In the ease of eompetitive inhibitors, the intereept on tlie 1/Cg axis inereases while tlie intereept of tlie 1/v axis remains unehanged by the addition of the inhibitor. Conversely, with a non-eompetitive inhibitor, only the 1/v axis intereept inereases. The effeet of eompetitive inhibitors ean be reversed by inereasing the substrate eoneentration. Where the enzyme or the enzyme substrate eomplex is made inaetive, a non-eompetitive inhibitor deereases of the enzyme, but remains eonstant. 

Plot both sets of data as a Lineweaver-Burk plot for competitive inhibition (see Fig. 

Figure 8-9. Lineweaver-Burk plot of competitive inhibition. Note the complete relief of inhibition at high [S] (ie, low 1 /[S]).

Enzymes can be used not only for the determination of substrates but also for the analysis of enzyme inhibitors. In this type of sensors the response of the detectable species will decrease in the presence of the analyte. The inhibitor may affect the vmax or KM values. Competitive inhibitors, which bind to the same active site than the substrate, will increase the KM value, reflected by a change on the slope of the Lineweaver-Burke plot but will not change vmax. Non-competitive inhibitors, i.e. those that bind to another site of the protein, do not affect KM but produce a decrease in vmax. For instance, the acetylcholinesterase enzyme is inhibited by carbamate and organophosphate pesticides and has been widely used for the development of optical fiber sensors for these compounds based on different chemical transduction schemes (hydrolysis of a colored substrate, pH changes). 

At low concentrations of substrate ([S] < Km), the enzyme is predominantly in the E form. The competitive inhibitor can combine with E, so the presense of the inhibitor decreases the velocity when the substrate concentration is low. At low substrate concentration ([S] < Km), the velocity is just Vmay IKm. Since the inhibitor decreases the velocity and the velocity at low substrate concentration is proportional to Vmax/Km, the presence of the inhibitor affects the slopes of the Lineweaver-Burk plots the slope is just the reciprocal of Vmax/Km. Increasing the inhibitor concentration causes Km/Vmax to increase. The characteristic pattern of competitive inhibition can then be rationalized if you simply remember that a competitive inhibitor combines only with E. 

Figure 2.13 Lineweaver-Burk plot for no inhibitor, competitive, and noncompetitive inhibition.

Figure 10.4 Lineweaver-Burk plot illustrating comparison of competitive inhibition with no inhibition of enzyme activity...

As discussed above, the degree of inhibition is indicated by the ratio of k3/k and defines an inhibitor constant (Kj) [Eq. (3.19)], whose value reports the dissociation of the enzyme-inhibitor complex (El) [Eq. (3.20)]. Deriving the equation for competitive inhibition under steady-state conditions leads to Eq. (3.21). Reciprocal plots of 1/v versus 1/5 (Lineweaver-Burk plots) as a function of various inhibitor concentrations readily reveal competitive inhibition and define their characteristic properties (Fig. 3.5). Notice that Vmax does not change. Irrespective of how much competitive inhibitor is present, its effect can be overcome by adding a sufficient amount of substrate, i.e., substrate can be added until Vmax is reached. Also notice that K i does change with inhibitor concentration therefore the Km that is measured in the presence of inhibitor is an apparent Km- The true KM can only be obtained in the absence of inhibitor. 

For example, experimental data might reveal that a novel enzyme inhibitor causes a concentration-dependent increase in Km, with no effect on and with Lineweaver-Burk plots indicative of competitive inhibition. Flowever, even at very high inhibitor concentrations and very low substrate concentrations, it is observed that the degree of inhibition levels off when some 60% of activity still remains. Furthermore, it has been confirmed that only one enzyme is present, and all appropriate blank rates have been accounted for. It is clear that full competitive inhibition cannot account for such observations because complete inhibition can be attained at infinitely high concentrations of a full competitive inhibitor. Thus, it is likely that the inhibitor binds to the enzyme at an allosteric site. 

Full and partial competitive inhibitory mechanisms, (a) Reaction scheme for full competitive inhibition indicates binding of substrate and inhibitor to a common site, (b) Lineweaver-Burk plot for full competitive inhibition reveals a common intercept with the 1/v axis and an increase in slope to infinity at infinitely high inhibitor concentrations. In this example, Ki = 3 pM. (c) Replot of Lineweaver-Burk slopes from (b) is linear, confirming a full inhibitory mechanism, (d) Reaction scheme for partial competitive inhibition indicates binding of substrate and inhibitor to two mutually exclusive sites. The presence of inhibitor affects the affinity of enzyme for substrate and the presence of substrate affects the affinity of enzyme for inhibitor, both by a factor a. (e) Lineweaver-Burk plot for partial competitive inhibition reveals a common intercept with the 1/v axis and an increase in slope to a finite value at infinitely high inhibitor concentrations. In this example, Ki = 3 pM and = 4. (f) Replot of Lineweaver-Burk slopes from (e) is hyperbolic, confirming a partial inhibitory mechanism... 

