Lineweaver-Burk plot uncompetitive

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 9.7 Lineweaver-Burke plot representations ofcompetitive (a), noncompetitive (b), uncompetitive (c) and mixed (d) inhibition.

Full and partial uncompetitive inhibitory mechanisms, (a) Reaction scheme for full uncompetitive inhibition indicates ordered binding of substrate and inhibitor to two mutually exclusive sites. The presence of inhibitor prevents release of product, (b) Lineweaver-Burk plot for full uncompetitive inhibition reveals a series of parallel lines and an increase in the 1/v axis intercept to infinity at infinitely high inhibitor concentrations. In this example, Ki = 3 iulM. (c) Replot of Lineweaver-Burk slopes from (b) is linear, confirming a full inhibitory mechanism, (d) Reaction scheme for partial uncompetitive inhibition indicates random binding of substrate and inhibitor to two mutually exclusive sites. The presence of inhibitor alters the rate of release of product (by a factor P) and the affinity of enzyme for substrate (by a factor a) to an identical degree, while the presence of substrate alters the affinity of enzyme for inhibitor by a. (e) Lineweaver-Burk plot for partial uncompetitive inhibition reveals a series of parallel lines and an increase in the 1/v axis intercept to a finite value at infinitely high inhibitor concentrations. In this example, Ki = 3 iulM and a = = 0.5. (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. 

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. 

Figure 8.4 The Lineweaver-Burk plot (A) and the Hanes plot (B) of typical enzyme kinetics in presence of a competitve (a) noncompetive (b), mixed type (c) and uncompetitive (d) inhibitor.

Uncompetitive inhibitors can bind to the enzyme-substrate complex only, but not to the free enzyme molecule. The Lineweaver-Burk plots in such cases give parallel straight lines for activity-substrate concentration profiles, measured at different concentrations of the inhibitor (Figure 8.4), according to equation ... 

The slope of the Lineweaver-Burk plot is not altered by the presence of an uncompetitive inhibitor, but both intercepts change (Fig. S.I4). 

Uncompetitive inhibition has seldom been reported in studies of xenobiotic metabolism. It occurs when an inhibitor interacts with an enzyme-substrate complex but cannot interact with free enzyme. Both Km and Vmax change by the same ratio, giving rise to a family of parallel lines in a Lineweaver-Burke plot. 

Reversible inhibitors bind an enzyme through weak, intermolecular forces and establish an equilibrium of being bound or unbound to the enzyme. A competitive inhibitor binds at the active site and prevents the substrate from binding. A noncompetitive inhibitor binds an allosteric site on the enzyme and prevents conversion of the substrate to product. Uncompetitive inhibitors bind the enzyme-substrate complex and make it inactive. All three types of inhibitors show characteristic, distinctive features in a Lineweaver-Burk plot. 

An uncompetitive inhibitor is much like a noncompetitive inhibitor except that an uncompetitive inhibitor binds only the enzyme-substrate complex (Scheme 4.14). The inhibitor-bound ternary complex cannot form product. Uncompetitive inhibitors cause both Vmax and Km to decrease by the same factor (Figure 4.17). Because the slope of a Lineweaver-Burk plot is Km/Vmxi, the slope of the line of an inhibited enzyme is unchanged from the uninhibited enzyme.4... 

Figure 5 Lineweaver-Burk plots for determination of uncompetitive inhibitor kinetic constants, by Equation 6.

Figure 6 (a) Replot of data from Lineweaver-Burk plots (Fig. 5) for determination of K, values for uncompetitive inhibitors, (b) Alternative replot of data from Lineweaver-Burk plots (Fig. 12) for determination of Kj values for uncompetitive inhibitors. 

Figure 2.15. Lineweaver-Burk plots for uncompetitive inhibitors.

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... 

The Lineweaver-Burk plot is a diagnostic for uncompetitive inhibition. The slope of the lines is independent on the concentration of inhibitor, while the y-intersept increases with an increase of [I] (Figure 6.48). 

Noncompetitive inhibitors both increase the apparent Km and reduce the apparent Vmax of an enzyme-catalyzed reaction. Therefore, they affect both the slope and the y-intercept of a Lineweaver-Burk plot, as Figures 7-5 and 7-6 show. Uncompetitive inhibitors, because they reduce Vmax only, increase the reciprocal of Vmax. The lines of the reciprocal plot are parallel in this case. 

FIGURE 6.10 A Lineweaver-Burk plot for the case of uncompetitive inhibition at three concentrations of inhibitor. 

