Lineweaver-Burk plots, for inhibition competitive

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

In a Lineweaver-Burk plot for competitive inhibition, the lines intersect at the y-axis intercept, which is equal to 1/7. In a Lineweaver-Burk plot for noncompetitive inhibition, the lines intersect at the x-axis intercept, which is equal to - /K. ... 

In the Lineweaver-Burk plot for competitive inhibition in Fig. 1, an intercept on ordinate is a constant value (i/Vi), independent of the concentration of substrate or inhibitor. However, one can use the primary graph of i/t>o versus A to create the secondary plot, by plotting the slopes from die primary plots versus I. A replot of slopes versus I gives... 

Fig. 2.—Graphical Determination of the Maximum Velocity, V, and the Michaelis Constant, K . [(o) v against [S] (f>) t) against[S], Lineweaver—Burk plot (c) a Line-weaver—Burk plot for competitive inhibition (d) a Lineweaver—Burk plot for noncompetitive inhibition.]...

FIGURE 8.3 Lineweaver-Burk plots for competitive (2), noncompetitive (3), and mixed (4) inhibition, relative to the enzymatic reaction in the absence of inhibitors (1). 

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. 

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

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... 

Fig. 9.18. Lineweaver-Burk plots of competitive and pure noncompetitive inhibition. A. lAi versus 1/[S] in the presence of a competitive inhibitor. The competitive inhibitor alters the intersection on the abscissa. The new intersection is 1/K , p (also called 1/K ). A compietitive inhibitor does not affect B. 1/Vj versus 1/[S] in the presence of a pure noncompetitive inhibitor. The noncompetitive inhibitor alters the intersection on the ordinate, Wmax.app W niax> But docs not offcct 1/K j. A pure noncompetitive inhibitor binds to E and ES with the same affinity. If the inhibitor has different affinities for E and ES, the lines will intersect above or below the abscissa, and the noncompetitive inhibitor will change both the and the V, .

The kinetic results of noncompetitive inhibition differ from those of competitive inhibition. The Lineweaver-Burk plots for a reaction in the presence and absence of a noncompetitive inhibitor show that both the slope and the y intercept change for the inhibited reaction (Figure 6.13), without changing the x intercept. The value of decreases, but that of remains the same the inhibitor does not interfere with the binding of substrate to the active site. Increasing the substrate concentration cannot overcome noncompetitive inhibition because the inhibitor and substrate are not competing for the same site. 

FIGURE 6.8 A Lineweaver-Burk plot for the case of competitive enzyme inhibition at three concentrations of inhibitor. 

Strategy We draw a series of Lineweaver-Burk plots for different inhibitor concentrations. If the plots resemble those in Fig. 8.6a, then the inhibition is competitive. On the other hand, if the plots resemble those in Fig. 8.6c, then the inhibition is noncompetitive. To find K, we need to determine the slope at each value of [I], which is equal to aK lv, or Kulv + KM[I]/K f ,ax, then plot this slope against [1] the intercept at [1] = 0 is the value and the... 

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). 

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. 

Use the data in the table above to plot Michaelis-Menten, Lineweaver-Burke and Eadie-Hofstee graphs to determine Km and Vm DC values. State the type of inhibitor which is present. Calculate the K based on Equations 2.10 (for competitive inhibition) or 2.11 (non-competitive inhibition) asappropriate assuming the [I] = 10mmol/l. 

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. 

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 . ... 

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.

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. 

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. 

It can be seen from this equation that competitive inhibitors have no effect on the Vmax of the enzyme, but alter the apparent Km. In the presence of inhibitor, Km will be increased by a factor of (1 + /K ). Lineweaver-Burk plots constructed at various inhibitor concentrations provide a useful diagnostic for this type of inhibition. Figure 2.13 shows that identical y intercepts (l/Vmax) are obtained at different inhibitor concentrations, while x intercepts (reciprocal of apparent Km) decrease with increasing [I], and are equal to — / Km + [1]/ ). ... 

By using lineweaver-Burk plots the authors found that four xanthates exhibited different patterns of mixed, competitive, or uncompetitive inhibition. For the cresolase activity, 1 and 2 demonstrated uncompetitive inhibition but 3 and 4 exhibited competitive inhibition [43]. For the catecholase activity, 1 and 2 showed mixed inhibition but 3 and 4 showed competitive inhibition against tyrosinase [43]. The xanthates (compoimds 1, 2, 3 and 4) have been classified as potent inhibitors against tyrosinase due to their Ki values of 13.8,... 

D23.4 Refer to eqns 23.26 and 23.27, which are the analogues of the Michaelis-Menten and Lineweaver-Burk equations (23.21 and 23,22), as well as to Figure 23.13, There are three major modes of inhibition that give rise to distinctly different kinetic behavior (Figure 23.13), In competitive inhibition the inhibitor binds only to the active site of the enzyme and thereby inhibits the attachment of the substrate. This condition corresponds to a > 1 and a = 1 (because ESI does not form). The slope of the Lineweaver-Burk plot increases by a factor of a relative to the slope for data on the uninhibited enzyme (a = a = I), The y-intercept does not change as a result of competitive inhibition, In uncompetitive inhibition, the inhibitor binds to a site of the enzyme that is removed from the active site, but only if the substrate is already present. The inhibition occurs because ESI reduces the concentration of ES, the active type of the complex, In this case a = 1 (because El does not form) and or > 1. The y-intercepl of the Lineweaver-Burk plot increases by a factor of a relative to they-intercept for data on the uninhibited enzyme, but the slope does not change. In non-competitive inhibition, the inhibitor binds to a site other than the active site, and its presence reduces the ability of the substrate to bind to the active site. Inhibition occurs at both the E and ES sites. This condition corresponds to a > I and a > I. Both the slope and y-intercept... 

As shown in Fig. (11a), chitin-chitosan inhibited the pancreatic lipase activity dose-dependently between the concentrations of 6.25 p,g/ml and 200 ig/ml in the assay system, using triolein emulsified with lecithin. For characterization of the mechanism involved in the inhibition of pancreatic lipase by chitin-chitosan, the enzyme activity was assayed at various concentrations of lecithin-emulsified triolein and in the presence of increasing concentrations of chitin-chitosan. A Lineweaver-Burk plot of the data in Fig. (11 b) shows that chitin-chitosan was a competitive inhibitor. The Km and Vmax values of the lipase activity for lecithin-emulsified triolein were 6.06 ag/ml and 8.7 nmol/ml/min, respectively. The K value of chitin-chitosan on the lipase activity in lecithin-emulsified triolein was 17.6 M-g/ml. When triolein was emulsified with... 

Fig. 6. Competitive inhibition. The diagram shows the characteristic pattern of Lineweaver-Burk plots intersecting on the ordinate for data obtained with and without an added competitive inhibitor, I.

---