Partial uncompetitive inhibition does not resemble full uncompetitive inhibition in terms of having an ordered mechanism, but it instead represents a very specific form of partial mixed inhibition (discussed later). However, it is sometimes referred to as partial uncompetitive inhibition due to the parallel displacement of Lineweaver-Burk plots in the presence of inhibitor, and it is thus related to full uncompetitive inhibition in the same way that partial competitive inhibition is related to full competitive inhibition. 

Figure 1-8-7. Lineweaver-Burk Plot of Competitive inhibition...

In addition to the Lineweaver-Burk plot (see p.92), the Eadie-Hofstee plot is also commonly used. In this case, the velocity v is plotted against v /[A]. In this type of plot, Vmax corresponds to the intersection of the approximation lines with the v axis, while Km is derived from the gradient of the lines. Competitive and non-competitive inhibitors are also easily distinguishable in the Eadie-Hofstee plot. As mentioned earlier, competitive inhibitors only influence Km, and not Vmax- The lines obtained in the absence and presence of an inhibitor therefore intersect on the ordinate. Non-competitive inhibitors produce lines that have the same slope (llower level. Another type of inhibitor, not shown here, in which Vmax and lselective binding of the inhibitor to the EA complex. 

The Lineweaver-Burk plot is very useful for descriptions of type and effects of inhibitors Competitive inhibitors have the same intercept on the ordinate and different intercepts on abscissa, non-competitive inhibitors give the same intercept at the abscissa but different at the ordinate. In the case of (partially) inhibited reactions, the slope is larger than at the respective non-inhibited reaction. 

The results given in Table 3.3 are plotted as shown in Figure 3.9. This Lineweaver-Burk plot shows that the inhibition mechanism is competitive inhibition. From the line for the data without the inhibitor, and are obtained as 0.98gmolm and 9.1 mmol m s , respectively. From the slopes of the lines, is evaluated as 0.6 gmol m . ... 

A. Effect of a competitive inhibitor on the reaction velocity (v0) versus substrate [S] plot. B. Lineweaver-Burke plot of competitive inhibition of an enzyme. 

The inhibition effect of poly (vinyl alcohol) on the amylose hydrolysis was investigated. Figure 7 shows Lineweaver-Burk plots of the amylose hydrolysis rates catalyzed by the random copolymer in the presence of poly (vinyl alcohol). The reaction rate is found to decrease with increasing the concentration of poly (vinyl alcohol), and all of the straight lines obtained in the plots cross with each other at a point on the ordinate. This is a feature of the competitive inhibition in the enzymatic reactions. In the present reaction system, however, it is inferred to suggest that the copolymer and poly (vinyl alcohol) molecules competitively absorb the substrate molecules. The elementary reaction can be described in the most simplified form as in Equation 3 where Z, SI, and Kj[ are inhibitor, nonproductive complex, and inhibitor constant, respectively. Then the reaction rate is expressed with Equation 4. 

EDTA inhibited the Ca2+-free wild type 1,2-a-D-mannosidase. The inhibition was investigated by fixed concentrations of EDTA in the assay buffer and by varying the concentrations of substrate. A Lineweaver-Burk plot showed a pattern consistent with competitive inhibition (Figure 30) and gave a K, of 0.91 mM for EDTA. 

Fig. 2. The characteristics of competitive inhibition, (a) A competitive inhibitor competes with the substrate for binding at the active site (b) the enzyme can bind either substrate or the competitive inhibitor but not both (c) Lineweaver-Burk plot showing the effect of a competitive inhibitor on Km and Vmax.

An inhibition of an Mg2+ ATPase on corn root plasma membrane was also observed. Kinetic data on aluminum inhibition present a competitive pattern, as demonstrated by the Lineweaver-Burk plot with an apparent inhibition constant (K,) of 40 pM [44]. These results were obtained at pH 6.6. The authors suggested that the inhibition may be a result of either the formation of an inefficient substrate (Al-ATP) or an interaction directly with the enzyme structure. 

This equation predicts that the slope of a Lineweaver-Burk plot will increase with increasing inhibitor concentration, but the intercept on the l/v0 axis (1/Kmax) will not change. A series of plots for several experiments with different concentrations of inhibitor will all have the same l/u0 intercept as shown in Fig. 9-4(a), indicating that competitive inhibition does not alter Kmax. 

When benzyl alccdiol, 2,4-dinitrophenol, dioxane, etc. were added to the reaction system, the competitive inhibition was observed as inferred from the chai of the lineweaver-Burk plot (70). The fact that mutral molecules sudi as benzyl alccdiol competitively inhibit the catalysis, indicates that the hydrophobic nature of the catalytic site makes a major contributicxi to substrate binding. 

Compounds that resemble the substrate dosely may bind at or very close to the active site, but the inhibitor is not capable of being turned over catalytically. This form of inhibition, in which substrate and inhibitor compete for the same site, and where it is not possible for both to bind simultaneously, is called competitive inhibition (Figure 8-7). The rate equation for reaction in the presence of a competitive inhibitor, expressed in the form of the linearised double redprocal Lineweaver-Burk plot, is shown in Eqn. 8.27. 

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