Although enzyme reactions are highly specific, inhibition of the enzymes do occur. Inhibitors, substances that decrease the rate of an enzyme-catalyzed reaction, are classified as competitive, noncompetitive, uncompetitive, or mixed [4]. Each type can be characterized by deviation from the Lineweaver-Burk plot of the corresponding uninhibited reaction. Competitive inhibitors compete for the active sites with the substrate and slow down the enzyme reaction they increase Km but have no affect on Vmax- Noncompetitive inhibitors bind reversibly to the enzyme at a site different from the active site, but one that is necessary for the enzyme action. These inhibitors decrease Emax, but Km is unaffected. Uncompetitive inhibitors are known to bind reversibly to the enzyme-substrate complex to form an inactive enzyme-substrate-inhibitor complex. A decrease in Km and max by the same factor is observed (i.e., the Lineweaver-Burk plot is parallel to the plot of the uninhibited reaction). In mixed-type inhibitors, more than one of the foregoing mechanisms operate, and Km and Lmax values are both altered. 

Figure 5.10. Demonstration of the four basic types of inhibitions of enzyme kinetics in Lineweaver-Burk plots (see Fig. 4.24c) (a) competitive, (b) noncompetitive, (c) uncompetitive, and (d) substrate inhibition. The parameters and can be estimated from the intercepts and slope of the line with p — 0, where p = inhibitor concentration.

The three different types of inhibition—competitive, uncompetitive, and noncompetitive (mixed) inhibition—are shown on the Lineweaver-Burk plot ... 

Uncompetitive inhibitors bind only to a formed ES complex. This type of inhibition, characterized by equal effects on both V and K , is rare in one-substrate reactions but may occur as a type of product inhibition in reactions with multiple substrates and products. Figure 6.10 illustrates mechanistic and plot differences between the discussed inhibitions. Table 6.1 summarizes the effect of inhibitors on Lineweaver-Burk plot parameters. Graphical methods are available for the estimation of Aj, the inhibition constant. In competitive inhibition, for example jgjjjj... 

The Lineweaver-Burk plot is a diagnostic for uncompetitive inhibition. 

While investigating antibrowning agents, it was found [6] that dodecyl (C12) gaUate (3,4,5 trihydroxybenzoate) inhibits oxidation of L-3,4-dixydroxyphenylalanine (L-DOPA) catalyzed by tyrosinase. The Lineweaver-Burk plots (Fig. 6.51) revealed an uncompetitive inhibitor behavior of dodecyl gaUate. 

Inhibition experiments are performed by varying the concentration of substrate around the Km just as you would in an experiment to determine the Km and Vmax, except that the experiment is repeated at several different concentrations of an inhibitor. On a Lineweaver-Burk transformation (1/v vs. 1/[S]), each different inhibitor concentration will be represented as a different straight line (Fig. 8-6). The pattern that the lines make tells you the kind of inhibition. There are three possibilities (1) The inhibitor can affect only the slopes of the plot (competitive), (2) the inhibitor can affect only the y intercepts of the plot (uncompetitive), or (3) the inhibitor can affect both the slopes and the intercepts (noncompetitive). Plots are plots, and what s really important is not the pattern on a piece of paper but what the pattern tells us about the behavior of the enzyme. 

Figure 17.8. (a) Lineweaver-Burk, (b) Eadie-Hof-stee, and (c) Hanes-Woolf plots exhibiting uncompetitive inhibition patterns. The dashed line indicates the reaction in the absence of inhibitor, whereas the solid lines represent enzymatic reactions in the presence of increasing concentrationsof inhibitor. 

For each of the four types of inhibition of a Michaelis-Menten enzyme [competitive, Eq. (5.25) noncompetitive and mixed Eq. (5.29) and uncompetitive, Eq. (5.32)], derive the corresponding Lineweaver-Burk equations [Eqs. (5.26), and (5.30), respectively] and draw the characteristic plots that are the basis for the rapid visnal identification of which type of inhibition apphes when analyzing enzyme kinetic data. 

Fig. 3.5 Graphical representation of inhibition mechanisms in Lineweaver-Burke double reciprocal plots. Cl competitive inhibition NCI non-competitive inhibition UCI uncompetitive inhibition MTI mixed-type inhibition...

In the preceding sections, we have shown that all the rate equations, in the presence of a competitive, noncompetitive, or an uncompetitive inhibitor, have a form of a Michaelis-Menten equation, and can be linearized in the Lineweaver-Burk manner, in the fashion of Hanes, or in the form of Dixon plots ... 

FIGURE 1.3 Types of enzyme inhibition represented in both Lineweaver—Burk and Eadie-Hofstee plots (a) competitive, (b) noncompetitive, (c) uncompetitive, and (d) mixed type. 

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